Functional synapses are formed between human NTera2 (NT2N, hNT) neurons grown on astrocytesHartley, Rebecca S.; Margulis, Michael; Fishman, Paul S.; Lee, Virginia M.‐Y.; Tang, Cha‐Min
doi: 10.1002/(SICI)1096-9861(19990428)407:1<1::AID-CNE1>3.0.CO;2-Zpmid: 10213184
The formation of functional synapses is a late milestone of neuronal differentiation. The establishment of functional synapses can be used to assess neuronal characteristics of different cell lines. In the present study, we examined the in vitro conditions that influence the ability of human neurons derived from the NT2 cell line (NT2N neurons) to establish synapses. The morphologic, immunologic, and electrophysiologic characteristics of these synapses was examined. In the absence of astrocytes, NT2N neurons rarely formed synapses and their action potentials were weak and uncommon. In contrast, when plated on primary astrocytes, NT2N neurons were able to form both glutamatergic excitatory (71%) and GABAergic inhibitory (29%) functional synapses whose properties (kinetics, ion selectivity, pharmacology, and ultrastructure) were similar to those of synapses of neurons in primary cultures. In addition, coculture of NT2N neurons with astrocytes modified the morphology of the neurons and extended their in vitro viability to more than 1 year. Because astrocyte‐conditioned medium did not produce these effects, we infer that direct contact between NT2N neurons and astrocytes is required. These results suggest that NT2N neurons are similar to primary neurons in their synaptogenesis and their requirement for glial support for optimal survival and maturation. This system provides a model for further investigations into the neurobiology of synapses formed by human neurons. J. Comp. Neurol. 407:1–10, 1999. © 1999 Wiley‐Liss, Inc.
Distribution of the P2X2 receptor subunit of the ATP‐gated ion channels in the rat central nervous systemKanjhan, Refik; Housley, Gary D.; Burton, Lucille D.; Christie, David L.; Kippenberger, Andree; Thorne, Peter R.; Luo, Lin; Ryan, Allen F.
doi: 10.1002/(SICI)1096-9861(19990428)407:1<11::AID-CNE2>3.0.CO;2-Rpmid: N/A
The distribution of the P2X2 receptor subunit of the adenosine 5′‐triphosphate (ATP)‐gated ion channels was examined in the adult rat central nervous system (CNS) by using P2X2 receptor‐specific antisera and riboprobe‐based in situ hybridisation. P2X2 receptor mRNA expression matched the P2X2 receptor protein localisation. An extensive expression pattern was observed, including: olfactory bulb, cerebral cortex, hippocampus, habenula, thalamic and subthalamic nuclei, caudate putamen, posteromedial amygdalo‐hippocampal and amygdalo‐cortical nuclei, substantia nigra pars compacta, ventromedial and arcuate hypothalamic nuclei, supraoptic nucleus, tuberomammillary nucleus, mesencephalic trigeminal nucleus, dorsal raphe, locus coeruleus, medial parabrachial nucleus, tegmental areas, pontine nuclei, red nucleus, lateral superior olive, cochlear nuclei, spinal trigeminal nuclei, cranial motor nuclei, ventrolateral medulla, area postrema, nucleus of solitary tract, and cerebellar cortex. In the spinal cord, P2X2 receptor expression was highest in the dorsal horn, with significant neuronal labeling in the ventral horn and intermediolateral cell column. The identification of extensive P2X2 receptor immunoreactivity and mRNA distribution within the CNS demonstrated here provides a basis for the P2X receptor antagonist pharmacology reported in electrophysiological studies. These data support the role for extracellular ATP acting as a fast neurotransmitter at pre‐ and postsynaptic sites in processes such as sensory transmission, sensory‐motor integration, motor and autonomic control, and in neuronal phenomena such as long‐term potentiation (LTP) and depression (LTD). Additionally, labelling of neuroglia and fibre tracts supports a diverse role for extracellular ATP in CNS homeostasis. J. Comp. Neurol. 407:11–32, 1999. © 1999 Wiley‐Liss, Inc.
Immunohistochemical localization of subtype 4a metabotropic glutamate receptors in the rat and mouse basal gangliaBradley, Stefania Risso; Standaert, David G.; Rhodes, Kenneth J.; Rees, Howard D.; Testa, Claudia M.; Levey, Allan I.; Conn, P. Jeffrey
doi: 10.1002/(SICI)1096-9861(19990428)407:1<33::AID-CNE3>3.0.CO;2-Gpmid: N/A
Recent studies suggest that metabotropic glutamate receptors (mGluRs) may play a significant role in regulating basal ganglia functions. In this study, we investigated the localization of mGluR4a protein in the mouse and rat basal ganglia. Polyclonal antibodies that specifically react with the metabotropic glutamate receptor subtype mGluR4a were produced and characterized by Western blot analysis. These antibodies recognized a native protein in wild‐type mouse brain with a molecular weight similar to the molecular weight of the band from a mGluR4a‐transfected cell line. The immunoreactivity was absent in brains of knockout mice deficient in mGluR4. mGluR4a immunoreactivity was most intense in the molecular layer of the cerebellum. We also found a striking mGluR4a immunoreactivity in globus pallidus, and moderate staining in substantia nigra pars reticulata and entopeduncular nucleus. Moderate to low mGluR4a immunoreactivity was present in striatum and other brain regions, including hippocampus, neocortex, and thalamus. Double labeling with mGluR4a antibodies and antibodies to either a dendritic marker or a marker of presynaptic terminals suggest a localization of mGluR4a on presynaptic terminals. Immunocytochemistry at electron microscopy level confirmed these results, revealing that in the globus pallidus, mGluR4a is mainly localized in presynaptic sites in axonal elements. Finally, quinolinic acid lesion of striatal projection neurons decreased mGluR4a immunoreactivity in globus pallidus, suggesting a localization of mGluR4a on striatopallidal terminals. These data support the hypothesis that mGluR4a serves as a presynaptic heteroreceptor in the globus pallidus, where it may play an important role in regulating g‐amino‐n‐butyric acid (GABA) release from striatopallidal terminals. J. Comp. Neurol. 407:33–46, 1999. © 1999 Wiley‐Liss, Inc.
Mapping glutamatergic drive in the vertebrate retina with a channel‐permeant organic cationMarc, Robert E.
doi: 10.1002/(SICI)1096-9861(19990428)407:1<47::AID-CNE4>3.0.CO;2-0pmid: 10213187
Patterns of neuronal excitation in complex populations can be mapped anatomically by activating ionotropic glutamate receptors in the presence of 1‐amino‐4‐guanidobutane (AGB), a channel‐permeant guanidinium analogue. Intracellular AGB signals were trapped with conventional glutaraldehyde fixation and were detected by probing registered serial thin sections with anti‐AGB and anti‐amino acid immunoglobulins, revealing both the accumulated AGB and the characteristic neurochemical signatures of individual cells. In isolated rabbit retina, both glutamate and the ionotropic glutamate receptor agonists α‐amino‐3‐hydroxyl‐5‐methylisoxazole‐4‐propionic acid (AMPA), kainic acid (KA), and N‐methyl‐D‐aspartic acid (NMDA) activated permeation of AGB into retinal neurons in dose‐dependent and pharmacologically specific modes. Horizontal cells and bipolar cells were dominated by AMPA/KA receptor activation with little or no evidence of NMDA receptor involvement. Strong NMDA activation of AGB permeation was restricted to subsets of the amacrine and ganglion cell populations. Threshold agonist doses for the most responsive cell groups (AMPA, 300 nm; KA, 2 μM; NMDA, 63 μm; glutamate, 1 mM) were similar to values obtained from electrophysiological and neurotransmitter release measures. The threshold for activation of AGB permeation by exogenous glutamate was shifted to <200 μM in the presence of the glutamate transporter antagonist dihydrokainate, indicating substantial spatial buffering of extracellular glutamate levels in vitro. Agonist‐activated permeation of AGB into neurons persisted under blockades of Na+‐dependent transporters, voltage‐activated Ca+ and Na+ channels, and ionotropic γ‐aminobutyric acid and glycine receptors. Cholinergic agonists evoked no permeation. J. Comp. Neurol. 407:47–64, 1999. © 1999 Wiley‐Liss, Inc.
Kainate activation of horizontal, bipolar, amacrine, and ganglion cells in the rabbit retinaMarc, Robert E.
doi: 10.1002/(SICI)1096-9861(19990428)407:1<65::AID-CNE5>3.0.CO;2-1pmid: N/A
Patterns of excitation in populations of retinal bipolar, amacrine, and ganglion cells were mapped by activating α‐amino‐3‐hydroxyl‐5‐methylisoxazole‐4‐propionic acid (AMPA) and kainate (KA) receptors with KA in the presence of the channel‐permeant guanidinium analogue 1‐amino‐4‐guanidobutane (AGB). Registered serial thin sections were probed with immunoglobulins targeting AGB, glutamate, glycine, and γ‐aminobutyric acid (GABA) to visualize KA‐evoked responses and the neurochemical signatures of distinct cell types. OFF‐center cone bipolar cells and both type A and type B horizontal cells were strongly activated by KA. ON‐center cone bipolar cells displayed weak AGB signals that arose at least partially, if not entirely, from coupling with KA‐responsive glycinergic amacrine cells, whereas rod bipolar cells exhibited no detectable AGB permeation after KA activation. GABA‐positive amacrine cells displayed a range of KA responses, some possessing little AGB signal even after strong KA activation, whereas all identifiable glycine‐positive amacrine cells were driven by KA. Quantitative agonist responsivities of cells in the ganglion cell layer revealed that starburst amacrine cells are the most KA‐responsive cell type in that layer. Ganglion cells varied in KA responsivity across morphologic subtypes, with a large α‐like ganglion cell group the being the most KA responsive. Some ganglion cells displayed weak KA responses, even with saturating doses, that may have been be due to an absence of AMPA/KA receptors or to the existence of AGB‐impermeant AMPA/KA receptor complexes. J. Comp. Neurol. 407:65–76, 1999. © 1999 Wiley‐Liss, Inc.
p75NTR immunoreactivity in the rat dentate gyrus is mostly within presynaptic profiles but is also found in some astrocytic and postsynaptic profilesDougherty, Karen D.; Milner, Teresa A.
doi: 10.1002/(SICI)1096-9861(19990428)407:1<77::AID-CNE6>3.0.CO;2-Spmid: 10213189
To localize neurotrophin binding sites within the rat dentate gyrus, the distribution of low‐affinity p75 neurotrophin receptor (p75NTR) immunoreactivity (IR) was examined by using antiserum raised against the cytoplasmic domain of the receptor. Semiquantitative electron microscopic examination of p75NTR‐labeled sections showed that most p75NTR‐labeled profiles were axons and axon terminals (72% from a total of 3,975); p75NTR‐IR was observed throughout the extent of these structures and was not limited to the plasmalemmal surface. Axons and axon terminals containing p75NTR‐IR were distributed in approximately equal proportions across the hilus, infragranular zone, and the inner, middle, and outer molecular layers; significantly fewer p75NTR‐labeled profiles were observed in the granule cell layer. Axon terminals containing p75NTR‐IR, which made synapses (296 of 552), formed equal proportions of symmetric and asymmetric synapses, primarily with the shafts and spines of dendrites. The remainder of the p75NTR‐labeled terminals apposed unlabeled somata and dendrites without forming synapses in the single sections analyzed. In addition, p75NTR‐IR was contained within some astrocytes (17.5% of 3,975) and dendritic shafts (3%) and spines (5%). Within dendritic spines, p75NTR‐IR was most often associated with the plasmalemmal surface near postsynaptic densities; in dendritic shafts, p75NTR labeling was associated with microfilaments distant from the plasmalemma. Most p75NTR‐labeled dendritic profiles were located in the molecular layer, and some originated from granule cells. Moreover, in some granule cell somata (<1% of 3,975), p75NTR‐IR was associated with endosomes. The primary localization of p75NTR‐IR to presynaptic structures in the dentate gyrus, presumably arising from medial septal/diagonal band neurons, agrees with previous reports. However, p75NTR‐IR within some astrocytes, somata, and dendritic structures suggests that this receptor may also be involved in controlling local neurotrophin levels and possibly modulating the viability of local hippocampal cell populations. J. Comp. Neurol. 407:77–91, 1999. © 1999 Wiley‐Liss, Inc.
Relationship between laminar topology and retinotopy in the rhesus lateral geniculate nucleus: Results from a functional atlasErwin, Ed; Baker, Frank H.; Busen, William F.; Malpeli, Joseph G.
doi: 10.1002/(SICI)1096-9861(19990428)407:1<92::AID-CNE7>3.0.CO;2-1pmid: N/A
The primary focus of this paper is the abrupt transition that occurs midway through the rhesus lateral geniculate nucleus (LGN) from six layers posteriorly (conventionally numbered 1–6, ventral to dorsal) to four layers anteriorly. At this transition, layers 4 and 6 fuse into a single layer, as do layers 3 and 5, requiring an inversion of the stacking order of the cell categories making up layers 4 and 5. To understand the topology of this transition and its relationship to geniculate retinotopy, we have created a functional atlas of a rhesus LGN that affords three‐dimensional views of morphology and retinotopy at a resolution of 25 μm. The projection of the path of the transition into visual space is highly biased toward lower visual fields, intersecting the upper vertical meridian at 6.4°, the horizonal meridian at 15.4°, and the lower vertical meridian at 25.0°. Between inclinations of −31° and 55°, layers 3 and 5 merge through an elongated tear in layer 4 that subsumes the optic disk gap and extends medially and laterally; elsewhere, layers 4 and 6 merge through a tear in layer 5. These tears cause substantial violations of retinotopy and laminar integrity, so the inversion of layers 4 and 5 requires that the forces establishing retinotopy and grouping by cell class be locally overcome during morphogenesis. The transition and associated tears are evaluated in the context of recent computational models of geniculate morphogenesis. We have also used the atlas to estimate the borders of the binocular (55 ≈ 62°) and monocular (91 ≈ 97°) visual fields. Files containing the atlas are made publicly available on a website. J. Comp. Neurol. 407:92–102, 1999. © 1999 Wiley‐Liss, Inc.
Distribution of aromatase, estrogen receptor, and androgen receptor mRNA in the forebrain of songbirds and nonsongbirdsMetzdorf, Reinhold; Gahr, Manfred; Fusani, Leonida
doi: 10.1002/(SICI)1096-9861(19990428)407:1<115::AID-CNE9>3.0.CO;2-Wpmid: N/A
Androgens and estrogens are crucial for the differentiation and function of the vocal control system of songbirds. A major source of estrogens in songbirds is the cerebral aromatization of circulating testosterone by aromatase (ARO). In the vocal control system, songbirds have a unique estrogen receptor (ER)‐containing area, the nucleus hyperstriatalis ventrale pars caudale (HVC) of the caudal neostriatum. Work in the zebra finch has demonstrated ARO expression adjacent to but not in the HVC. Compared with other songbirds, such as the canary, the HVC of adult zebra finches contains only few ERs. To determine whether the disjunctive distribution of ERs and ARO in the forebrain is a songbird‐specific feature, the authors investigated ARO and ER mRNA expression in songbirds (canary, house sparrow, and zebra finch) and in nonsongbirds (budgerigar, ring dove, swift, grey partridge, and barn owl) of five avian orders. In addition, the coexpression of androgen receptor (AR) and ARO mRNAs was studied. Preoptic hypothalamic areas showed similar expression of ARO in all species. In the caudal neostriatum, ARO, AR, and ER transcripts were found only in songbirds. ARO and ER mRNA expression in the caudal forebrain was spatially separated, i.e., the HVC contained ER mRNA but very little or no ARO mRNA, and the caudomedial neostriatum contained high levels of ARO mRNA but few if any ERs. ARO and AR mRNAs, however, were coexpressed in the caudomedial neostriatum. The coexpression of ARO mRNA with AR mRNA but not with ER mRNA was found in further brain areas, such as the nucleus posterior lateralis hypothalami. The area‐specific coexpression of AR, ER, and ARO suggests various possibilities for the steroid‐dependent regulation of ARO and for the role of ARO in controlling AR‐ and ER‐dependent mechanisms. J. Comp. Neurol. 407:115–129, 1999. © 1999 Wiley‐Liss, Inc.
Subnuclear organization of the rat habenular complexesAndres, Karl Hermann; Düring, Monika Von; Veh, Rüdiger W.
doi: 10.1002/(SICI)1096-9861(19990428)407:1<130::AID-CNE10>3.0.CO;2-8pmid: 10213193
The habenular complexes represent phylogenetically constant structures in the diencephalon of all vertebrates. Available evidence suggests that this area is engaged in a variety of important biological functions, such as reproductive behaviors, central pain processing, nutrition, sleep‐wake cycles, stress responses, and learning. Based on Nissl‐stained sections, one medial nucleus and two lateral nuclei (divisions) have been widely accepted in the rat. Cytochemical, hodologic, and functional studies suggest a considerably more complex subnuclear structure. To improve our knowledge of the precise structural composition of the habenular complexes, we have systematically investigated their fine ultrastructure in the rat. Based on the detailed analysis of complete series of large, semithin sections supplemented with electron photomicrographs of selected fields, clear criteria for the delineation of five distinct subnuclei of the medial and ten subnuclei of the lateral habenular complexes were elaborated for the first time. All 15 subnuclei were reconstructed, and their dimensions were determined. A medial and lateral stria medullaris were described. Different roots of the fasciculus retroflexus were differentiated within the medial and lateral habenular complexes. The topographical relationships with respect to the adjacent habenular areas as well as to the neighboring thalamic nuclei were identified and demonstrated. The new understanding of the subnuclear organization of the habenular complexes certainly will facilitate further functional investigations. Whether the newly identified subnuclei finally will be recognized as functionally distinct awaits ongoing immunocytochemical, hodologic, and functional studies. J. Comp. Neurol. 407:130–150, 1999. © 1999 Wiley‐Liss, Inc.