Cortical Circuits of Callosal GABAergic Neurons

Cortical Circuits of Callosal GABAergic Neurons Abstract Anatomical studies have shown that the majority of callosal axons are glutamatergic. However, a small proportion of callosal axons are also immunoreactive for glutamic acid decarboxylase, an enzyme required for gamma-aminobutyric acid (GABA) synthesis and a specific marker for GABAergic neurons. Here, we test the hypothesis that corticocortical parvalbumin-expressing (CC-Parv) neurons connect the two hemispheres of multiple cortical areas, project through the corpus callosum, and are a functional part of the local cortical circuit. Our investigation of this hypothesis takes advantage of viral tracing and optogenetics to determine the anatomical and electrophysiological properties of CC-Parv neurons of the mouse auditory, visual, and motor cortices. We found a direct inhibitory pathway made up of parvalbumin-expressing (Parv) neurons which connects corresponding cortical areas (CC-Parv neurons → contralateral cortex). Like other Parv cortical neurons, these neurons provide local inhibition onto nearby pyramidal neurons and receive thalamocortical input. These results demonstrate a previously unknown long-range inhibitory circuit arising from a genetically defined type of GABAergic neuron that is engaged in interhemispheric communication. auditory, callosum, inhibition, long-range GABAergic neurons, motor cortex, Parv neurons, visual Introduction It is a well-established principle of the cortical circuit organization that excitation is both local and long-range, whereas inhibition is exclusively local, but this is not true; inhibition can be long-range as well. While much progress has been made in understanding the local cortical circuit organization of GABAergic interneurons (Buzsaki 1984; Freund and Buzsaki 1996; Ali et al. 1999; Holmgren et al. 2003; Pouille and Scanziani 2004; Silberberg and Markram 2007; Pouille et al. 2009; Stokes and Isaacson 2010; Hayut et al. 2011; Pouille et al. 2013; Crandall and Connors 2016), much less has been learned about the cortical and extracortical input organization of long-range GABAergic neurons. Anatomical studies using retrograde tracers and immunohistochemistry have proposed that between 1% and 10% of the cortical GABAergic neurons in rodents, cats, and monkeys also give rise to long-range corticocortical projections (McDonald and Burkhalter 1993; Tomioka et al. 2005; Higo et al. 2007; Tomioka and Rockland 2007; Higo et al. 2009). It has been previously demonstrated that some axons of the corpus callosum, important for transfer of sensory information between the right and left cerebral hemispheres, are immunoreactive for glutamic acid decarboxylase, an enzyme required for gamma-aminobutyric acid (GABA) synthesis and a specific marker for GABAergic neurons (Fabri and Manzoni 2004). Additionally, anatomical studies have demonstrated the presence of callosal nonpyramidal neuron projections (Code and Winer 1985; Hughes and Peters 1990; Peters et al. 1990; Hughes and Peters 1992; Gonchar et al. 1995), but the cells of origin and physiological function of these GABAergic projections were not explored. A growing body of evidence suggests that many of the long-range GABAergic projections arise from somatostatin-expressing neurons (Tomioka et al. 2005; Higo et al. 2007; Tomioka and Rockland 2007; Higo et al. 2009; McDonald et al. 2012; Melzer et al. 2012; Rock et al. 2016). However, it has been recently suggested that parvalbumin-expressing (Parv) neurons  may contribute to long-range GABAergic projections as well (Jinno and Kosaka 2004; Lee et al. 2014). The present study focused on four main goals: (1) determine if Parv neurons send long-range GABAergic projections to the contralateral cortex via the corpus callosum; (2) describe the anatomical and electrophysiological properties of these corticocortical Parv neurons (CC-Parv neurons); (3) determine the local (ipsilateral) inhibitory connectivity pattern of CC-Parv neurons; and (4) investigate the thalamic inputs onto CC-Parv neurons. Our approach consisted of viral tracing and optogenetics methods to investigate these questions. Using these manipulations, we found a direct inhibitory pathway made up of Parv neurons which connects corresponding cortical areas (CC-Parv neurons → contralateral cortex). Our data show that these neurons provide local inhibition onto nearby pyramidal neurons and are recipients of thalamocortical input. Overall, we describe a previously unknown long-range inhibitory circuit and attribute a specific function to a genetically defined type of GABAergic neuron in interhemispheric communication. Methods All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Texas at San Antonio. Procedures followed animal welfare guidelines set by the National Institutes of Health. Mice used in this experiment were housed in a vivarium maintaining a 12 h light/dark schedule and given ad libidum access to mouse chow and water. Transgenic Mouse Lines The following mouse lines were used in this study: Parv-Cre: B6;129P2-Pvalbtm1(cre)Arbr/J, The Jackson Laboratory stock number 008069; ROSA-tdTomato reporter: B6.CG.Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, The Jackson Laboratory, stock number 007914. Parv-Cre female mice were crossed with a ROSA-tdTomato reporter male mouse to generate a Parv-Cre-tdTomato line (parvalbumin-containing neurons expressed both Cre and tdTomato). Stereotaxic Injections Basic Surgical Procedures Mice were initially anesthetized with isoflurane (3–5%; 1 L/min O2 flow) in preparation for the stereotaxic injections detailed in the sections below. The mice were head-fixed on a stereotaxic frame (Model 1900; Kopf Instruments) using nonrupture ear bars. Anesthesia was maintained at 1–1.5% isoflurane for the duration of the surgery. A warming pad was used to maintain body temperature during the procedure. Standard aseptic technique was followed for all surgical procedures. Injections were performed using a pressure injector (Nanoject II; Drummond Scientific) mounted on the stereotaxic frame. Injections were delivered through a borosilicate glass injection pipette (Wiretrol II; Drummond Scientific) with a taper length of ~30 mm and a tip diameter of ~50 μm. After lowering it to the target injection depth, the glass pipette remained in place 5–10 min both before and after the injection was made. Both male and female Parv-Cre or Parv-Cre-tdTomato mice, P31–40 at the time of the injection, were utilized in these experiments. Retrograde Labeling of CC-Parv Neurons CC-Parv neurons in the auditory cortex (AC) were retrogradely labeled with green fluorescent protein (GFP) using AAV1.CAG.Flex.eGFP.WPRE.bGH (AAV1.GFP.Flex; University of Pennsylvania Vector Core) stereotaxically injected into the right AC of Parv-Cre mice (12 mice from 7 litters). Injections were performed as above, with the following parameters: stereotaxic coordinates for the AC injection site were 2.45 mm posterior and 4.3 mm lateral to bregma. Approximately 50 nL of AAV1.GFP.Flex was delivered between two depths in the AC, 1.0 mm and 0.75 mm below the surface of the brain, over the course of 5 min. CC-Parv neurons in the visual cortex (VC; 3 mice from 1 litter) and motor cortex (MC; 4 mice from 1 litter) were also retrogradely labeled with GFP as in the AC or with tdTomato using AAV1.CAG.Flex.tdTomato.WPRE.bGH (AAV1.tdTomato.Flex; University of Pennsylvania Vector Core) for recording intrinsic properties and morphological reconstruction. Stereotaxic coordinates for VC were 2.8 mm posterior and 2.25 mm lateral to bregma. Approximately 50 nL of AAV1.GFP.Flex or AAV1.tdTomato.Flex was delivered between two depths in the VC, 0.75 mm and 0.5 mm below the surface of the brain, over the course of 5 min. Stereotaxic coordinates for MC were 1.1 mm anterior and 1.6 mm lateral to bregma. Approximately 50 nL of AAV1.GFP.Flex or AAV1.tdTomato.Flex was delivered between two depths in the MC, 0.85 mm and 0.5 mm below the surface of the brain, over the course of 5 min. Retrograde Transfection of CC-Parv Neurons with Channelrhodopsin-2 CC-Parv neurons in the AC were retrogradely transfected with channelrhodopsin-2 (ChR2) using AAV1.CAGGS.Flex.ChR2-tdTomato.WPRE.SV40 (AAV1.ChR2.Flex; University of Pennsylvania Vector Core) stereotaxically injected into the right AC of Parv-Cre mice (5 mice from 4 litters). Injections were performed in the same manner and using the same AC stereotaxic coordinates as described previously. Retrograde Labeling of CC-Parv Neurons and Transfection of Thalamic Axons with ChR2 CC-Parv neurons in the AC were retrogradely labeled with GFP in the same manner and using the same AC stereotaxic coordinates as described previously in Parv-Cre mice (7 animals from 5 litters). In these same animals, thalamocortical projections were transfected with ChR2 using AAV9.CAG.hChR2.tdTomato (AAV9.ChR2; University of North Carolina Vector Core) stereotaxically injected into the left medial geniculate body (MGB) with the following parameters: stereotaxic coordinates for the left MGB injection site were 3.2 mm posterior and 2.0 mm lateral to bregma. Approximately 80–100 nL of AAV.ChR2 was delivered at 3.2 mm ventral to bregma over the course of 10 min. In vitro Slice Preparation and Recordings Slice preparation and electrophysiological recordings were performed as previously described (Rock and Apicella 2015; Rock et al. 2016). We allowed 2–4 weeks for expression of ChR2 or GFP. Mice were anesthetized with isoflurane and decapitated. Coronal slices (300 μm) containing the area of interest (AC, VC, or MC) were sectioned on a vibratome (VT1200S; Leica) in a chilled cutting solution containing the following (in mM): 100 choline chloride, 25 NaHCO3, 25 d-glucose, 11.6 sodium ascorbate, 7 MgSO4, 3.1 sodium pyruvate, 2.5 KCl, 1.25 NaH2PO4, and 0.5 CaCl2. These slices were incubated in oxygenated artificial cerebrospinal fluid (ACSF) in a submerged chamber at 35–37 °C for 30 min and then at room temperature (21–25 °C) until recordings were performed. ACSF contained the following (in mM): 126 NaCl, 26 NaHCO3, 10 d-glucose, 2.5 KCl, 2 CaCl2, 1.25 NaH2PO4, and 1 MgCl2; osmolarity was ~290 Osm/L. Whole-cell recordings were performed in 31–33 °C ACSF. Thin-walled borosilicate glass pipettes (Warner Instruments) were pulled on a vertical pipette puller (PC-10; Narishige) and typically were in the range of 3–5 MΩ resistance when filled with a cesium-based intracellular solution, which contained the following (in mM): 110 CsOH, 100 d-gluconic acid, 10 CsCl2, 10 HEPES, 10 phosphocreatine, 1 EGTA, 1 ATP, and 0.3–0.5% biocytin. Inhibitory postsynaptic currents (IPSCs) were recorded in the voltage-clamp configuration with a holding potential of 0 mV (the calculated reversal potential for glutamatergic excitatory conductances). Intrinsic properties were recorded in the current-clamp configuration using a potassium-based intracellular solution at 31–33 °C. Potassium-based intracellular solution contained the following (in mM): 120 potassium gluconate, 20 KCl, 10 HEPES, 10 phosphocreatine, 4 ATP, 0.3 GTP, 0.2 EGTA, and 0.3–0.5% biocytin. Signals were sampled at 10 kHz and filtered at 4 kHz. Pharmacological blockers used were CPP (5 μM; Tocris Bioscience), NBQX (10 μM; Abcam), and Gabazine (25 μM; Abcam). To block disynaptic and polysynaptic responses and to examine direct thalamocortical monosynaptic inputs onto CC-Parv neurons, we applied both TTX (1 μM; Tocris Bioscience) and 4-AP (100 μM; Sigma-Aldrich). Hardware control and data acquisition were performed by Ephus (www.ephus.org) (Suter et al. 2010). ChR2 Photostimulation CC-Parv somata transfected with ChR2 were present in the left AC following injection of AAV1.ChR2.Flex in the right AC. Because of variability both in ChR2 expression levels (number of ChR2 molecules per transfected neuron) and in transfection efficiency (number of ChR2-expressing neurons per animal), to minimize the variability from experiment to experiment, we performed the electrophysiological recording in the same slices containing the AC and with the highest density of ChR2-transfected axons. We recorded IPSCs from nearby pyramidal neurons during photoactivation of the CC-Parv ChR2-transfected neurons and axon terminals. A 470 nm wavelength blue LED (CoolLED pE excitation system) passed through a GFP filter cube (Endow GFP/EGFP longpass, C-156625; Chroma) and a 60× water-immersion objective was used for photoactivation of ChR2. Distribution of CC-Parv Neurons in AC CC-Parv neurons in the AC were retrogradely labeled with GFP using AAV1.GFP.Flex stereotaxically injected into the right AC of Parv-Cre-tdTomato mice (3 mice from 1 litter). Injections were performed in the same manner and using the same auditory cortex stereotaxic coordinates as described previously. Three weeks following the injection, these mice were deeply anesthetized with 5% isoflurane and transcardially perfused with phosphate-buffered saline (PBS), followed by 10% buffered formalin (Sigma-Aldrich). The brain was carefully removed and fixed overnight. The following day, the fixed brain was sliced into 200-μm thick sections on a vibrating microtome. Following six washes in PBS, the slices were mounted on microscope slides with Fluoromount-G (Southern Biotech). Confocal images were taken with a Zeiss LSM-710 microscope and a 40× oil-immersion objective. Images were rotated, cropped, and the brightness/contrast was adjusted in ImageJ (National Institutes of Health). Scored sections (9 slices, 3 slices from each of 3 mice) contained a 100-μm wide portion of AC spanning from the pia to the white matter. The distance between the pia and the white matter was normalized to 1000 μm, and then the cortex was divided into layers based on the following thicknesses: L1, 0–150 μm; L2/3, 151–375 μm; L4, 376–500 μm; L5, 501–750 μm; and L6, 751–1000 μm. Layer thicknesses in the AC were similar to the ones used in a previous study (Ji et al. 2016). Counts of parvalbumin neurons expressing tdTomato only and of CC-Parv neurons coexpressing GFP and tdTomato were made by hand. All of the GFP-expressing neurons from these sections were found to coexpress tdTomato. VGluT2 Immunoreactivity on CC-Parv Dendrites CC-Parv neurons in the AC were retrogradely labeled with tdTomato using AAV1.tdTomato.Flex stereotaxically injected into the right AC of Parv-Cre mice (2 mice from 1 litter). Three weeks following the injection, these mice were deeply anesthetized with 5% isoflurane and transcardially perfused with PBS, followed by 10% buffered formalin. The brain was carefully removed and fixed overnight. The following day, the fixed brain was sliced into 50-μm thick sections on a vibrating microtome. The sections were then incubated in 0.3% Triton X-100 and 5% normal goat serum (NGS) in PBS at room temperature for 2 h on a shaking platform. Following this blocking stage, the slices were incubated in the primary antibody (guinea pig Anti-VGLUT2, 1:15 000; Millipore) with 0.1% Triton X-100 and 1% NGS in PBS overnight at 4 °C. The next day, the slices were washed 3× with PBS and incubated for 3 h at room temperature on a shaking platform with the secondary antibody (goat anti-guinea pig IgG-conjugated Alexa 488, 1:500; Life Technologies) with 0.1% Triton X-100 and 1% NGS in PBS. Following three washes in PBS, the slices were mounted on microscope slides with Fluoromount-G. Confocal images were taken with a Zeiss LSM-710 microscope and a 100× oil-immersion objective to document appositions of tdTomato CC-Parv dendrites and Alexa 488 VGluT2 puncta. Images were rotated, cropped, and the brightness/contrast was adjusted in ImageJ. Histology During whole-cell recordings, neurons were filled with an internal solution containing 0.3–0.5% biocytin. Filled neurons were held for at least 20 min, and then the slices were fixed in a formalin solution at 4 °C until ready for processing. The slices were washed 6× in PBS and placed in a 4% streptavidin (Alexa Fluor 488 or 594; Life Technologies) solution with 0.3% Triton X-100 in PBS. Slices were allowed to incubate in this solution at 4 °C overnight, then washed 6× in PBS and mounted with Fluoromount-G on a glass microscope slide. Confocal images were taken with a Zeiss LSM-710 microscope at varying magnifications (3×–63×). Individual high magnification images were stitched together, when necessary, using XuvStitch software (XuvTools). Image adjustment was performed in ImageJ for brightness/contrast corrections and pseudocoloring. Neurons were morphologically reconstructed in three dimensions using the Simple Neurite Tracer plugin for ImageJ (Longair et al. 2011). Data Analysis Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. In the text, data are indicated as group averages (± s.e.m.). Data analysis was performed offline using custom MATLAB (MathWorks) routines. Results Anatomical Characterization of Long-Range Callosal Parv Neurons To visualize long-range GABAergic projections originating in the cortex and terminating in the contralateral cortex, we conditionally expressed GFP in Parv interneurons by injecting AAV1.GFP.Flex into the right auditory cortex (AC) of Parv-Cre transgenic mice (Taniguchi et al. 2011). Using this virus, long-range callosal Parv neurons (from this point forward referred to as CC-Parv neurons) were retrogradely labeled with GFP. Somata of CC-Parv neurons were present in all layers of the AC, and GFP-positive axons were visible in coronal sections of the corpus callosum in these mice (Fig. 1a–d). Next, to determine the proportion of CC-Parv neurons, we injected AAV1.GFP.Flex into the right AC of Parv-Cre-tdTomato transgenic mice (Fig. 2). GFP was colocalized with Parv/tdTomato-expressing neurons in the AC (Fig. 2b). We found that CC-Parv neurons can account for up to 42% of the entire Parv population (601 Parv neurons, 253 CC-Parv neurons; n = 9 slices, 3 mice; Fig. 2c,d). These data show that a large proportion of Parv neurons, arising from all layers of the AC, project to the contralateral AC via the corpus callosum. Figure 1. View largeDownload slide Characterization of CC-Parv neurons in the mouse auditory cortex. (a) Schematic depicting injection site using the Parv-Cre transgenic mouse line to retrogradely transfect CC-Parv neurons in the contralateral auditory cortex with GFP. (b) Bright-field (left) and epifluorescence (right) images of a slice containing the auditory cortex injection site for AAV1.GFP.Flex. (c) Bright-field (left) and epifluorescence (middle) images of a slice containing the left auditory cortex showing expression of GFP following injection of AAV1.GFP.Flex into the right auditory cortex. Right: confocal epifluorescence image of CC-Parv GFP-positive neurons. (d) Bright-field (left) and epifluorescence (middle) of CC-Parv GFP-positive axons in the corpus callosum. The arrows indicate the location of GFP-positive axons. Right: confocal higher magnification epifluorescence image of CC-Parv GFP-positive axons in the callosum. Figure 1. View largeDownload slide Characterization of CC-Parv neurons in the mouse auditory cortex. (a) Schematic depicting injection site using the Parv-Cre transgenic mouse line to retrogradely transfect CC-Parv neurons in the contralateral auditory cortex with GFP. (b) Bright-field (left) and epifluorescence (right) images of a slice containing the auditory cortex injection site for AAV1.GFP.Flex. (c) Bright-field (left) and epifluorescence (middle) images of a slice containing the left auditory cortex showing expression of GFP following injection of AAV1.GFP.Flex into the right auditory cortex. Right: confocal epifluorescence image of CC-Parv GFP-positive neurons. (d) Bright-field (left) and epifluorescence (middle) of CC-Parv GFP-positive axons in the corpus callosum. The arrows indicate the location of GFP-positive axons. Right: confocal higher magnification epifluorescence image of CC-Parv GFP-positive axons in the callosum. Figure 2. View largeDownload slide Laminar distribution and proportion of CC-Parv neurons in the mouse auditory cortex. (a) Schematic depicting injection site using the Parv-Cre-tdTomato transgenic mouse line to identify the laminar distribution of CC-Parv neurons and their proportion compared with the overall Parv population in the auditory cortex. Right auditory cortex: AAV1.GFP.Flex injection site; left auditory cortex: yellow CC-Parv somata coexpressing GFP and tdTomato; green CC-Parv GFP-positive axons; red Parv tdTomato-positive neurons. (b) Left: epifluorescence confocal image of a coronal section containing the left auditory cortex with Parv tdTomato-positive neurons. Middle: epifluorescence confocal image of CC-Parv GFP-positive neurons. Right: merged epifluorescence confocal image of Parv tdTomato-positive neurons and CC-Parv GFP-positive neurons. Layers are indicated on the right. (c) The laminar distribution and ratio of Parv-expressing interneurons and CC-Parv neurons. For the quantification of parvalbumin neurons expressing tdTomato and of CC-Parv neurons coexpressing GFP and tdTomato, the distance between the pia and white matter was normalized to 1000 μm, and the cortex was divided into layers based on the following thicknesses: L1, 0–150 μm; L2/3, 151–375 μm; L4, 376–500 μm; L5, 501–750 μm; and L6, 751–1000 μm. (d) Summary plot of the ratio of CC-Parv neurons versus the total population of Parv-expressing neurons. Figure 2. View largeDownload slide Laminar distribution and proportion of CC-Parv neurons in the mouse auditory cortex. (a) Schematic depicting injection site using the Parv-Cre-tdTomato transgenic mouse line to identify the laminar distribution of CC-Parv neurons and their proportion compared with the overall Parv population in the auditory cortex. Right auditory cortex: AAV1.GFP.Flex injection site; left auditory cortex: yellow CC-Parv somata coexpressing GFP and tdTomato; green CC-Parv GFP-positive axons; red Parv tdTomato-positive neurons. (b) Left: epifluorescence confocal image of a coronal section containing the left auditory cortex with Parv tdTomato-positive neurons. Middle: epifluorescence confocal image of CC-Parv GFP-positive neurons. Right: merged epifluorescence confocal image of Parv tdTomato-positive neurons and CC-Parv GFP-positive neurons. Layers are indicated on the right. (c) The laminar distribution and ratio of Parv-expressing interneurons and CC-Parv neurons. For the quantification of parvalbumin neurons expressing tdTomato and of CC-Parv neurons coexpressing GFP and tdTomato, the distance between the pia and white matter was normalized to 1000 μm, and the cortex was divided into layers based on the following thicknesses: L1, 0–150 μm; L2/3, 151–375 μm; L4, 376–500 μm; L5, 501–750 μm; and L6, 751–1000 μm. (d) Summary plot of the ratio of CC-Parv neurons versus the total population of Parv-expressing neurons. Anatomical and Electrophysiological Properties of Callosal Parv Neurons To determine the anatomical and electrophysiological properties of CC-Parv neurons, we once again injected AAV1.GFP.Flex into the right AC of Parv-Cre mice (Fig. 1a). This approach allowed us to visually identify and record from CC-Parv neurons using whole-cell patch clamp. We verified their identity based on the comparison with electrophysiological properties of Parv interneurons (for review, see Hu et al. 2014). These properties include a narrow action potential and high rheobase (the smallest current step evoking an action potential) (Fig. 3a, c, e, g, inset; action potentials in CC-Parv neurons, shown in black, are very narrow). The responses to current steps in CC-Parv neurons were typical for Parv interneurons (Fig. 3; notice the high current step needed to generate action potentials). To characterize the electrophysiological properties of CC-Parv neurons, we recorded  45 GFP-labeled neurons spanning from layer 2/3 to layer 6 in the AC (Table 1). Table 1 Electrophysiological properties of CC-Parv neurons   CC-Parv L2/3  CC-Parv L4  CC-Parv L5  CC-Parv L6  n = 11  n = 10  n = 14  n = 11  Resting potential (mV)  −80.5 ± 1.3  −78.4 ± 1.2  −76.5 ± 0.9  −77.5 ± 1.5  Input resistance (MΩ)  125.6 ± 10.8  106.5 ± 7.2  117.8 ± 7.8  114.7 ± 8.7  Membrane time constant (ms)  0.58 ± 0.04  0.6 ± 0.03  0.6 ± 0.06  0.6 ± 0.004  Rheobase (pA)  268.2 ± 23.6  320 ± 32.6  271.4 ± 22.6  270 ± 18.6  After hyperpolarization (mV)  −11.6 ± 2.2  −14.7 ± 1.6  −13.2 ± 1.2  −12.5 ± 0.9  Action potential threshold (mV)  −36.4 ± 1.2  −38.2 ± 2.2  −40.1 ± 1.4  −40.7 ± 1.7  Action potential height (mV)  35.2 ± 1.8  31.9 ± 2.9  39.5 ± 2.6  40.5 ± 3.1  Action potential width (ms)  0.46 ± 0.01  0.46 ± 0.01  0.46 ± 0.08  0.47 ± 0.01  F/I slope (Hz/pA step)  0.7 ± 0.05  0.7 ± 0.08  0.72 ± 0.04  0.77 ± 0.05  Spike frequency adaptation (third/fifth)  1 ± 0.05  1 ± 0.02  0.97 ± 0.08  1 ± 0.04    CC-Parv L2/3  CC-Parv L4  CC-Parv L5  CC-Parv L6  n = 11  n = 10  n = 14  n = 11  Resting potential (mV)  −80.5 ± 1.3  −78.4 ± 1.2  −76.5 ± 0.9  −77.5 ± 1.5  Input resistance (MΩ)  125.6 ± 10.8  106.5 ± 7.2  117.8 ± 7.8  114.7 ± 8.7  Membrane time constant (ms)  0.58 ± 0.04  0.6 ± 0.03  0.6 ± 0.06  0.6 ± 0.004  Rheobase (pA)  268.2 ± 23.6  320 ± 32.6  271.4 ± 22.6  270 ± 18.6  After hyperpolarization (mV)  −11.6 ± 2.2  −14.7 ± 1.6  −13.2 ± 1.2  −12.5 ± 0.9  Action potential threshold (mV)  −36.4 ± 1.2  −38.2 ± 2.2  −40.1 ± 1.4  −40.7 ± 1.7  Action potential height (mV)  35.2 ± 1.8  31.9 ± 2.9  39.5 ± 2.6  40.5 ± 3.1  Action potential width (ms)  0.46 ± 0.01  0.46 ± 0.01  0.46 ± 0.08  0.47 ± 0.01  F/I slope (Hz/pA step)  0.7 ± 0.05  0.7 ± 0.08  0.72 ± 0.04  0.77 ± 0.05  Spike frequency adaptation (third/fifth)  1 ± 0.05  1 ± 0.02  0.97 ± 0.08  1 ± 0.04  Figure 3. View largeDownload slide Electrophysiological properties of long-range CC-Parv neurons in the mouse auditory cortex. (a) Response recorded from a layer 2/3 CC-Parv neuron in the auditory cortex during injection of a hyperpolarizing current (1 s, −200 pA pulse) and a train of action potentials recorded during injection of a depolarizing current (1 s, 400 pA pulse). Top inset: single action potential from a L2/3 CC-Parv neuron. (b) Summary plot of Vrest: resting membrane potential; Rin: input resistance; Tau: membrane time constant; Rheobase: the smallest current step evoking an action potential; AHP: after hyperpolarization; AP thr: action potential threshold; AP height: action potential height; AP width: action potential width; F/I slope; and SFA: spikes frequency adaptation from layer 2/3 CC-Parv neurons in the auditory cortex (n = 11; animals n = 8). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. (c) Same as in panel (a), for a layer 4 CC-Parv neuron. (d) Same as in panel (b), for layer 4 CC-Parv neurons (n = 10; animals n = 6). (e) Same as in panel (a), for a layer 5 CC-Parv neuron. (f) Same as in panel (b), for layer 5 CC-Parv neurons (n = 14; animals n = 7). (g) Same as in panel (a), for a layer 6 CC-Parv neuron. (h) Same as in panel (b), for layer 6 CC-Parv neurons (n = 10; animals n = 7). Figure 3. View largeDownload slide Electrophysiological properties of long-range CC-Parv neurons in the mouse auditory cortex. (a) Response recorded from a layer 2/3 CC-Parv neuron in the auditory cortex during injection of a hyperpolarizing current (1 s, −200 pA pulse) and a train of action potentials recorded during injection of a depolarizing current (1 s, 400 pA pulse). Top inset: single action potential from a L2/3 CC-Parv neuron. (b) Summary plot of Vrest: resting membrane potential; Rin: input resistance; Tau: membrane time constant; Rheobase: the smallest current step evoking an action potential; AHP: after hyperpolarization; AP thr: action potential threshold; AP height: action potential height; AP width: action potential width; F/I slope; and SFA: spikes frequency adaptation from layer 2/3 CC-Parv neurons in the auditory cortex (n = 11; animals n = 8). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. (c) Same as in panel (a), for a layer 4 CC-Parv neuron. (d) Same as in panel (b), for layer 4 CC-Parv neurons (n = 10; animals n = 6). (e) Same as in panel (a), for a layer 5 CC-Parv neuron. (f) Same as in panel (b), for layer 5 CC-Parv neurons (n = 14; animals n = 7). (g) Same as in panel (a), for a layer 6 CC-Parv neuron. (h) Same as in panel (b), for layer 6 CC-Parv neurons (n = 10; animals n = 7). Statistical analysis to compare the electrophysiological properties of CC-Parv neurons revealed no difference between CC-Parv neurons located in different cortical layers. Confocal images of biocytin-filled CC-Parv neurons showed that they are similar to basket-cell interneurons in their morphology and send an axonal projection towards/into the subcortical white matter (Fig. 4). These data show that CC-Parv neuronal morphology and intrinsic electrophysiological properties resemble those of the Parv basket-cell interneurons. Figure 4. View largeDownload slide Morphological properties of long-range CC-Parv neurons in the mouse auditory cortex. (a) Morphological reconstruction of a layer 4 CC-Parv neuron (dendrites: white; axon: red). (b) Same as in panel (a), for a layer 5 CC-Parv neuron. Figure 4. View largeDownload slide Morphological properties of long-range CC-Parv neurons in the mouse auditory cortex. (a) Morphological reconstruction of a layer 4 CC-Parv neuron (dendrites: white; axon: red). (b) Same as in panel (a), for a layer 5 CC-Parv neuron. Do Callosal Parv Neurons Inhibit Pyramidal Neurons Locally? To determine the connectivity pattern of CC-Parv neurons onto pyramidal neurons in the AC, we used an optogenetic approach in which we conditionally and retrogradely expressed ChR2 (Nagel et al. 2003) in CC-Parv neurons by injecting AAV1.ChR2.Flex into the right AC of Parv-Cre transgenic mice (Fig. 5a–d). By expressing ChR2, mammalian neurons can be excited with high temporal resolution (Boyden et al. 2005; Cardin et al. 2009; Apicella et al. 2012; Rock and Apicella 2015; Rock et al. 2016). Three to four weeks following viral transfection, we recorded from the left AC in cell-attached mode and triggered photo-evoked action potentials from CC-Parv neurons (Fig. 5f). Blue light stimulation (470 nm) caused CC-Parv-neurons to fire action potentials (n = 10). We used the trough of the photo-evoked action potential to determine the onset of the action potential (3.8 ± 0.4 ms, n = 10). The morphological and the electrical identity of patched neurons were confirmed after photostimulation by breaking in and recording the neurons’ electrical properties in current-clamp mode. At the same time, neurons were filled with biocytin and subsequently stained for light-microscopic morphological identification (Fig. 5e). Figure 5. View largeDownload slide Photostimulation of auditory CC-Parv neurons elicits direct inhibition onto local pyramidal neurons. (a) Schematic depicting injection site using the Parv-Cre transgenic mouse line to retrogradely transfect CC-Parv neurons in the contralateral auditory cortex with ChR2. Right auditory cortex: AAV1.ChR2.Flex injection site. Left auditory cortex: red CC-Parv ChR2-tdTomato-positive neurons and callosum axons. (b) Bright-field (left) and epifluorescence (right) images of a slice containing the auditory cortex injection site for AAV1.ChR2.Flex. (c) Bright-field (left) and epifluorescence (right) images of a slice containing the left auditory cortex showing expression of ChR2 following injection of AAV1.ChR2.Flex into the right auditory cortex. (d) Experimental paradigm for photostimulating ChR2-positive CC-Parv neurons while recording from pyramidal neurons. (e) High-resolution image of a layer 5 biocytin-labeled CC-Parv and pyramidal neuron. (f) Left: example of responses recorded in cell-attached mode during photoactivation of CC-Parv neurons. Right: plot of onset latencies recorded in CC-Parv neurons (n = 10; animals n = 5). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. (g) Example of IPSCs recorded at 0 mV from a pyramidal neuron before (red trace) and after application of ionotropic glutamate receptor antagonists (NBQX 10 μM, CPP 5 μM: magenta trace) and GABAA receptor antagonist (Gabazine 25 μM: black trace). (h) Left: plot of onset latencies recorded in pyramidal neurons (n = 10; animals n = 5). Middle: plot of IPSCs peaks calculated for pyramidal neurons. Right: plot of IPSCs charge transfer calculated for individual IPSCs for pyramidal neurons. Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. (i) Left: example of IPSCs (black trace) and rising time course (red trace) recorded at 0 mV from a pyramidal neuron. Right: plot of IPSCs rising time course recorded in pyramidal neurons (n = 15; animals n = 8). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. (j) Left: example of IPSCs (black trace) and decay time course (amber trace) recorded at 0 mV from a pyramidal neuron. Right: plot of IPSCs decay time course recorded in pyramidal neurons (n = 15; animals n = 8). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. Figure 5. View largeDownload slide Photostimulation of auditory CC-Parv neurons elicits direct inhibition onto local pyramidal neurons. (a) Schematic depicting injection site using the Parv-Cre transgenic mouse line to retrogradely transfect CC-Parv neurons in the contralateral auditory cortex with ChR2. Right auditory cortex: AAV1.ChR2.Flex injection site. Left auditory cortex: red CC-Parv ChR2-tdTomato-positive neurons and callosum axons. (b) Bright-field (left) and epifluorescence (right) images of a slice containing the auditory cortex injection site for AAV1.ChR2.Flex. (c) Bright-field (left) and epifluorescence (right) images of a slice containing the left auditory cortex showing expression of ChR2 following injection of AAV1.ChR2.Flex into the right auditory cortex. (d) Experimental paradigm for photostimulating ChR2-positive CC-Parv neurons while recording from pyramidal neurons. (e) High-resolution image of a layer 5 biocytin-labeled CC-Parv and pyramidal neuron. (f) Left: example of responses recorded in cell-attached mode during photoactivation of CC-Parv neurons. Right: plot of onset latencies recorded in CC-Parv neurons (n = 10; animals n = 5). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. (g) Example of IPSCs recorded at 0 mV from a pyramidal neuron before (red trace) and after application of ionotropic glutamate receptor antagonists (NBQX 10 μM, CPP 5 μM: magenta trace) and GABAA receptor antagonist (Gabazine 25 μM: black trace). (h) Left: plot of onset latencies recorded in pyramidal neurons (n = 10; animals n = 5). Middle: plot of IPSCs peaks calculated for pyramidal neurons. Right: plot of IPSCs charge transfer calculated for individual IPSCs for pyramidal neurons. Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. (i) Left: example of IPSCs (black trace) and rising time course (red trace) recorded at 0 mV from a pyramidal neuron. Right: plot of IPSCs rising time course recorded in pyramidal neurons (n = 15; animals n = 8). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. (j) Left: example of IPSCs (black trace) and decay time course (amber trace) recorded at 0 mV from a pyramidal neuron. Right: plot of IPSCs decay time course recorded in pyramidal neurons (n = 15; animals n = 8). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. To determine the synaptic properties of CC-Parv projections onto local pyramidal neurons, we photoactivated CC-Parv ChR2-positive neurons by flashing blue light for 2–5 ms during whole-cell recordings from pyramidal neurons (Fig. 5d,e, g–j; n = 15). Because of the variability of ChR2 expression levels (number of ChR2 molecules per transfected neuron) and transfection efficiency (number of ChR2-expressing neurons per animal), we adjusted the intensity of the blue light to evoke inhibitory postsynaptic currents (IPSCs) with a single peak. IPSCs (Fig. 5g, red trace) were isolated by applying a command potential of 0 mV (the calculated reversal potential for glutamatergic excitatory conductance). The latency of the photo-evoked IPSCs response was 4.0 ± 0.2 ms (Fig. 5h, left). This latency is consistent with the IPSCs being the result of a monosynaptic inhibitory input from CC-Parv neurons and not a cortical feedback inhibitory network recruited by cortical projections. Blocking excitatory neurotransmission by application of glutamate receptor antagonists NBQX and CPP did not abolish the photo-evoked IPSCs (Fig. 5g, magenta trace; n = 5). In contrast, blocking inhibitory neurotransmission by application of Gabazine (Fig. 5g, black trace; n = 5) completely abolished the photo-evoked IPSCs, confirming that they were elicited by direct cortical inhibitory transmission. Basic biophysical properties for photo-evoked IPSCs (n = 15) included (Fig. 5h–j): peak, 126 ± 13 pA; charge, 2.03 ± 0.195 × 104 pC; rise time, 0.0075 ± 0.001 ms; and decay time, 13.95 ± 1.26 ms. These data reveal that a large proportion of pyramidal neurons receive direct inhibitory input driven by local CC-Parv neurons, but does not exclude the possibility that long-range axons form the contralateral CC-Parv neurons also contribute to the photo-evoked IPSCs. Do Callosal Parv Neurons Receive Thalamocortical Input? To determine the connectivity pattern of thalamocortical input onto CC-Parv neurons in the AC, we used an optogenetic approach in which we conditionally and retrogradely expressed GFP in CC-Parv neurons by injecting AAV1.GFP.Flex into the right AC and expressed ChR2 in thalamocortical projections by injecting AAV9.ChR2 in the left MGB of Parv-Cre mice (Fig. 6a–c). We performed whole-cell voltage-clamp recordings from visually identified layers 4 and 5 CC-Parv neurons in the left AC during photoactivation of thalamic projections (Fig. 6d). To block disynaptic and polysynaptic responses and to examine direct thalamocortical monosynaptic inputs onto CC-Parv neurons, we applied both TTX and 4-AP (Petreanu et al. 2009; Cruikshank et al. 2010; Ji et al. 2016). We isolated excitatory postsynaptic currents (EPSCs) by applying a command potential of −70 mV (the calculated reversal potential for GABAergic inhibitory conductance). Basic biophysical properties for photo-evoked EPSCs onto layer 4 CC-Parv neurons (n = 9) included (Fig. 6e): onset, 2.1 ± 0.2 ms; peak, 100.4 ± 12.2 pA; and charge, 6.82 ± 1.37 × 103 pC. Basic biophysical properties for photo-evoked EPSCs onto layer 5 CC-Parv neurons (n = 11) included (Fig. 6e): onset, 2.3 ± 0.2 ms; peak, 105.4 ± 11.9 pA; and charge, 5.44 ± 0.737 × 103 pC. Figure 6. View largeDownload slide Photostimulation of thalamic projections elicit monosynaptic excitatory inputs onto CC-Parv neurons. (a) Experimental paradigm for photostimulating ChR2-positive thalamic projections and retrograde viral labeling of CC-Parv neurons. Top, auditory cortex: AAV1.GFP.Flex injection site. Bottom, MGB: red AAV9.ChR2 injection site. (b) Bright-field (top) and epifluorescence (bottom) images of a slice containing the MGB injection site for AAV9.ChR2. (c) tdTomato epifluorescence (left) and GFP epifluorescence (right) images of a slice containing the left auditory cortex showing expression of ChR2-tdTomato thalamic axons following injection of AAV9.ChR2 into the left MGB and GFP-positive CC-Parv neurons following injection of AAV1.GFP.Flex into the right auditory cortex. (d) Top: example of EPSCs recorded at −70 mV from a layer 4 pyramidal neuron during photostimulation of ChR2-positve thalamic axons after application of TTX (1 μM) and 4-AP (100 μM). Bottom: example of EPSCs recorded at −70 mV from a layer 5 pyramidal neuron during photostimulation of ChR2-positve thalamic axons after application of TTX (1 μM) and 4-AP (100 μM). (e) Top row: for layer 4 CC-Parv neurons (n = 9; animals n = 6): left, plot of onset latencies; middle, plot of IPSCs peaks; right, plot of EPSCs charge transfer calculated for individual EPSCs. Bottom row: for layer 5 CC-Parv neurons (n = 11; animals n = 9): same as top row. Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. Figure 6. View largeDownload slide Photostimulation of thalamic projections elicit monosynaptic excitatory inputs onto CC-Parv neurons. (a) Experimental paradigm for photostimulating ChR2-positive thalamic projections and retrograde viral labeling of CC-Parv neurons. Top, auditory cortex: AAV1.GFP.Flex injection site. Bottom, MGB: red AAV9.ChR2 injection site. (b) Bright-field (top) and epifluorescence (bottom) images of a slice containing the MGB injection site for AAV9.ChR2. (c) tdTomato epifluorescence (left) and GFP epifluorescence (right) images of a slice containing the left auditory cortex showing expression of ChR2-tdTomato thalamic axons following injection of AAV9.ChR2 into the left MGB and GFP-positive CC-Parv neurons following injection of AAV1.GFP.Flex into the right auditory cortex. (d) Top: example of EPSCs recorded at −70 mV from a layer 4 pyramidal neuron during photostimulation of ChR2-positve thalamic axons after application of TTX (1 μM) and 4-AP (100 μM). Bottom: example of EPSCs recorded at −70 mV from a layer 5 pyramidal neuron during photostimulation of ChR2-positve thalamic axons after application of TTX (1 μM) and 4-AP (100 μM). (e) Top row: for layer 4 CC-Parv neurons (n = 9; animals n = 6): left, plot of onset latencies; middle, plot of IPSCs peaks; right, plot of EPSCs charge transfer calculated for individual EPSCs. Bottom row: for layer 5 CC-Parv neurons (n = 11; animals n = 9): same as top row. Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. To further characterize that thalamic projections are a source of excitatory input onto CC-Parv neurons, we used VGluT2 immunoreactivity for the identification of presynaptic terminals of thalamocortical inputs. VGluT2 is a vesicular glutamate transporter and it has been demonstrated in the neocortex that almost all the VGluT2 immunoreactivity belongs to thalamic axons (Kaneko and Fujiyama 2002). First, we conditionally expressed tdTomato in CC-Parv neurons by injecting AAV1.tdTomato.Flex into the right AC of Parv-Cre mice. After incubating slices with a VGluT2 antibody and a secondary antibody conjugated with Alexa 488, we then performed confocal microscopic analysis to examine the appositions of VGluT2-positive green puncta on CC-Parv-tdTomato neurons. Only images which contained CC-Parv dendrites were used for this analysis in order to minimize the identification of spurious appositions. We found that VGluT2-positive puncta were apposed to the dendrites of the CC-Parv neurons (Fig. 7; n = 11 neurons). Figure 7. View largeDownload slide Confocal laser-scanning microscope analysis of putative thalamic synaptic input onto CC-Parv neurons in the mouse auditory cortex. (a) Left: confocal tdTomato epifluorescence image of CC-Parv tdTomato-positive dendrites (magenta). Middle: epifluorescence image of VGluT2-positive axons varicosities (green). Right: merged epifluorescence image of CC-Parv tdTomato-positive dendrites and VGluT2-positive axons varicosities. The arrows indicate putative appositions of VGluT2-positive axon varicosities with tdTomato dendrites of CC-Parv neurons. (b) Same as in panel (a), for a different CC-Parv neuron dendrite. Figure 7. View largeDownload slide Confocal laser-scanning microscope analysis of putative thalamic synaptic input onto CC-Parv neurons in the mouse auditory cortex. (a) Left: confocal tdTomato epifluorescence image of CC-Parv tdTomato-positive dendrites (magenta). Middle: epifluorescence image of VGluT2-positive axons varicosities (green). Right: merged epifluorescence image of CC-Parv tdTomato-positive dendrites and VGluT2-positive axons varicosities. The arrows indicate putative appositions of VGluT2-positive axon varicosities with tdTomato dendrites of CC-Parv neurons. (b) Same as in panel (a), for a different CC-Parv neuron dendrite. Taken together, these data reveal that both layer 4 and layer 5 CC-Parv-neurons receive direct thalamocortical input. The Callosal Parv Projection is a General Feature of the Cortex We next asked whether CC-Parv projections are a general feature of the corticocortical circuit organization, or if they are specific to the AC. To test this, we used a similar viral tracing approach in a different sensory area, the VC. We also investigated the existence of CC-Parv projections in the MC, a cortical area which is not involved in sensory processing like the AC and the VC, but instead is involved in the planning, control, and execution of voluntary movements. We used the same methods as in the AC to conditionally and retrogradely express either GFP or tdTomato in CC-Parv neurons in the VC (Figure 8) and MC (Figure 9) using a viral injection of AAV1.GFP.Flex or AAV1.tdTomato.Flex in the contralateral cortex. As before, we observed local transfection in the VC and MC (Figs. 8b and  9b), as well as GFP-positive (or tdTomato-positive) somata present in all layers of the contralateral cortices (Figs. 8c and  9c). GFP-positive (or tdTomato-positive) axons were visible in coronal sections of the corpus callosum in these mice (Figs. 8d and  9d). Figure 8. View largeDownload slide Characterization of long-range CC-Parv neurons in the mouse VC. (a) Schematic depicting injection site using the Parv-Cre transgenic mouse line to retrogradely transfect CC-Parv somata in the contralateral VC with GFP. (b) Bright-field (left) and epifluorescence (right) images of a slice containing the VC injection site for AAV1.GFP.Flex. (c) Bright-field (left) and epifluorescence (right) images of a slice containing the left VC showing expression of GFP following injection of AAV1.GFP.Flex into the right VC. (d) Confocal higher magnification epifluorescence image of GFP fluorescence of VC CC-Parv axons in the callosum. The arrows indicate the location of GFP-positive axons. (e) Morphological reconstruction of a layer 5 biocytin-labeled CC-Parv neuron in the VC (dendrites: white; axon: red). (f) Response recorded from a layer 5 CC-Parv neuron in the VC during injection of a hyperpolarizing current (1 s, −200 pA pulse) and a train of action potentials recorded during injection of a depolarizing current (1 s, 400 pA pulse). Top inset: single action potential from a VC CC-Parv neuron. (g) Summary plot of Vrest: resting membrane potential; Rin: input resistance; Tau: membrane time constant; Rheobase: the smallest current step evoking an action potential; AHP: after hyperpolarization; AP thr: action potential threshold; AP height: action potential height; AP width: action potential width; F/I slope; and SFA: spikes frequency adaptation from CC-Parv neurons in the VC (n = 13; animals n = 3). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. Figure 8. View largeDownload slide Characterization of long-range CC-Parv neurons in the mouse VC. (a) Schematic depicting injection site using the Parv-Cre transgenic mouse line to retrogradely transfect CC-Parv somata in the contralateral VC with GFP. (b) Bright-field (left) and epifluorescence (right) images of a slice containing the VC injection site for AAV1.GFP.Flex. (c) Bright-field (left) and epifluorescence (right) images of a slice containing the left VC showing expression of GFP following injection of AAV1.GFP.Flex into the right VC. (d) Confocal higher magnification epifluorescence image of GFP fluorescence of VC CC-Parv axons in the callosum. The arrows indicate the location of GFP-positive axons. (e) Morphological reconstruction of a layer 5 biocytin-labeled CC-Parv neuron in the VC (dendrites: white; axon: red). (f) Response recorded from a layer 5 CC-Parv neuron in the VC during injection of a hyperpolarizing current (1 s, −200 pA pulse) and a train of action potentials recorded during injection of a depolarizing current (1 s, 400 pA pulse). Top inset: single action potential from a VC CC-Parv neuron. (g) Summary plot of Vrest: resting membrane potential; Rin: input resistance; Tau: membrane time constant; Rheobase: the smallest current step evoking an action potential; AHP: after hyperpolarization; AP thr: action potential threshold; AP height: action potential height; AP width: action potential width; F/I slope; and SFA: spikes frequency adaptation from CC-Parv neurons in the VC (n = 13; animals n = 3). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. Figure 9. View largeDownload slide Characterization of long-range CC-Parv neurons in the mouse MC. (a) Schematic depicting injection site using the Parv-Cre transgenic mouse line to retrogradely transfect CC-Parv somata in the contralateral MC with tdTomato. (b) Bright-field (left) and epifluorescence (right) images of a slice containing the MC injection site for AAV1.tdTomato.Flex. (c) Bright-field (left) and epifluorescence (right) images of a slice containing the left MC showing expression of tdTomato following injection of AAV1.tdTomato.Flex into the right MC. (d) Confocal higher magnification epifluorescence image of tdTomato fluorescence of MC CC-Parv axons in the callosum. The arrows indicate the location of tdTomato-positive axons. (e) Morphological reconstruction of a layer 5 biocytin-labeled CC-Parv neuron in the MC (dendrites: white; axon: red). (f) Response recorded from a layer 5 CC-Parv neuron in the MC during injection of a hyperpolarizing current (1 s, −200 pA pulse) and a train of action potentials recorded during injection of a depolarizing current (1 s, 400 pA pulse). Top inset: single action potential from a MC CC-Parv neuron. (g) Summary plot of Vrest: resting membrane potential; Rin: input resistance; Tau: membrane time constant; Rheobase: the smallest current step evoking an action potential; AHP: after hyperpolarization; AP thr: action potential threshold; AP height: action potential height; AP width: action potential width; F/I slope; and SFA: spikes frequency adaptation from CC-Parv neurons in the MC (n = 14; animals n = 4). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. Figure 9. View largeDownload slide Characterization of long-range CC-Parv neurons in the mouse MC. (a) Schematic depicting injection site using the Parv-Cre transgenic mouse line to retrogradely transfect CC-Parv somata in the contralateral MC with tdTomato. (b) Bright-field (left) and epifluorescence (right) images of a slice containing the MC injection site for AAV1.tdTomato.Flex. (c) Bright-field (left) and epifluorescence (right) images of a slice containing the left MC showing expression of tdTomato following injection of AAV1.tdTomato.Flex into the right MC. (d) Confocal higher magnification epifluorescence image of tdTomato fluorescence of MC CC-Parv axons in the callosum. The arrows indicate the location of tdTomato-positive axons. (e) Morphological reconstruction of a layer 5 biocytin-labeled CC-Parv neuron in the MC (dendrites: white; axon: red). (f) Response recorded from a layer 5 CC-Parv neuron in the MC during injection of a hyperpolarizing current (1 s, −200 pA pulse) and a train of action potentials recorded during injection of a depolarizing current (1 s, 400 pA pulse). Top inset: single action potential from a MC CC-Parv neuron. (g) Summary plot of Vrest: resting membrane potential; Rin: input resistance; Tau: membrane time constant; Rheobase: the smallest current step evoking an action potential; AHP: after hyperpolarization; AP thr: action potential threshold; AP height: action potential height; AP width: action potential width; F/I slope; and SFA: spikes frequency adaptation from CC-Parv neurons in the MC (n = 14; animals n = 4). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. Using the same approach described above, we next performed whole-cell patch-clamp recordings from visually identified CC-Parv neurons in VC and MC. Confocal images of biocytin-filled CC-Parv neurons in VC and MC showed that they are similar to basket-cell interneurons in their morphology and send an axonal projection towards/into the subcortical white matter (Figs. 8e and  9e). We verified their identity based on the comparison with electrophysiological properties of Parv interneurons (for review, see Hu et al. 2014). These properties include a narrow action potential and high rheobase (the smallest current step evoking an action potential) (Figs. 8f and  9f, top; inset; action potentials in CC-Parv neurons, shown in black, are very narrow). The responses to current steps in CC-Parv neurons were typical for Parv interneurons (Figs. 8f and  9f; notice the high current step needed to generate action potentials). Basic electrophysiological properties for CC-Parv neurons in VC (n = 11) included (Fig. 8g): resting membrane potential, −77.5 ± 0.8 mV; input resistance, 103.9 ± 5.2 MΩ; membrane time constant, 0.53 ± 0.02 ms; rheobase, 338.4 ± 18.0 pA; after hyperpolarization, −10.5 ± 0.9 mV; action potential threshold, −41.7 ± 1.8 mV; action potential height, 43.4 ± 2.6 mV; action potential width, 0.42 ± 0.05 ms; F/I slope, 0.8 ± 0.1 Hz/pA step; and spike frequency adaptation (third/fifth), 1.1 ± 0.03. Basic electrophysiological properties for CC-Parv neurons in MC (n = 14) included (Fig. 9g): resting membrane potential, −76.0 ± 0.8 mV; input resistance, 73.9 ± 3.8 MΩ; membrane time constant, 0.6 ± 0.08 ms; rheobase, 307.7 ± 18.6 pA; after hyperpolarization, −11.7 ± 0.9 mV; action potential threshold, −39.1 ± 1.2 mV; action potential height, 38.7 ± 1.9 mV; action potential width, 0.42 ± 0.04 ms; F/I slope, 0.8 ± 0.09 Hz/pA step; and spike frequency adaptation (third/fifth), 1.1 ± 0.1. Taken together, these results indicate that CC-Parv neurons can be found in both VC and MC, as well as in AC, and they project via the corpus callosum to the opposite hemisphere. Discussion In this study, we test the hypothesis that a class of Parv neurons makes up a callosal inhibitory projection in the mouse. Our results support this hypothesis and additionally conclude that the auditory, visual, and motor cortices send long-range GABAergic projections to their corresponding cortex in the contralateral hemisphere via these CC-Parv neurons (Fig. 10). Because of its presence in three such disparate cortical areas, this would suggest that the callosal Parv projection is likely a general feature of the interhemispheric network of the cortex. Figure 10. View largeDownload slide Summary diagram: CC-Parv neurons callosal projection. Auditory, visual, and motor CC-Parv projections modulate the activity of local pyramidal neurons by direct inhibition. Green lines: excitatory inputs from thalamic projections; red line: ipsi- and contralateral inhibitory input from CC-Parv neurons. Figure 10. View largeDownload slide Summary diagram: CC-Parv neurons callosal projection. Auditory, visual, and motor CC-Parv projections modulate the activity of local pyramidal neurons by direct inhibition. Green lines: excitatory inputs from thalamic projections; red line: ipsi- and contralateral inhibitory input from CC-Parv neurons. GABAergic neurons are the primary source of inhibition in the adult brain and they represent a minority of all cortical neurons (10–15% in rodents) (Meyer et al. 2011), but are composed of a heterogeneous cell population (for review, see Ascoli et al. 2008; Xu et al. 2010; Rudy et al. 2011). Since the observation by Ramon y Cajal (1988) that a large number of cells with “short-axons” are present in the brain, GABAergic neurons have been considered to project by and large locally in the cortex, and for this reason are often referred to as “interneurons.” However, recent studies (Melzer et al. 2012; Lee et al. 2014; Tomioka et al. 2015; Basu et al. 2016; Rock et al. 2016) are demonstrating that long-range GABAergic projections may be more prevalent than previously assumed. The main finding of the present study is that a large proportion of the entire Parv neuronal population projects through the corpus callosum to connect the two hemispheres of AC, VC, and MC. In addition, our results indicate that the somata of callosally projecting CC-Parv neurons span all layers of the cortex, while the somata of callosally projecting pyramidal neurons are located predominately in layers 2–3 (about 80% in rodents), layer 5 (about 20%) and, to a lesser extent, layer 6 (Jacobson and Trojanowski 1974; Fame et al. 2011). Despite the large number of long-range Parv neurons identified in this study, our approach is limited by caveats (limited injection volume and variability in transfection which leads to incomplete coverage of the cortical area) that preclude us from determining the absolute number and ratio of long-range CC-Parv versus short-range Parv neurons. Our findings, together with previous studies, in which long-range GABAergic projections were found to connect different brain areas in different species both ipsi- and contralaterally (Buhl and Singer 1989; McDonald and Burkhalter 1993; Tomioka et al. 2005; Apergis-Schoute et al. 2007; Higo et al. 2007; Tamamaki and Tomioka 2010; Rock et al. 2016), support a new notion in which long-range GABAergic neurons can be considered a large fraction in many cortical areas of the total GABAergic population. Moreover, this will lead to a new concept in which not only glutamatergic cortical neurons are carrying information to far stations (for review, see Tremblay et al. 2016), but also long-range GABAergic neurons. Our results are in line with recent findings that long-range GABAergic neurons in the neocortex belong to a number of molecular groups, including Parv, somatostatin-expressing, and vasoactive intestinal polypeptide-expressing neurons (Tamamaki and Tomioka 2010; Lee et al. 2014; Tomioka et al. 2015; Rock et al. 2016). Based on these previous findings and because GABAergic neurons are the most heterogeneous class of neurons in the neocortex, we predict that other cell-types may contribute to the GABAergic callosal projection. Future experiments will provide insight about the complexity of the cell-type composition of the long-range callosal GABAergic projections. Morphologically, CC-Parv neurons resemble other Parv basket-cells. Particularly, CC-Parv neurons have dendrites that often span multiple layers, allowing the basket-cells to receive input from different afferent pathways (Xu and Callaway 2009; Helmstaedter et al. 2009a, 2009b; Bagnall et al. 2011; Kubota et al. 2011; Tukker et al. 2013). The axons of Parv neurons are also very puzzling. Parv neurons have a massive axonal arborization (Sik et al. 1995; Karube et al. 2004) that generates a divergent inhibitory output (Packer and Yuste 2011; Bezaire and Soltesz 2013) that often spans multiple layers of the cortex, forming translaminar inhibitory circuits (Helmstaedter et al. 2008, 2009a, 2009b; Bortone et al. 2014; Pluta et al. 2015). Similarly, the axons of CC-Parv neurons innervate local pyramidal neurons in the AC. A large proportion of pyramidal neurons receive direct inhibitory input driven by CC-Parv neurons. The fast IPSCs rise time recorded from pyramidal neurons after photoactivation of CC-Parv neurons suggests that these neurons, as already demonstrated for the basket-cells, innervate the perisomatic domain of pyramidal neurons (Pouille and Scanziani 2001). However, this remains to be demonstrated morphologically. In addition, AAV1.Flex viral vectors (Atasoy et al. 2008) exhibit both anterograde and retrograde transfection capabilities (Rothermel et al. 2013; Rock et al. 2016). For this reason, we cannot exclude the possibility that long-range axons from the contralateral cortex also contribute to the photo-evoked inhibitory inputs onto pyramidal neurons. Numerous questions still need to be addressed in regard to long-range inhibitory circuits in the cortex. An important line of investigation will be to determine how CC-Parv neurons may differ from short-axon Parv interneurons. For example, are CC-Parv neurons receiving highly convergent inputs from pyramidal neurons and inhibitory inputs primarily from the Parv neurons in a similar connectivity pattern as short-axon Parv interneurons (Xu and Callaway 2009; Apicella et al. 2012; Pfeffer et al. 2013)? Do CC-Parv neurons form inhibitory synapses that are depressing as reported for basket-cells (Reyes et al. 1998)? Are CC-Parv neurons electrically coupled, and if so, are they only coupled with the cells expressing the same molecular markers (Amitai et al. 2002; Christie et al. 2005)? Experimental evidence indicates that basket-cells are involved in thalamic-feedforward inhibition both in vitro (Agmon and Connors 1991, 1992; Porter et al. 2001; Cruikshank et al. 2002; Beierlein et al. 2003; Gabernet et al. 2005; Rose and Metherate 2005; Inoue and Imoto 2006; Sun et al. 2006; Barkat et al. 2011; Schiff and Reyes 2012; Ji et al. 2016) and in vivo (Moore and Nelson 1998; Li et al. 2013a, 2013b; Lien and Scanziani 2013). Our findings establish that both layers 4 and 5 CC-Parv neurons are also innervated by thalamic axons. Our results are consistent with the data from somatosensory (Constantinople and Bruno 2013) and auditory cortex (Sun et al. 2013) supporting the growing view that the layer 4 is not a mandatory hub for the distribution of cortical activity, but thalamic inputs can activate also deep layers of the cortex such as layer 5. Therefore, both layers 4 and 5 CC-Parv neurons can have access to the same sensory information but can alter the behavioral outcome by differentially controlling the output of pyramidal neurons projecting to different brain areas. Moreover, our finding that CC-Parv neurons respond to thalamic activity could imply a mechanism of coincidence detection (Pouille and Scanziani 2001), in which CC-Parv neurons produce a feedforward somatic inhibition across the local pyramidal neurons in the AC. Since homotopic projections are the strongest of all the callosal projections (Rouiller et al. 1991; Lee and Winer 2008), the presence of thalamic-feedforward interhemispheric inhibition may thus serve as a means to maintain timing across these areas, and therefore refine sensory information about auditory stimuli between the two hemispheres. Indeed, basket-cells have been proposed to contribute to the phasing of pyramidal neurons during oscillations (Bartos et al. 2007; Buzsaki and Wang 2012). Therefore, we can speculate that CC-Parv neurons can be involved in the synchronization of neuronal activity between distant brain areas (for review, see Uhlhaas and Singer 2012). However, previous studies have shown that long-range GABAergic neurons preferentially innervate inhibitory neurons in the target area (Melzer et al. 2012; Basu et al. 2016). Due to both anterograde and retrograde transfection capabilities of the virus used, our study cannot determine whether CC-Parv neurons are involved in interhemispheric inhibition or disinhibition. Taken together, our results establish a previously unknown thalamo-corticocortical long-range inhibitory circuit (thalamus → CC-Parv inhibitory projections → contralateral AC). This finding suggests that the timing and ratio of excitation and inhibition provided by the callosal projection via glutamatergic and GABAergic axons may underlie long-distance synchronization. Notes We are indebted to G. Gaufo for help with confocal imaging. 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Cortical Circuits of Callosal GABAergic Neurons

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Abstract

Abstract Anatomical studies have shown that the majority of callosal axons are glutamatergic. However, a small proportion of callosal axons are also immunoreactive for glutamic acid decarboxylase, an enzyme required for gamma-aminobutyric acid (GABA) synthesis and a specific marker for GABAergic neurons. Here, we test the hypothesis that corticocortical parvalbumin-expressing (CC-Parv) neurons connect the two hemispheres of multiple cortical areas, project through the corpus callosum, and are a functional part of the local cortical circuit. Our investigation of this hypothesis takes advantage of viral tracing and optogenetics to determine the anatomical and electrophysiological properties of CC-Parv neurons of the mouse auditory, visual, and motor cortices. We found a direct inhibitory pathway made up of parvalbumin-expressing (Parv) neurons which connects corresponding cortical areas (CC-Parv neurons → contralateral cortex). Like other Parv cortical neurons, these neurons provide local inhibition onto nearby pyramidal neurons and receive thalamocortical input. These results demonstrate a previously unknown long-range inhibitory circuit arising from a genetically defined type of GABAergic neuron that is engaged in interhemispheric communication. auditory, callosum, inhibition, long-range GABAergic neurons, motor cortex, Parv neurons, visual Introduction It is a well-established principle of the cortical circuit organization that excitation is both local and long-range, whereas inhibition is exclusively local, but this is not true; inhibition can be long-range as well. While much progress has been made in understanding the local cortical circuit organization of GABAergic interneurons (Buzsaki 1984; Freund and Buzsaki 1996; Ali et al. 1999; Holmgren et al. 2003; Pouille and Scanziani 2004; Silberberg and Markram 2007; Pouille et al. 2009; Stokes and Isaacson 2010; Hayut et al. 2011; Pouille et al. 2013; Crandall and Connors 2016), much less has been learned about the cortical and extracortical input organization of long-range GABAergic neurons. Anatomical studies using retrograde tracers and immunohistochemistry have proposed that between 1% and 10% of the cortical GABAergic neurons in rodents, cats, and monkeys also give rise to long-range corticocortical projections (McDonald and Burkhalter 1993; Tomioka et al. 2005; Higo et al. 2007; Tomioka and Rockland 2007; Higo et al. 2009). It has been previously demonstrated that some axons of the corpus callosum, important for transfer of sensory information between the right and left cerebral hemispheres, are immunoreactive for glutamic acid decarboxylase, an enzyme required for gamma-aminobutyric acid (GABA) synthesis and a specific marker for GABAergic neurons (Fabri and Manzoni 2004). Additionally, anatomical studies have demonstrated the presence of callosal nonpyramidal neuron projections (Code and Winer 1985; Hughes and Peters 1990; Peters et al. 1990; Hughes and Peters 1992; Gonchar et al. 1995), but the cells of origin and physiological function of these GABAergic projections were not explored. A growing body of evidence suggests that many of the long-range GABAergic projections arise from somatostatin-expressing neurons (Tomioka et al. 2005; Higo et al. 2007; Tomioka and Rockland 2007; Higo et al. 2009; McDonald et al. 2012; Melzer et al. 2012; Rock et al. 2016). However, it has been recently suggested that parvalbumin-expressing (Parv) neurons  may contribute to long-range GABAergic projections as well (Jinno and Kosaka 2004; Lee et al. 2014). The present study focused on four main goals: (1) determine if Parv neurons send long-range GABAergic projections to the contralateral cortex via the corpus callosum; (2) describe the anatomical and electrophysiological properties of these corticocortical Parv neurons (CC-Parv neurons); (3) determine the local (ipsilateral) inhibitory connectivity pattern of CC-Parv neurons; and (4) investigate the thalamic inputs onto CC-Parv neurons. Our approach consisted of viral tracing and optogenetics methods to investigate these questions. Using these manipulations, we found a direct inhibitory pathway made up of Parv neurons which connects corresponding cortical areas (CC-Parv neurons → contralateral cortex). Our data show that these neurons provide local inhibition onto nearby pyramidal neurons and are recipients of thalamocortical input. Overall, we describe a previously unknown long-range inhibitory circuit and attribute a specific function to a genetically defined type of GABAergic neuron in interhemispheric communication. Methods All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Texas at San Antonio. Procedures followed animal welfare guidelines set by the National Institutes of Health. Mice used in this experiment were housed in a vivarium maintaining a 12 h light/dark schedule and given ad libidum access to mouse chow and water. Transgenic Mouse Lines The following mouse lines were used in this study: Parv-Cre: B6;129P2-Pvalbtm1(cre)Arbr/J, The Jackson Laboratory stock number 008069; ROSA-tdTomato reporter: B6.CG.Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, The Jackson Laboratory, stock number 007914. Parv-Cre female mice were crossed with a ROSA-tdTomato reporter male mouse to generate a Parv-Cre-tdTomato line (parvalbumin-containing neurons expressed both Cre and tdTomato). Stereotaxic Injections Basic Surgical Procedures Mice were initially anesthetized with isoflurane (3–5%; 1 L/min O2 flow) in preparation for the stereotaxic injections detailed in the sections below. The mice were head-fixed on a stereotaxic frame (Model 1900; Kopf Instruments) using nonrupture ear bars. Anesthesia was maintained at 1–1.5% isoflurane for the duration of the surgery. A warming pad was used to maintain body temperature during the procedure. Standard aseptic technique was followed for all surgical procedures. Injections were performed using a pressure injector (Nanoject II; Drummond Scientific) mounted on the stereotaxic frame. Injections were delivered through a borosilicate glass injection pipette (Wiretrol II; Drummond Scientific) with a taper length of ~30 mm and a tip diameter of ~50 μm. After lowering it to the target injection depth, the glass pipette remained in place 5–10 min both before and after the injection was made. Both male and female Parv-Cre or Parv-Cre-tdTomato mice, P31–40 at the time of the injection, were utilized in these experiments. Retrograde Labeling of CC-Parv Neurons CC-Parv neurons in the auditory cortex (AC) were retrogradely labeled with green fluorescent protein (GFP) using AAV1.CAG.Flex.eGFP.WPRE.bGH (AAV1.GFP.Flex; University of Pennsylvania Vector Core) stereotaxically injected into the right AC of Parv-Cre mice (12 mice from 7 litters). Injections were performed as above, with the following parameters: stereotaxic coordinates for the AC injection site were 2.45 mm posterior and 4.3 mm lateral to bregma. Approximately 50 nL of AAV1.GFP.Flex was delivered between two depths in the AC, 1.0 mm and 0.75 mm below the surface of the brain, over the course of 5 min. CC-Parv neurons in the visual cortex (VC; 3 mice from 1 litter) and motor cortex (MC; 4 mice from 1 litter) were also retrogradely labeled with GFP as in the AC or with tdTomato using AAV1.CAG.Flex.tdTomato.WPRE.bGH (AAV1.tdTomato.Flex; University of Pennsylvania Vector Core) for recording intrinsic properties and morphological reconstruction. Stereotaxic coordinates for VC were 2.8 mm posterior and 2.25 mm lateral to bregma. Approximately 50 nL of AAV1.GFP.Flex or AAV1.tdTomato.Flex was delivered between two depths in the VC, 0.75 mm and 0.5 mm below the surface of the brain, over the course of 5 min. Stereotaxic coordinates for MC were 1.1 mm anterior and 1.6 mm lateral to bregma. Approximately 50 nL of AAV1.GFP.Flex or AAV1.tdTomato.Flex was delivered between two depths in the MC, 0.85 mm and 0.5 mm below the surface of the brain, over the course of 5 min. Retrograde Transfection of CC-Parv Neurons with Channelrhodopsin-2 CC-Parv neurons in the AC were retrogradely transfected with channelrhodopsin-2 (ChR2) using AAV1.CAGGS.Flex.ChR2-tdTomato.WPRE.SV40 (AAV1.ChR2.Flex; University of Pennsylvania Vector Core) stereotaxically injected into the right AC of Parv-Cre mice (5 mice from 4 litters). Injections were performed in the same manner and using the same AC stereotaxic coordinates as described previously. Retrograde Labeling of CC-Parv Neurons and Transfection of Thalamic Axons with ChR2 CC-Parv neurons in the AC were retrogradely labeled with GFP in the same manner and using the same AC stereotaxic coordinates as described previously in Parv-Cre mice (7 animals from 5 litters). In these same animals, thalamocortical projections were transfected with ChR2 using AAV9.CAG.hChR2.tdTomato (AAV9.ChR2; University of North Carolina Vector Core) stereotaxically injected into the left medial geniculate body (MGB) with the following parameters: stereotaxic coordinates for the left MGB injection site were 3.2 mm posterior and 2.0 mm lateral to bregma. Approximately 80–100 nL of AAV.ChR2 was delivered at 3.2 mm ventral to bregma over the course of 10 min. In vitro Slice Preparation and Recordings Slice preparation and electrophysiological recordings were performed as previously described (Rock and Apicella 2015; Rock et al. 2016). We allowed 2–4 weeks for expression of ChR2 or GFP. Mice were anesthetized with isoflurane and decapitated. Coronal slices (300 μm) containing the area of interest (AC, VC, or MC) were sectioned on a vibratome (VT1200S; Leica) in a chilled cutting solution containing the following (in mM): 100 choline chloride, 25 NaHCO3, 25 d-glucose, 11.6 sodium ascorbate, 7 MgSO4, 3.1 sodium pyruvate, 2.5 KCl, 1.25 NaH2PO4, and 0.5 CaCl2. These slices were incubated in oxygenated artificial cerebrospinal fluid (ACSF) in a submerged chamber at 35–37 °C for 30 min and then at room temperature (21–25 °C) until recordings were performed. ACSF contained the following (in mM): 126 NaCl, 26 NaHCO3, 10 d-glucose, 2.5 KCl, 2 CaCl2, 1.25 NaH2PO4, and 1 MgCl2; osmolarity was ~290 Osm/L. Whole-cell recordings were performed in 31–33 °C ACSF. Thin-walled borosilicate glass pipettes (Warner Instruments) were pulled on a vertical pipette puller (PC-10; Narishige) and typically were in the range of 3–5 MΩ resistance when filled with a cesium-based intracellular solution, which contained the following (in mM): 110 CsOH, 100 d-gluconic acid, 10 CsCl2, 10 HEPES, 10 phosphocreatine, 1 EGTA, 1 ATP, and 0.3–0.5% biocytin. Inhibitory postsynaptic currents (IPSCs) were recorded in the voltage-clamp configuration with a holding potential of 0 mV (the calculated reversal potential for glutamatergic excitatory conductances). Intrinsic properties were recorded in the current-clamp configuration using a potassium-based intracellular solution at 31–33 °C. Potassium-based intracellular solution contained the following (in mM): 120 potassium gluconate, 20 KCl, 10 HEPES, 10 phosphocreatine, 4 ATP, 0.3 GTP, 0.2 EGTA, and 0.3–0.5% biocytin. Signals were sampled at 10 kHz and filtered at 4 kHz. Pharmacological blockers used were CPP (5 μM; Tocris Bioscience), NBQX (10 μM; Abcam), and Gabazine (25 μM; Abcam). To block disynaptic and polysynaptic responses and to examine direct thalamocortical monosynaptic inputs onto CC-Parv neurons, we applied both TTX (1 μM; Tocris Bioscience) and 4-AP (100 μM; Sigma-Aldrich). Hardware control and data acquisition were performed by Ephus (www.ephus.org) (Suter et al. 2010). ChR2 Photostimulation CC-Parv somata transfected with ChR2 were present in the left AC following injection of AAV1.ChR2.Flex in the right AC. Because of variability both in ChR2 expression levels (number of ChR2 molecules per transfected neuron) and in transfection efficiency (number of ChR2-expressing neurons per animal), to minimize the variability from experiment to experiment, we performed the electrophysiological recording in the same slices containing the AC and with the highest density of ChR2-transfected axons. We recorded IPSCs from nearby pyramidal neurons during photoactivation of the CC-Parv ChR2-transfected neurons and axon terminals. A 470 nm wavelength blue LED (CoolLED pE excitation system) passed through a GFP filter cube (Endow GFP/EGFP longpass, C-156625; Chroma) and a 60× water-immersion objective was used for photoactivation of ChR2. Distribution of CC-Parv Neurons in AC CC-Parv neurons in the AC were retrogradely labeled with GFP using AAV1.GFP.Flex stereotaxically injected into the right AC of Parv-Cre-tdTomato mice (3 mice from 1 litter). Injections were performed in the same manner and using the same auditory cortex stereotaxic coordinates as described previously. Three weeks following the injection, these mice were deeply anesthetized with 5% isoflurane and transcardially perfused with phosphate-buffered saline (PBS), followed by 10% buffered formalin (Sigma-Aldrich). The brain was carefully removed and fixed overnight. The following day, the fixed brain was sliced into 200-μm thick sections on a vibrating microtome. Following six washes in PBS, the slices were mounted on microscope slides with Fluoromount-G (Southern Biotech). Confocal images were taken with a Zeiss LSM-710 microscope and a 40× oil-immersion objective. Images were rotated, cropped, and the brightness/contrast was adjusted in ImageJ (National Institutes of Health). Scored sections (9 slices, 3 slices from each of 3 mice) contained a 100-μm wide portion of AC spanning from the pia to the white matter. The distance between the pia and the white matter was normalized to 1000 μm, and then the cortex was divided into layers based on the following thicknesses: L1, 0–150 μm; L2/3, 151–375 μm; L4, 376–500 μm; L5, 501–750 μm; and L6, 751–1000 μm. Layer thicknesses in the AC were similar to the ones used in a previous study (Ji et al. 2016). Counts of parvalbumin neurons expressing tdTomato only and of CC-Parv neurons coexpressing GFP and tdTomato were made by hand. All of the GFP-expressing neurons from these sections were found to coexpress tdTomato. VGluT2 Immunoreactivity on CC-Parv Dendrites CC-Parv neurons in the AC were retrogradely labeled with tdTomato using AAV1.tdTomato.Flex stereotaxically injected into the right AC of Parv-Cre mice (2 mice from 1 litter). Three weeks following the injection, these mice were deeply anesthetized with 5% isoflurane and transcardially perfused with PBS, followed by 10% buffered formalin. The brain was carefully removed and fixed overnight. The following day, the fixed brain was sliced into 50-μm thick sections on a vibrating microtome. The sections were then incubated in 0.3% Triton X-100 and 5% normal goat serum (NGS) in PBS at room temperature for 2 h on a shaking platform. Following this blocking stage, the slices were incubated in the primary antibody (guinea pig Anti-VGLUT2, 1:15 000; Millipore) with 0.1% Triton X-100 and 1% NGS in PBS overnight at 4 °C. The next day, the slices were washed 3× with PBS and incubated for 3 h at room temperature on a shaking platform with the secondary antibody (goat anti-guinea pig IgG-conjugated Alexa 488, 1:500; Life Technologies) with 0.1% Triton X-100 and 1% NGS in PBS. Following three washes in PBS, the slices were mounted on microscope slides with Fluoromount-G. Confocal images were taken with a Zeiss LSM-710 microscope and a 100× oil-immersion objective to document appositions of tdTomato CC-Parv dendrites and Alexa 488 VGluT2 puncta. Images were rotated, cropped, and the brightness/contrast was adjusted in ImageJ. Histology During whole-cell recordings, neurons were filled with an internal solution containing 0.3–0.5% biocytin. Filled neurons were held for at least 20 min, and then the slices were fixed in a formalin solution at 4 °C until ready for processing. The slices were washed 6× in PBS and placed in a 4% streptavidin (Alexa Fluor 488 or 594; Life Technologies) solution with 0.3% Triton X-100 in PBS. Slices were allowed to incubate in this solution at 4 °C overnight, then washed 6× in PBS and mounted with Fluoromount-G on a glass microscope slide. Confocal images were taken with a Zeiss LSM-710 microscope at varying magnifications (3×–63×). Individual high magnification images were stitched together, when necessary, using XuvStitch software (XuvTools). Image adjustment was performed in ImageJ for brightness/contrast corrections and pseudocoloring. Neurons were morphologically reconstructed in three dimensions using the Simple Neurite Tracer plugin for ImageJ (Longair et al. 2011). Data Analysis Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. In the text, data are indicated as group averages (± s.e.m.). Data analysis was performed offline using custom MATLAB (MathWorks) routines. Results Anatomical Characterization of Long-Range Callosal Parv Neurons To visualize long-range GABAergic projections originating in the cortex and terminating in the contralateral cortex, we conditionally expressed GFP in Parv interneurons by injecting AAV1.GFP.Flex into the right auditory cortex (AC) of Parv-Cre transgenic mice (Taniguchi et al. 2011). Using this virus, long-range callosal Parv neurons (from this point forward referred to as CC-Parv neurons) were retrogradely labeled with GFP. Somata of CC-Parv neurons were present in all layers of the AC, and GFP-positive axons were visible in coronal sections of the corpus callosum in these mice (Fig. 1a–d). Next, to determine the proportion of CC-Parv neurons, we injected AAV1.GFP.Flex into the right AC of Parv-Cre-tdTomato transgenic mice (Fig. 2). GFP was colocalized with Parv/tdTomato-expressing neurons in the AC (Fig. 2b). We found that CC-Parv neurons can account for up to 42% of the entire Parv population (601 Parv neurons, 253 CC-Parv neurons; n = 9 slices, 3 mice; Fig. 2c,d). These data show that a large proportion of Parv neurons, arising from all layers of the AC, project to the contralateral AC via the corpus callosum. Figure 1. View largeDownload slide Characterization of CC-Parv neurons in the mouse auditory cortex. (a) Schematic depicting injection site using the Parv-Cre transgenic mouse line to retrogradely transfect CC-Parv neurons in the contralateral auditory cortex with GFP. (b) Bright-field (left) and epifluorescence (right) images of a slice containing the auditory cortex injection site for AAV1.GFP.Flex. (c) Bright-field (left) and epifluorescence (middle) images of a slice containing the left auditory cortex showing expression of GFP following injection of AAV1.GFP.Flex into the right auditory cortex. Right: confocal epifluorescence image of CC-Parv GFP-positive neurons. (d) Bright-field (left) and epifluorescence (middle) of CC-Parv GFP-positive axons in the corpus callosum. The arrows indicate the location of GFP-positive axons. Right: confocal higher magnification epifluorescence image of CC-Parv GFP-positive axons in the callosum. Figure 1. View largeDownload slide Characterization of CC-Parv neurons in the mouse auditory cortex. (a) Schematic depicting injection site using the Parv-Cre transgenic mouse line to retrogradely transfect CC-Parv neurons in the contralateral auditory cortex with GFP. (b) Bright-field (left) and epifluorescence (right) images of a slice containing the auditory cortex injection site for AAV1.GFP.Flex. (c) Bright-field (left) and epifluorescence (middle) images of a slice containing the left auditory cortex showing expression of GFP following injection of AAV1.GFP.Flex into the right auditory cortex. Right: confocal epifluorescence image of CC-Parv GFP-positive neurons. (d) Bright-field (left) and epifluorescence (middle) of CC-Parv GFP-positive axons in the corpus callosum. The arrows indicate the location of GFP-positive axons. Right: confocal higher magnification epifluorescence image of CC-Parv GFP-positive axons in the callosum. Figure 2. View largeDownload slide Laminar distribution and proportion of CC-Parv neurons in the mouse auditory cortex. (a) Schematic depicting injection site using the Parv-Cre-tdTomato transgenic mouse line to identify the laminar distribution of CC-Parv neurons and their proportion compared with the overall Parv population in the auditory cortex. Right auditory cortex: AAV1.GFP.Flex injection site; left auditory cortex: yellow CC-Parv somata coexpressing GFP and tdTomato; green CC-Parv GFP-positive axons; red Parv tdTomato-positive neurons. (b) Left: epifluorescence confocal image of a coronal section containing the left auditory cortex with Parv tdTomato-positive neurons. Middle: epifluorescence confocal image of CC-Parv GFP-positive neurons. Right: merged epifluorescence confocal image of Parv tdTomato-positive neurons and CC-Parv GFP-positive neurons. Layers are indicated on the right. (c) The laminar distribution and ratio of Parv-expressing interneurons and CC-Parv neurons. For the quantification of parvalbumin neurons expressing tdTomato and of CC-Parv neurons coexpressing GFP and tdTomato, the distance between the pia and white matter was normalized to 1000 μm, and the cortex was divided into layers based on the following thicknesses: L1, 0–150 μm; L2/3, 151–375 μm; L4, 376–500 μm; L5, 501–750 μm; and L6, 751–1000 μm. (d) Summary plot of the ratio of CC-Parv neurons versus the total population of Parv-expressing neurons. Figure 2. View largeDownload slide Laminar distribution and proportion of CC-Parv neurons in the mouse auditory cortex. (a) Schematic depicting injection site using the Parv-Cre-tdTomato transgenic mouse line to identify the laminar distribution of CC-Parv neurons and their proportion compared with the overall Parv population in the auditory cortex. Right auditory cortex: AAV1.GFP.Flex injection site; left auditory cortex: yellow CC-Parv somata coexpressing GFP and tdTomato; green CC-Parv GFP-positive axons; red Parv tdTomato-positive neurons. (b) Left: epifluorescence confocal image of a coronal section containing the left auditory cortex with Parv tdTomato-positive neurons. Middle: epifluorescence confocal image of CC-Parv GFP-positive neurons. Right: merged epifluorescence confocal image of Parv tdTomato-positive neurons and CC-Parv GFP-positive neurons. Layers are indicated on the right. (c) The laminar distribution and ratio of Parv-expressing interneurons and CC-Parv neurons. For the quantification of parvalbumin neurons expressing tdTomato and of CC-Parv neurons coexpressing GFP and tdTomato, the distance between the pia and white matter was normalized to 1000 μm, and the cortex was divided into layers based on the following thicknesses: L1, 0–150 μm; L2/3, 151–375 μm; L4, 376–500 μm; L5, 501–750 μm; and L6, 751–1000 μm. (d) Summary plot of the ratio of CC-Parv neurons versus the total population of Parv-expressing neurons. Anatomical and Electrophysiological Properties of Callosal Parv Neurons To determine the anatomical and electrophysiological properties of CC-Parv neurons, we once again injected AAV1.GFP.Flex into the right AC of Parv-Cre mice (Fig. 1a). This approach allowed us to visually identify and record from CC-Parv neurons using whole-cell patch clamp. We verified their identity based on the comparison with electrophysiological properties of Parv interneurons (for review, see Hu et al. 2014). These properties include a narrow action potential and high rheobase (the smallest current step evoking an action potential) (Fig. 3a, c, e, g, inset; action potentials in CC-Parv neurons, shown in black, are very narrow). The responses to current steps in CC-Parv neurons were typical for Parv interneurons (Fig. 3; notice the high current step needed to generate action potentials). To characterize the electrophysiological properties of CC-Parv neurons, we recorded  45 GFP-labeled neurons spanning from layer 2/3 to layer 6 in the AC (Table 1). Table 1 Electrophysiological properties of CC-Parv neurons   CC-Parv L2/3  CC-Parv L4  CC-Parv L5  CC-Parv L6  n = 11  n = 10  n = 14  n = 11  Resting potential (mV)  −80.5 ± 1.3  −78.4 ± 1.2  −76.5 ± 0.9  −77.5 ± 1.5  Input resistance (MΩ)  125.6 ± 10.8  106.5 ± 7.2  117.8 ± 7.8  114.7 ± 8.7  Membrane time constant (ms)  0.58 ± 0.04  0.6 ± 0.03  0.6 ± 0.06  0.6 ± 0.004  Rheobase (pA)  268.2 ± 23.6  320 ± 32.6  271.4 ± 22.6  270 ± 18.6  After hyperpolarization (mV)  −11.6 ± 2.2  −14.7 ± 1.6  −13.2 ± 1.2  −12.5 ± 0.9  Action potential threshold (mV)  −36.4 ± 1.2  −38.2 ± 2.2  −40.1 ± 1.4  −40.7 ± 1.7  Action potential height (mV)  35.2 ± 1.8  31.9 ± 2.9  39.5 ± 2.6  40.5 ± 3.1  Action potential width (ms)  0.46 ± 0.01  0.46 ± 0.01  0.46 ± 0.08  0.47 ± 0.01  F/I slope (Hz/pA step)  0.7 ± 0.05  0.7 ± 0.08  0.72 ± 0.04  0.77 ± 0.05  Spike frequency adaptation (third/fifth)  1 ± 0.05  1 ± 0.02  0.97 ± 0.08  1 ± 0.04    CC-Parv L2/3  CC-Parv L4  CC-Parv L5  CC-Parv L6  n = 11  n = 10  n = 14  n = 11  Resting potential (mV)  −80.5 ± 1.3  −78.4 ± 1.2  −76.5 ± 0.9  −77.5 ± 1.5  Input resistance (MΩ)  125.6 ± 10.8  106.5 ± 7.2  117.8 ± 7.8  114.7 ± 8.7  Membrane time constant (ms)  0.58 ± 0.04  0.6 ± 0.03  0.6 ± 0.06  0.6 ± 0.004  Rheobase (pA)  268.2 ± 23.6  320 ± 32.6  271.4 ± 22.6  270 ± 18.6  After hyperpolarization (mV)  −11.6 ± 2.2  −14.7 ± 1.6  −13.2 ± 1.2  −12.5 ± 0.9  Action potential threshold (mV)  −36.4 ± 1.2  −38.2 ± 2.2  −40.1 ± 1.4  −40.7 ± 1.7  Action potential height (mV)  35.2 ± 1.8  31.9 ± 2.9  39.5 ± 2.6  40.5 ± 3.1  Action potential width (ms)  0.46 ± 0.01  0.46 ± 0.01  0.46 ± 0.08  0.47 ± 0.01  F/I slope (Hz/pA step)  0.7 ± 0.05  0.7 ± 0.08  0.72 ± 0.04  0.77 ± 0.05  Spike frequency adaptation (third/fifth)  1 ± 0.05  1 ± 0.02  0.97 ± 0.08  1 ± 0.04  Figure 3. View largeDownload slide Electrophysiological properties of long-range CC-Parv neurons in the mouse auditory cortex. (a) Response recorded from a layer 2/3 CC-Parv neuron in the auditory cortex during injection of a hyperpolarizing current (1 s, −200 pA pulse) and a train of action potentials recorded during injection of a depolarizing current (1 s, 400 pA pulse). Top inset: single action potential from a L2/3 CC-Parv neuron. (b) Summary plot of Vrest: resting membrane potential; Rin: input resistance; Tau: membrane time constant; Rheobase: the smallest current step evoking an action potential; AHP: after hyperpolarization; AP thr: action potential threshold; AP height: action potential height; AP width: action potential width; F/I slope; and SFA: spikes frequency adaptation from layer 2/3 CC-Parv neurons in the auditory cortex (n = 11; animals n = 8). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. (c) Same as in panel (a), for a layer 4 CC-Parv neuron. (d) Same as in panel (b), for layer 4 CC-Parv neurons (n = 10; animals n = 6). (e) Same as in panel (a), for a layer 5 CC-Parv neuron. (f) Same as in panel (b), for layer 5 CC-Parv neurons (n = 14; animals n = 7). (g) Same as in panel (a), for a layer 6 CC-Parv neuron. (h) Same as in panel (b), for layer 6 CC-Parv neurons (n = 10; animals n = 7). Figure 3. View largeDownload slide Electrophysiological properties of long-range CC-Parv neurons in the mouse auditory cortex. (a) Response recorded from a layer 2/3 CC-Parv neuron in the auditory cortex during injection of a hyperpolarizing current (1 s, −200 pA pulse) and a train of action potentials recorded during injection of a depolarizing current (1 s, 400 pA pulse). Top inset: single action potential from a L2/3 CC-Parv neuron. (b) Summary plot of Vrest: resting membrane potential; Rin: input resistance; Tau: membrane time constant; Rheobase: the smallest current step evoking an action potential; AHP: after hyperpolarization; AP thr: action potential threshold; AP height: action potential height; AP width: action potential width; F/I slope; and SFA: spikes frequency adaptation from layer 2/3 CC-Parv neurons in the auditory cortex (n = 11; animals n = 8). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. (c) Same as in panel (a), for a layer 4 CC-Parv neuron. (d) Same as in panel (b), for layer 4 CC-Parv neurons (n = 10; animals n = 6). (e) Same as in panel (a), for a layer 5 CC-Parv neuron. (f) Same as in panel (b), for layer 5 CC-Parv neurons (n = 14; animals n = 7). (g) Same as in panel (a), for a layer 6 CC-Parv neuron. (h) Same as in panel (b), for layer 6 CC-Parv neurons (n = 10; animals n = 7). Statistical analysis to compare the electrophysiological properties of CC-Parv neurons revealed no difference between CC-Parv neurons located in different cortical layers. Confocal images of biocytin-filled CC-Parv neurons showed that they are similar to basket-cell interneurons in their morphology and send an axonal projection towards/into the subcortical white matter (Fig. 4). These data show that CC-Parv neuronal morphology and intrinsic electrophysiological properties resemble those of the Parv basket-cell interneurons. Figure 4. View largeDownload slide Morphological properties of long-range CC-Parv neurons in the mouse auditory cortex. (a) Morphological reconstruction of a layer 4 CC-Parv neuron (dendrites: white; axon: red). (b) Same as in panel (a), for a layer 5 CC-Parv neuron. Figure 4. View largeDownload slide Morphological properties of long-range CC-Parv neurons in the mouse auditory cortex. (a) Morphological reconstruction of a layer 4 CC-Parv neuron (dendrites: white; axon: red). (b) Same as in panel (a), for a layer 5 CC-Parv neuron. Do Callosal Parv Neurons Inhibit Pyramidal Neurons Locally? To determine the connectivity pattern of CC-Parv neurons onto pyramidal neurons in the AC, we used an optogenetic approach in which we conditionally and retrogradely expressed ChR2 (Nagel et al. 2003) in CC-Parv neurons by injecting AAV1.ChR2.Flex into the right AC of Parv-Cre transgenic mice (Fig. 5a–d). By expressing ChR2, mammalian neurons can be excited with high temporal resolution (Boyden et al. 2005; Cardin et al. 2009; Apicella et al. 2012; Rock and Apicella 2015; Rock et al. 2016). Three to four weeks following viral transfection, we recorded from the left AC in cell-attached mode and triggered photo-evoked action potentials from CC-Parv neurons (Fig. 5f). Blue light stimulation (470 nm) caused CC-Parv-neurons to fire action potentials (n = 10). We used the trough of the photo-evoked action potential to determine the onset of the action potential (3.8 ± 0.4 ms, n = 10). The morphological and the electrical identity of patched neurons were confirmed after photostimulation by breaking in and recording the neurons’ electrical properties in current-clamp mode. At the same time, neurons were filled with biocytin and subsequently stained for light-microscopic morphological identification (Fig. 5e). Figure 5. View largeDownload slide Photostimulation of auditory CC-Parv neurons elicits direct inhibition onto local pyramidal neurons. (a) Schematic depicting injection site using the Parv-Cre transgenic mouse line to retrogradely transfect CC-Parv neurons in the contralateral auditory cortex with ChR2. Right auditory cortex: AAV1.ChR2.Flex injection site. Left auditory cortex: red CC-Parv ChR2-tdTomato-positive neurons and callosum axons. (b) Bright-field (left) and epifluorescence (right) images of a slice containing the auditory cortex injection site for AAV1.ChR2.Flex. (c) Bright-field (left) and epifluorescence (right) images of a slice containing the left auditory cortex showing expression of ChR2 following injection of AAV1.ChR2.Flex into the right auditory cortex. (d) Experimental paradigm for photostimulating ChR2-positive CC-Parv neurons while recording from pyramidal neurons. (e) High-resolution image of a layer 5 biocytin-labeled CC-Parv and pyramidal neuron. (f) Left: example of responses recorded in cell-attached mode during photoactivation of CC-Parv neurons. Right: plot of onset latencies recorded in CC-Parv neurons (n = 10; animals n = 5). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. (g) Example of IPSCs recorded at 0 mV from a pyramidal neuron before (red trace) and after application of ionotropic glutamate receptor antagonists (NBQX 10 μM, CPP 5 μM: magenta trace) and GABAA receptor antagonist (Gabazine 25 μM: black trace). (h) Left: plot of onset latencies recorded in pyramidal neurons (n = 10; animals n = 5). Middle: plot of IPSCs peaks calculated for pyramidal neurons. Right: plot of IPSCs charge transfer calculated for individual IPSCs for pyramidal neurons. Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. (i) Left: example of IPSCs (black trace) and rising time course (red trace) recorded at 0 mV from a pyramidal neuron. Right: plot of IPSCs rising time course recorded in pyramidal neurons (n = 15; animals n = 8). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. (j) Left: example of IPSCs (black trace) and decay time course (amber trace) recorded at 0 mV from a pyramidal neuron. Right: plot of IPSCs decay time course recorded in pyramidal neurons (n = 15; animals n = 8). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. Figure 5. View largeDownload slide Photostimulation of auditory CC-Parv neurons elicits direct inhibition onto local pyramidal neurons. (a) Schematic depicting injection site using the Parv-Cre transgenic mouse line to retrogradely transfect CC-Parv neurons in the contralateral auditory cortex with ChR2. Right auditory cortex: AAV1.ChR2.Flex injection site. Left auditory cortex: red CC-Parv ChR2-tdTomato-positive neurons and callosum axons. (b) Bright-field (left) and epifluorescence (right) images of a slice containing the auditory cortex injection site for AAV1.ChR2.Flex. (c) Bright-field (left) and epifluorescence (right) images of a slice containing the left auditory cortex showing expression of ChR2 following injection of AAV1.ChR2.Flex into the right auditory cortex. (d) Experimental paradigm for photostimulating ChR2-positive CC-Parv neurons while recording from pyramidal neurons. (e) High-resolution image of a layer 5 biocytin-labeled CC-Parv and pyramidal neuron. (f) Left: example of responses recorded in cell-attached mode during photoactivation of CC-Parv neurons. Right: plot of onset latencies recorded in CC-Parv neurons (n = 10; animals n = 5). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. (g) Example of IPSCs recorded at 0 mV from a pyramidal neuron before (red trace) and after application of ionotropic glutamate receptor antagonists (NBQX 10 μM, CPP 5 μM: magenta trace) and GABAA receptor antagonist (Gabazine 25 μM: black trace). (h) Left: plot of onset latencies recorded in pyramidal neurons (n = 10; animals n = 5). Middle: plot of IPSCs peaks calculated for pyramidal neurons. Right: plot of IPSCs charge transfer calculated for individual IPSCs for pyramidal neurons. Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. (i) Left: example of IPSCs (black trace) and rising time course (red trace) recorded at 0 mV from a pyramidal neuron. Right: plot of IPSCs rising time course recorded in pyramidal neurons (n = 15; animals n = 8). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. (j) Left: example of IPSCs (black trace) and decay time course (amber trace) recorded at 0 mV from a pyramidal neuron. Right: plot of IPSCs decay time course recorded in pyramidal neurons (n = 15; animals n = 8). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. To determine the synaptic properties of CC-Parv projections onto local pyramidal neurons, we photoactivated CC-Parv ChR2-positive neurons by flashing blue light for 2–5 ms during whole-cell recordings from pyramidal neurons (Fig. 5d,e, g–j; n = 15). Because of the variability of ChR2 expression levels (number of ChR2 molecules per transfected neuron) and transfection efficiency (number of ChR2-expressing neurons per animal), we adjusted the intensity of the blue light to evoke inhibitory postsynaptic currents (IPSCs) with a single peak. IPSCs (Fig. 5g, red trace) were isolated by applying a command potential of 0 mV (the calculated reversal potential for glutamatergic excitatory conductance). The latency of the photo-evoked IPSCs response was 4.0 ± 0.2 ms (Fig. 5h, left). This latency is consistent with the IPSCs being the result of a monosynaptic inhibitory input from CC-Parv neurons and not a cortical feedback inhibitory network recruited by cortical projections. Blocking excitatory neurotransmission by application of glutamate receptor antagonists NBQX and CPP did not abolish the photo-evoked IPSCs (Fig. 5g, magenta trace; n = 5). In contrast, blocking inhibitory neurotransmission by application of Gabazine (Fig. 5g, black trace; n = 5) completely abolished the photo-evoked IPSCs, confirming that they were elicited by direct cortical inhibitory transmission. Basic biophysical properties for photo-evoked IPSCs (n = 15) included (Fig. 5h–j): peak, 126 ± 13 pA; charge, 2.03 ± 0.195 × 104 pC; rise time, 0.0075 ± 0.001 ms; and decay time, 13.95 ± 1.26 ms. These data reveal that a large proportion of pyramidal neurons receive direct inhibitory input driven by local CC-Parv neurons, but does not exclude the possibility that long-range axons form the contralateral CC-Parv neurons also contribute to the photo-evoked IPSCs. Do Callosal Parv Neurons Receive Thalamocortical Input? To determine the connectivity pattern of thalamocortical input onto CC-Parv neurons in the AC, we used an optogenetic approach in which we conditionally and retrogradely expressed GFP in CC-Parv neurons by injecting AAV1.GFP.Flex into the right AC and expressed ChR2 in thalamocortical projections by injecting AAV9.ChR2 in the left MGB of Parv-Cre mice (Fig. 6a–c). We performed whole-cell voltage-clamp recordings from visually identified layers 4 and 5 CC-Parv neurons in the left AC during photoactivation of thalamic projections (Fig. 6d). To block disynaptic and polysynaptic responses and to examine direct thalamocortical monosynaptic inputs onto CC-Parv neurons, we applied both TTX and 4-AP (Petreanu et al. 2009; Cruikshank et al. 2010; Ji et al. 2016). We isolated excitatory postsynaptic currents (EPSCs) by applying a command potential of −70 mV (the calculated reversal potential for GABAergic inhibitory conductance). Basic biophysical properties for photo-evoked EPSCs onto layer 4 CC-Parv neurons (n = 9) included (Fig. 6e): onset, 2.1 ± 0.2 ms; peak, 100.4 ± 12.2 pA; and charge, 6.82 ± 1.37 × 103 pC. Basic biophysical properties for photo-evoked EPSCs onto layer 5 CC-Parv neurons (n = 11) included (Fig. 6e): onset, 2.3 ± 0.2 ms; peak, 105.4 ± 11.9 pA; and charge, 5.44 ± 0.737 × 103 pC. Figure 6. View largeDownload slide Photostimulation of thalamic projections elicit monosynaptic excitatory inputs onto CC-Parv neurons. (a) Experimental paradigm for photostimulating ChR2-positive thalamic projections and retrograde viral labeling of CC-Parv neurons. Top, auditory cortex: AAV1.GFP.Flex injection site. Bottom, MGB: red AAV9.ChR2 injection site. (b) Bright-field (top) and epifluorescence (bottom) images of a slice containing the MGB injection site for AAV9.ChR2. (c) tdTomato epifluorescence (left) and GFP epifluorescence (right) images of a slice containing the left auditory cortex showing expression of ChR2-tdTomato thalamic axons following injection of AAV9.ChR2 into the left MGB and GFP-positive CC-Parv neurons following injection of AAV1.GFP.Flex into the right auditory cortex. (d) Top: example of EPSCs recorded at −70 mV from a layer 4 pyramidal neuron during photostimulation of ChR2-positve thalamic axons after application of TTX (1 μM) and 4-AP (100 μM). Bottom: example of EPSCs recorded at −70 mV from a layer 5 pyramidal neuron during photostimulation of ChR2-positve thalamic axons after application of TTX (1 μM) and 4-AP (100 μM). (e) Top row: for layer 4 CC-Parv neurons (n = 9; animals n = 6): left, plot of onset latencies; middle, plot of IPSCs peaks; right, plot of EPSCs charge transfer calculated for individual EPSCs. Bottom row: for layer 5 CC-Parv neurons (n = 11; animals n = 9): same as top row. Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. Figure 6. View largeDownload slide Photostimulation of thalamic projections elicit monosynaptic excitatory inputs onto CC-Parv neurons. (a) Experimental paradigm for photostimulating ChR2-positive thalamic projections and retrograde viral labeling of CC-Parv neurons. Top, auditory cortex: AAV1.GFP.Flex injection site. Bottom, MGB: red AAV9.ChR2 injection site. (b) Bright-field (top) and epifluorescence (bottom) images of a slice containing the MGB injection site for AAV9.ChR2. (c) tdTomato epifluorescence (left) and GFP epifluorescence (right) images of a slice containing the left auditory cortex showing expression of ChR2-tdTomato thalamic axons following injection of AAV9.ChR2 into the left MGB and GFP-positive CC-Parv neurons following injection of AAV1.GFP.Flex into the right auditory cortex. (d) Top: example of EPSCs recorded at −70 mV from a layer 4 pyramidal neuron during photostimulation of ChR2-positve thalamic axons after application of TTX (1 μM) and 4-AP (100 μM). Bottom: example of EPSCs recorded at −70 mV from a layer 5 pyramidal neuron during photostimulation of ChR2-positve thalamic axons after application of TTX (1 μM) and 4-AP (100 μM). (e) Top row: for layer 4 CC-Parv neurons (n = 9; animals n = 6): left, plot of onset latencies; middle, plot of IPSCs peaks; right, plot of EPSCs charge transfer calculated for individual EPSCs. Bottom row: for layer 5 CC-Parv neurons (n = 11; animals n = 9): same as top row. Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. To further characterize that thalamic projections are a source of excitatory input onto CC-Parv neurons, we used VGluT2 immunoreactivity for the identification of presynaptic terminals of thalamocortical inputs. VGluT2 is a vesicular glutamate transporter and it has been demonstrated in the neocortex that almost all the VGluT2 immunoreactivity belongs to thalamic axons (Kaneko and Fujiyama 2002). First, we conditionally expressed tdTomato in CC-Parv neurons by injecting AAV1.tdTomato.Flex into the right AC of Parv-Cre mice. After incubating slices with a VGluT2 antibody and a secondary antibody conjugated with Alexa 488, we then performed confocal microscopic analysis to examine the appositions of VGluT2-positive green puncta on CC-Parv-tdTomato neurons. Only images which contained CC-Parv dendrites were used for this analysis in order to minimize the identification of spurious appositions. We found that VGluT2-positive puncta were apposed to the dendrites of the CC-Parv neurons (Fig. 7; n = 11 neurons). Figure 7. View largeDownload slide Confocal laser-scanning microscope analysis of putative thalamic synaptic input onto CC-Parv neurons in the mouse auditory cortex. (a) Left: confocal tdTomato epifluorescence image of CC-Parv tdTomato-positive dendrites (magenta). Middle: epifluorescence image of VGluT2-positive axons varicosities (green). Right: merged epifluorescence image of CC-Parv tdTomato-positive dendrites and VGluT2-positive axons varicosities. The arrows indicate putative appositions of VGluT2-positive axon varicosities with tdTomato dendrites of CC-Parv neurons. (b) Same as in panel (a), for a different CC-Parv neuron dendrite. Figure 7. View largeDownload slide Confocal laser-scanning microscope analysis of putative thalamic synaptic input onto CC-Parv neurons in the mouse auditory cortex. (a) Left: confocal tdTomato epifluorescence image of CC-Parv tdTomato-positive dendrites (magenta). Middle: epifluorescence image of VGluT2-positive axons varicosities (green). Right: merged epifluorescence image of CC-Parv tdTomato-positive dendrites and VGluT2-positive axons varicosities. The arrows indicate putative appositions of VGluT2-positive axon varicosities with tdTomato dendrites of CC-Parv neurons. (b) Same as in panel (a), for a different CC-Parv neuron dendrite. Taken together, these data reveal that both layer 4 and layer 5 CC-Parv-neurons receive direct thalamocortical input. The Callosal Parv Projection is a General Feature of the Cortex We next asked whether CC-Parv projections are a general feature of the corticocortical circuit organization, or if they are specific to the AC. To test this, we used a similar viral tracing approach in a different sensory area, the VC. We also investigated the existence of CC-Parv projections in the MC, a cortical area which is not involved in sensory processing like the AC and the VC, but instead is involved in the planning, control, and execution of voluntary movements. We used the same methods as in the AC to conditionally and retrogradely express either GFP or tdTomato in CC-Parv neurons in the VC (Figure 8) and MC (Figure 9) using a viral injection of AAV1.GFP.Flex or AAV1.tdTomato.Flex in the contralateral cortex. As before, we observed local transfection in the VC and MC (Figs. 8b and  9b), as well as GFP-positive (or tdTomato-positive) somata present in all layers of the contralateral cortices (Figs. 8c and  9c). GFP-positive (or tdTomato-positive) axons were visible in coronal sections of the corpus callosum in these mice (Figs. 8d and  9d). Figure 8. View largeDownload slide Characterization of long-range CC-Parv neurons in the mouse VC. (a) Schematic depicting injection site using the Parv-Cre transgenic mouse line to retrogradely transfect CC-Parv somata in the contralateral VC with GFP. (b) Bright-field (left) and epifluorescence (right) images of a slice containing the VC injection site for AAV1.GFP.Flex. (c) Bright-field (left) and epifluorescence (right) images of a slice containing the left VC showing expression of GFP following injection of AAV1.GFP.Flex into the right VC. (d) Confocal higher magnification epifluorescence image of GFP fluorescence of VC CC-Parv axons in the callosum. The arrows indicate the location of GFP-positive axons. (e) Morphological reconstruction of a layer 5 biocytin-labeled CC-Parv neuron in the VC (dendrites: white; axon: red). (f) Response recorded from a layer 5 CC-Parv neuron in the VC during injection of a hyperpolarizing current (1 s, −200 pA pulse) and a train of action potentials recorded during injection of a depolarizing current (1 s, 400 pA pulse). Top inset: single action potential from a VC CC-Parv neuron. (g) Summary plot of Vrest: resting membrane potential; Rin: input resistance; Tau: membrane time constant; Rheobase: the smallest current step evoking an action potential; AHP: after hyperpolarization; AP thr: action potential threshold; AP height: action potential height; AP width: action potential width; F/I slope; and SFA: spikes frequency adaptation from CC-Parv neurons in the VC (n = 13; animals n = 3). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. Figure 8. View largeDownload slide Characterization of long-range CC-Parv neurons in the mouse VC. (a) Schematic depicting injection site using the Parv-Cre transgenic mouse line to retrogradely transfect CC-Parv somata in the contralateral VC with GFP. (b) Bright-field (left) and epifluorescence (right) images of a slice containing the VC injection site for AAV1.GFP.Flex. (c) Bright-field (left) and epifluorescence (right) images of a slice containing the left VC showing expression of GFP following injection of AAV1.GFP.Flex into the right VC. (d) Confocal higher magnification epifluorescence image of GFP fluorescence of VC CC-Parv axons in the callosum. The arrows indicate the location of GFP-positive axons. (e) Morphological reconstruction of a layer 5 biocytin-labeled CC-Parv neuron in the VC (dendrites: white; axon: red). (f) Response recorded from a layer 5 CC-Parv neuron in the VC during injection of a hyperpolarizing current (1 s, −200 pA pulse) and a train of action potentials recorded during injection of a depolarizing current (1 s, 400 pA pulse). Top inset: single action potential from a VC CC-Parv neuron. (g) Summary plot of Vrest: resting membrane potential; Rin: input resistance; Tau: membrane time constant; Rheobase: the smallest current step evoking an action potential; AHP: after hyperpolarization; AP thr: action potential threshold; AP height: action potential height; AP width: action potential width; F/I slope; and SFA: spikes frequency adaptation from CC-Parv neurons in the VC (n = 13; animals n = 3). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. Figure 9. View largeDownload slide Characterization of long-range CC-Parv neurons in the mouse MC. (a) Schematic depicting injection site using the Parv-Cre transgenic mouse line to retrogradely transfect CC-Parv somata in the contralateral MC with tdTomato. (b) Bright-field (left) and epifluorescence (right) images of a slice containing the MC injection site for AAV1.tdTomato.Flex. (c) Bright-field (left) and epifluorescence (right) images of a slice containing the left MC showing expression of tdTomato following injection of AAV1.tdTomato.Flex into the right MC. (d) Confocal higher magnification epifluorescence image of tdTomato fluorescence of MC CC-Parv axons in the callosum. The arrows indicate the location of tdTomato-positive axons. (e) Morphological reconstruction of a layer 5 biocytin-labeled CC-Parv neuron in the MC (dendrites: white; axon: red). (f) Response recorded from a layer 5 CC-Parv neuron in the MC during injection of a hyperpolarizing current (1 s, −200 pA pulse) and a train of action potentials recorded during injection of a depolarizing current (1 s, 400 pA pulse). Top inset: single action potential from a MC CC-Parv neuron. (g) Summary plot of Vrest: resting membrane potential; Rin: input resistance; Tau: membrane time constant; Rheobase: the smallest current step evoking an action potential; AHP: after hyperpolarization; AP thr: action potential threshold; AP height: action potential height; AP width: action potential width; F/I slope; and SFA: spikes frequency adaptation from CC-Parv neurons in the MC (n = 14; animals n = 4). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. Figure 9. View largeDownload slide Characterization of long-range CC-Parv neurons in the mouse MC. (a) Schematic depicting injection site using the Parv-Cre transgenic mouse line to retrogradely transfect CC-Parv somata in the contralateral MC with tdTomato. (b) Bright-field (left) and epifluorescence (right) images of a slice containing the MC injection site for AAV1.tdTomato.Flex. (c) Bright-field (left) and epifluorescence (right) images of a slice containing the left MC showing expression of tdTomato following injection of AAV1.tdTomato.Flex into the right MC. (d) Confocal higher magnification epifluorescence image of tdTomato fluorescence of MC CC-Parv axons in the callosum. The arrows indicate the location of tdTomato-positive axons. (e) Morphological reconstruction of a layer 5 biocytin-labeled CC-Parv neuron in the MC (dendrites: white; axon: red). (f) Response recorded from a layer 5 CC-Parv neuron in the MC during injection of a hyperpolarizing current (1 s, −200 pA pulse) and a train of action potentials recorded during injection of a depolarizing current (1 s, 400 pA pulse). Top inset: single action potential from a MC CC-Parv neuron. (g) Summary plot of Vrest: resting membrane potential; Rin: input resistance; Tau: membrane time constant; Rheobase: the smallest current step evoking an action potential; AHP: after hyperpolarization; AP thr: action potential threshold; AP height: action potential height; AP width: action potential width; F/I slope; and SFA: spikes frequency adaptation from CC-Parv neurons in the MC (n = 14; animals n = 4). Boxplots represent the median and interquartile range, and whiskers represent the minimum and maximum values. Using the same approach described above, we next performed whole-cell patch-clamp recordings from visually identified CC-Parv neurons in VC and MC. Confocal images of biocytin-filled CC-Parv neurons in VC and MC showed that they are similar to basket-cell interneurons in their morphology and send an axonal projection towards/into the subcortical white matter (Figs. 8e and  9e). We verified their identity based on the comparison with electrophysiological properties of Parv interneurons (for review, see Hu et al. 2014). These properties include a narrow action potential and high rheobase (the smallest current step evoking an action potential) (Figs. 8f and  9f, top; inset; action potentials in CC-Parv neurons, shown in black, are very narrow). The responses to current steps in CC-Parv neurons were typical for Parv interneurons (Figs. 8f and  9f; notice the high current step needed to generate action potentials). Basic electrophysiological properties for CC-Parv neurons in VC (n = 11) included (Fig. 8g): resting membrane potential, −77.5 ± 0.8 mV; input resistance, 103.9 ± 5.2 MΩ; membrane time constant, 0.53 ± 0.02 ms; rheobase, 338.4 ± 18.0 pA; after hyperpolarization, −10.5 ± 0.9 mV; action potential threshold, −41.7 ± 1.8 mV; action potential height, 43.4 ± 2.6 mV; action potential width, 0.42 ± 0.05 ms; F/I slope, 0.8 ± 0.1 Hz/pA step; and spike frequency adaptation (third/fifth), 1.1 ± 0.03. Basic electrophysiological properties for CC-Parv neurons in MC (n = 14) included (Fig. 9g): resting membrane potential, −76.0 ± 0.8 mV; input resistance, 73.9 ± 3.8 MΩ; membrane time constant, 0.6 ± 0.08 ms; rheobase, 307.7 ± 18.6 pA; after hyperpolarization, −11.7 ± 0.9 mV; action potential threshold, −39.1 ± 1.2 mV; action potential height, 38.7 ± 1.9 mV; action potential width, 0.42 ± 0.04 ms; F/I slope, 0.8 ± 0.09 Hz/pA step; and spike frequency adaptation (third/fifth), 1.1 ± 0.1. Taken together, these results indicate that CC-Parv neurons can be found in both VC and MC, as well as in AC, and they project via the corpus callosum to the opposite hemisphere. Discussion In this study, we test the hypothesis that a class of Parv neurons makes up a callosal inhibitory projection in the mouse. Our results support this hypothesis and additionally conclude that the auditory, visual, and motor cortices send long-range GABAergic projections to their corresponding cortex in the contralateral hemisphere via these CC-Parv neurons (Fig. 10). Because of its presence in three such disparate cortical areas, this would suggest that the callosal Parv projection is likely a general feature of the interhemispheric network of the cortex. Figure 10. View largeDownload slide Summary diagram: CC-Parv neurons callosal projection. Auditory, visual, and motor CC-Parv projections modulate the activity of local pyramidal neurons by direct inhibition. Green lines: excitatory inputs from thalamic projections; red line: ipsi- and contralateral inhibitory input from CC-Parv neurons. Figure 10. View largeDownload slide Summary diagram: CC-Parv neurons callosal projection. Auditory, visual, and motor CC-Parv projections modulate the activity of local pyramidal neurons by direct inhibition. Green lines: excitatory inputs from thalamic projections; red line: ipsi- and contralateral inhibitory input from CC-Parv neurons. GABAergic neurons are the primary source of inhibition in the adult brain and they represent a minority of all cortical neurons (10–15% in rodents) (Meyer et al. 2011), but are composed of a heterogeneous cell population (for review, see Ascoli et al. 2008; Xu et al. 2010; Rudy et al. 2011). Since the observation by Ramon y Cajal (1988) that a large number of cells with “short-axons” are present in the brain, GABAergic neurons have been considered to project by and large locally in the cortex, and for this reason are often referred to as “interneurons.” However, recent studies (Melzer et al. 2012; Lee et al. 2014; Tomioka et al. 2015; Basu et al. 2016; Rock et al. 2016) are demonstrating that long-range GABAergic projections may be more prevalent than previously assumed. The main finding of the present study is that a large proportion of the entire Parv neuronal population projects through the corpus callosum to connect the two hemispheres of AC, VC, and MC. In addition, our results indicate that the somata of callosally projecting CC-Parv neurons span all layers of the cortex, while the somata of callosally projecting pyramidal neurons are located predominately in layers 2–3 (about 80% in rodents), layer 5 (about 20%) and, to a lesser extent, layer 6 (Jacobson and Trojanowski 1974; Fame et al. 2011). Despite the large number of long-range Parv neurons identified in this study, our approach is limited by caveats (limited injection volume and variability in transfection which leads to incomplete coverage of the cortical area) that preclude us from determining the absolute number and ratio of long-range CC-Parv versus short-range Parv neurons. Our findings, together with previous studies, in which long-range GABAergic projections were found to connect different brain areas in different species both ipsi- and contralaterally (Buhl and Singer 1989; McDonald and Burkhalter 1993; Tomioka et al. 2005; Apergis-Schoute et al. 2007; Higo et al. 2007; Tamamaki and Tomioka 2010; Rock et al. 2016), support a new notion in which long-range GABAergic neurons can be considered a large fraction in many cortical areas of the total GABAergic population. Moreover, this will lead to a new concept in which not only glutamatergic cortical neurons are carrying information to far stations (for review, see Tremblay et al. 2016), but also long-range GABAergic neurons. Our results are in line with recent findings that long-range GABAergic neurons in the neocortex belong to a number of molecular groups, including Parv, somatostatin-expressing, and vasoactive intestinal polypeptide-expressing neurons (Tamamaki and Tomioka 2010; Lee et al. 2014; Tomioka et al. 2015; Rock et al. 2016). Based on these previous findings and because GABAergic neurons are the most heterogeneous class of neurons in the neocortex, we predict that other cell-types may contribute to the GABAergic callosal projection. Future experiments will provide insight about the complexity of the cell-type composition of the long-range callosal GABAergic projections. Morphologically, CC-Parv neurons resemble other Parv basket-cells. Particularly, CC-Parv neurons have dendrites that often span multiple layers, allowing the basket-cells to receive input from different afferent pathways (Xu and Callaway 2009; Helmstaedter et al. 2009a, 2009b; Bagnall et al. 2011; Kubota et al. 2011; Tukker et al. 2013). The axons of Parv neurons are also very puzzling. Parv neurons have a massive axonal arborization (Sik et al. 1995; Karube et al. 2004) that generates a divergent inhibitory output (Packer and Yuste 2011; Bezaire and Soltesz 2013) that often spans multiple layers of the cortex, forming translaminar inhibitory circuits (Helmstaedter et al. 2008, 2009a, 2009b; Bortone et al. 2014; Pluta et al. 2015). Similarly, the axons of CC-Parv neurons innervate local pyramidal neurons in the AC. A large proportion of pyramidal neurons receive direct inhibitory input driven by CC-Parv neurons. The fast IPSCs rise time recorded from pyramidal neurons after photoactivation of CC-Parv neurons suggests that these neurons, as already demonstrated for the basket-cells, innervate the perisomatic domain of pyramidal neurons (Pouille and Scanziani 2001). However, this remains to be demonstrated morphologically. In addition, AAV1.Flex viral vectors (Atasoy et al. 2008) exhibit both anterograde and retrograde transfection capabilities (Rothermel et al. 2013; Rock et al. 2016). For this reason, we cannot exclude the possibility that long-range axons from the contralateral cortex also contribute to the photo-evoked inhibitory inputs onto pyramidal neurons. Numerous questions still need to be addressed in regard to long-range inhibitory circuits in the cortex. An important line of investigation will be to determine how CC-Parv neurons may differ from short-axon Parv interneurons. For example, are CC-Parv neurons receiving highly convergent inputs from pyramidal neurons and inhibitory inputs primarily from the Parv neurons in a similar connectivity pattern as short-axon Parv interneurons (Xu and Callaway 2009; Apicella et al. 2012; Pfeffer et al. 2013)? Do CC-Parv neurons form inhibitory synapses that are depressing as reported for basket-cells (Reyes et al. 1998)? Are CC-Parv neurons electrically coupled, and if so, are they only coupled with the cells expressing the same molecular markers (Amitai et al. 2002; Christie et al. 2005)? Experimental evidence indicates that basket-cells are involved in thalamic-feedforward inhibition both in vitro (Agmon and Connors 1991, 1992; Porter et al. 2001; Cruikshank et al. 2002; Beierlein et al. 2003; Gabernet et al. 2005; Rose and Metherate 2005; Inoue and Imoto 2006; Sun et al. 2006; Barkat et al. 2011; Schiff and Reyes 2012; Ji et al. 2016) and in vivo (Moore and Nelson 1998; Li et al. 2013a, 2013b; Lien and Scanziani 2013). Our findings establish that both layers 4 and 5 CC-Parv neurons are also innervated by thalamic axons. Our results are consistent with the data from somatosensory (Constantinople and Bruno 2013) and auditory cortex (Sun et al. 2013) supporting the growing view that the layer 4 is not a mandatory hub for the distribution of cortical activity, but thalamic inputs can activate also deep layers of the cortex such as layer 5. Therefore, both layers 4 and 5 CC-Parv neurons can have access to the same sensory information but can alter the behavioral outcome by differentially controlling the output of pyramidal neurons projecting to different brain areas. Moreover, our finding that CC-Parv neurons respond to thalamic activity could imply a mechanism of coincidence detection (Pouille and Scanziani 2001), in which CC-Parv neurons produce a feedforward somatic inhibition across the local pyramidal neurons in the AC. Since homotopic projections are the strongest of all the callosal projections (Rouiller et al. 1991; Lee and Winer 2008), the presence of thalamic-feedforward interhemispheric inhibition may thus serve as a means to maintain timing across these areas, and therefore refine sensory information about auditory stimuli between the two hemispheres. Indeed, basket-cells have been proposed to contribute to the phasing of pyramidal neurons during oscillations (Bartos et al. 2007; Buzsaki and Wang 2012). Therefore, we can speculate that CC-Parv neurons can be involved in the synchronization of neuronal activity between distant brain areas (for review, see Uhlhaas and Singer 2012). However, previous studies have shown that long-range GABAergic neurons preferentially innervate inhibitory neurons in the target area (Melzer et al. 2012; Basu et al. 2016). Due to both anterograde and retrograde transfection capabilities of the virus used, our study cannot determine whether CC-Parv neurons are involved in interhemispheric inhibition or disinhibition. Taken together, our results establish a previously unknown thalamo-corticocortical long-range inhibitory circuit (thalamus → CC-Parv inhibitory projections → contralateral AC). This finding suggests that the timing and ratio of excitation and inhibition provided by the callosal projection via glutamatergic and GABAergic axons may underlie long-distance synchronization. Notes We are indebted to G. Gaufo for help with confocal imaging. 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