Functional expression of the peptide transporter PEPT2 in the mammalian enteric nervous systemRühl, Anne; Hoppe, Susanne; Frey, Isabelle; Daniel, Hannelore; Schemann, Michael
doi: 10.1002/cne.20617pmid: 16041713
The peptide transporter PEPT2 mediates transmembrane uptake of small peptides. So far, its expression has not been evidenced in the gastrointestinal tract. We have investigated peptide transport activity in the neuromuscular layers of the gastrointestinal tract by using the fluorescent tracer‐dipeptide β‐Ala‐Lys‐Nϵ‐7‐amino‐4‐methyl‐coumarin‐3‐acetic acid (Ala‐Lys‐AMCA). Whole‐mount preparations from mouse, rat, and guinea pig stomach and small and large intestine were incubated with Ala‐Lys‐AMCA in the presence or absence of the uptake‐inhibitors L‐histidine, D‐phenylalanyl‐L‐alanine (D‐Phe‐Ala), glycyl‐L‐sarcosine (Gly‐Sar), glycyl‐L‐glutamine (Gly‐Gln), benzylpenicillin, and cefadroxil. Fluorescence microscopy revealed that Ala‐Lys‐AMCA specifically accumulated in both ganglionic layers of the enteric nervous system (ENS) in all regions and species studied. This could be inhibited by Gly‐Sar, D‐Phe‐Ala, Gly‐Gln, and cefadroxil, but not by free histidine and benzylpenicillin, indicating uptake via PEPT2. Accordingly, dipeptide uptake was completely abolished in PEPT2‐deficient mice. Reverse transcriptase‐polymerase chain reaction analysis detected a PEPT2‐specific transcript in extracts from the ganglionic ENS layers of mouse small and large intestine, further proving that enteric dipeptide transport activity is specifically mediated via PEPT2. The cellular site of dipeptide uptake was immunohistochemically localized to enteric glial cells and tissue‐resident macrophages. In addition, dipeptide uptake occurred in a neurochemically defined subset of neurons in the guinea pig ENS. Our results constitute the first functional evidence for dipeptide transport activity in the ENS. PEPT2‐mediated dipeptide transport in enteric glia could contribute to the clearance of neuropeptides in the ENS. In addition, the fluorophore‐coupled dipeptide uptake via PEPT2 is a novel vital marker for glial cells in the ENS. J. Comp. Neurol. 490:1–11, 2005. © 2005 Wiley‐Liss, Inc.
Development of putative GABAergic neurons in the appendicularian urochordate Oikopleura dioicaSøviknes, Anne Mette; Chourrout, Daniel; Glover, Joel C.
doi: 10.1002/cne.20629pmid: 16041716
Studying the developing brain of urochordates can increase our understanding of brain evolution in the chordate lineage. To begin addressing regional patterns of neuronal differentiation in appendicularian urochordates, we examined the development of putative GABAergic neurons in Oikopleura dioica using GABA immunohistochemistry and in situ hybridization for the GABA‐synthesizing enzyme GAD. First, we assessed the developmental dynamics of neuron number and organization in the cerebral and caudal ganglia. We then identified and mapped the positions of putative GABAergic neurons using confocal microscopy. We found GAD mRNA‐positive and GABA‐immunopositive neurons in the first brain nerves and the cerebral and caudal ganglia, but not in the caudal nerve cord. In both ganglia GAD mRNA‐positive and GABA‐immunopositive neurons are found in the same characteristic intraganglionic locations. The differentiation of these GABAergic markers occurs first in the first brain nerves and the cerebral ganglion and then with a several‐hour delay in the caudal ganglion. In all three structures GAD mRNA expression appears 2–3 hours prior to GABA expression. In general, GABA is expressed by the same number of neurons as express GAD. Several discrepancies suggest differential regulation of the GABAergic phenotype in different neurons, however. Our results show that the GABAergic phenotype has a stereotyped pattern of expression along the anteroposterior axis of the CNS. Given recent genome sequencing and developmental patterning gene studies in this species, the GABAergic neurons in O. dioica provide a good model for assessing, at the invertebrate–vertebrate transition, the molecular mechanisms that specify the GABAergic phenotype. J. Comp. Neurol. 490:12–28, 2005. © 2005 Wiley‐Liss, Inc.
Connexin45 mediates gap junctional coupling of bistratified ganglion cells in the mouse retinaSchubert, Timm; Maxeiner, Stephan; Krüger, Olaf; Willecke, Klaus; Weiler, Reto
doi: 10.1002/cne.20621pmid: 16041717
Direction selectivity, a key feature of visual perception, originates in the retina and is transmitted by bistratified ganglion cells that, in the rabbit retina, exhibit a particular coupling pattern. We intracellularly labeled ganglion cells in different transgenic mouse lines, allowing a morphological classification of bistratified ganglion cells, an analysis of their coupling pattern, and the molecular identification of the connexins responsible for the coupling. Based on dendritic characteristics including co‐fasciculation with the dendrites of cholinergic starburst amacrine cells, we were able to distinguish three types of bistratified ganglion cells. Two of these co‐fasciculate with starburst amacrine cells and exhibit a specific homologous coupling pattern. Connexin45 (Cx45) appears to be the major component of the gap junctional channels because tracer coupling is absent in Cx45‐deficient animals whereas it persists in Cx36‐deficient animals. It is speculated that the transjunctional voltage dependence of Cx45 channels could support the transmission of direction selectivity. J. Comp. Neurol. 490:29–39, 2005. © 2005 Wiley‐Liss, Inc.
Definition and novel connections of the entopallium in the pigeon (Columba livia)Krützfeldt, Nils O.E.; Wild, J. Martin
doi: 10.1002/cne.20627pmid: 16041718
The avian entopallium (E) is the major thalamorecipient zone, within the telencephalon, of the tectofugal visual system. Because of discrepancies concerning the structure of this nuclear mass in pigeons, and in light of recent evidence concerning entopallial projections in other avian species, we here redefine and chart some novel entopallial projections in the pigeon by using a combination of cytochrome oxidase (CO) activity, calcium binding protein immunohistochemistry (CBPi), normal histology, and tract tracing. We show that 1) E is defined by the accurate overlap of CO activity and the dense terminations of thalamic (rotundal) efferents; 2) the perientopallium (Ep), E's overlying belt region, receives a relatively sparse rotundal input and is a major source of projections to wider regions of the hemisphere; and 3) E can be subdivided into internal (Ei) and external (Ex) portions on the basis of normal histology, CBPi, and differential projections. Thus, Ei, but not Ex, makes a reciprocal connection with a distinct nucleus in the ventrolateral mesopallium and is a major source of projections to the lateral striatum. These findings suggest the necessity for a revision of the original proposal of a strictly serial flow of visual information through the entopallial complex and further regions of the hemisphere and also require a modification of the long‐standing view that E is comparable to only one specific lamina (IV) of extrastriate visual cortex of mammals (Karten and Hodos [1970] J. Comp. Neurol. 140:35–51; Shimizu and Karten [1990] In: Finlay BL, et al., editors. The neocortex. New York: Plenum Press. p 75–86). Rather, E appears to be composed of a variety of neuronal types possibly equivalent to those in several neocortical laminae. J. Comp. Neurol. 490:40–56, 2005. © 2005 Wiley‐Liss, Inc.
Novel insect orcokinins: Characterization and neuronal distribution in the brains of selected dicondylian insectsHofer, Sabine; Dircksen, Heinrich; Tollbäck, Petter; Homberg, Uwe
doi: 10.1002/cne.20650pmid: 16041719
Orcokinins are a family of myotropic neuropeptides identified in various decapod crustaceans and recently in a cockroach. Their presence in the crustacean nervous system and hemolymph suggests that they act as hormones and as locally acting neuromodulators. To provide further evidence for the existence of orcokinins in insects, we identified a novel orcokinin‐related peptide in the locust Schistocerca gregaria and used an antiserum against Asn13‐orcokinin for immunostaining in the brains of selected dicondylian insects, including a silverfish, three polyneopteran species (a cockroach and two locusts), and three endopterygote species (a moth, a bee, and a fly). As analyzed by MALDI‐TOF spectrometry and nanoelectrospray Q‐TOF, the locust orcokinin is a novel tetradecapeptide with striking sequence similarity to crustacean orcokinins. Orcokinin immunostaining was widespread and occurred in similar patterns in the brain of the silverfish and the polyneopteran species. Prominent immunostaining was detected in the optic lobe, especially in the medulla and in the accessory medulla, in local interneurons of the antennal lobe, and in extrinsic and intrinsic mushroom‐body neurons. All parts of the central complex and many other areas of the brains were densely stained. In the silverfish, the cockroach, and the locusts, processes in the corpora cardiaca showed orcokinin immunoreactivity, suggesting that orcokinins also serve a hormonal role. In contrast to the case in polyneopteran species, immunostaining was completely lacking in the brains of the honeybee, fruitfly, and sphinx moth. This indicates that orcokinins either are modified considerably or may be completely absent in the brains of endopterygote insects. J. Comp. Neurol. 490:57–71, 2005. © 2005 Wiley‐Liss, Inc.
Synapse composition and organization following chronic activity blockade in cultured hippocampal neuronsHarms, Kimberly J.; Craig, Ann Marie
doi: 10.1002/cne.20635pmid: 16041714
Activity plays multiple roles in the expression of synaptic plasticity, and has been shown to regulate the localization of both neurotransmitter receptors and downstream signaling machinery. However, the role of activity in central synapse formation and organization is incompletely understood. Some studies indicate that synapse formation can occur in the absence of synaptic activity, while others indicate that activity is required for synapse maintenance and receptor recruitment. In addition, the effects of long‐term blockade of transmission generally, rather than blockade of specific receptors, on postsynaptic protein complement has been poorly characterized. In order to address the role of activity in synapse formation and postsynaptic specialization, we used tetanus toxin to chronically cleave VAMP2 and inhibit SNARE‐mediated neurotransmitter release in cultured hippocampal neurons. Although these neurons are deficient in synaptic release, they are of normal size and morphology. In addition, both excitatory and inhibitory synapses form along their processes with normal density. These synapses have a remarkably similar cellular and molecular organization compared to controls, and are capable of recruiting postsynaptic scaffolding proteins, GABA, and glutamate receptors. Subcellular enrichment of synaptic proteins into specialized domains also appears intact. These data indicate that global activity inhibition is insufficient to disrupt central synapse formation or organization. J. Comp. Neurol. 490:72–84, 2005. © 2005 Wiley‐Liss, Inc.
Developmental modifications of olivocerebellar topography: The granuloprival cerebellum reveals multiple routes from the inferior oliveFournier, Betty; Lohof, Ann M.; Bower, Adrian J.; Mariani, Jean; Sherrard, Rachel M.
doi: 10.1002/cne.20648pmid: 16041715
Correct function of neural circuits depends on highly organized neuronal connections, refined from less precise projections through synaptic elimination, collateral regression, or neuronal death. We examined regressive phenomena that define olivocerebellar topography during maturation from Purkinje cell polyinnervation to monoinnervation. We used bilateral retrograde tracing to determine the source of olivocerebellar afferents to posterior vermis lobules VII–VIII in a model of retained immature Purkinje cell polyinnervation, the granuloprival cerebellum. In controls, labelled neurons were found only in the contralateral inferior olive (ION) clustered in a small ventromedial locus that is congruent with known olivocerebellar topography. In granuloprival animals, olivary labelling appeared more dispersed and was present in homologous ipsilateral regions. Double‐labelled neurons were never seen. Retrograde tracing following unilateral olivocerebellar transection in adult granuloprival rats revealed: 1) the origin of the normal (remaining) path projecting through the contralateral inferior peduncle was more localized than in irradiated nonpedunculotomized rats, 2) a small double‐crossed path, and 3) a projection that ascends the peduncle ipsilateral to the ION of origin, part of which crosses the midline within the cerebellum. Electrophysiological and immunohistochemical assessment in the neonatal cerebellum revealed that transcommissural paths are not present during development but sprout within the irradiated cerebellum. Therefore, the olivocerebellar projection in the granuloprival rat, as a model of the immature path, shows parasagittal organization similar to that of controls in its normally crossed path but possesses additional abnormal projections. Thus, maturation of olivocerebellar topography involves removal of whole developmental paths to define laterality plus synapse elimination within largely predefined parasagittal zones. J. Comp. Neurol. 490:85–97, 2005. © 2005 Wiley‐Liss, Inc.