GABA(C) receptors are expressed in GABAergic and non-GABAergic neurons of the rat superior colliculus and visual cortex.

GABA(C) receptors are enriched in the upper grey layers of the mammalian superior colliculus and contribute to synaptic processing. Electrophysiological data suggested that the GABA(C) receptor ρ subunits are expressed by GABAergic interneurons which represent about half of the neurons in the stratum griseum superficiale (SGS). Combining in situ hybridization for ρ2 receptor mRNA and the glutamic acid decarboxylase GAD-65 mRNA confirmed this assumption. A majority of ρ-labeled neurons in SGS and pretectum are GABAergic. Combining in situ hybridization with immunohistochemistry for the two projection neuron markers calbindin and parvalbumin revealed that a few ρ2 mRNA expressing cells coexpressed calbindin, but not parvalbumin. In visual cortex, ρ2 mRNA was present in pyramidal neurons and parvalbumin-containing interneurons. The results show that in the SGS primarily GABAergic neurons express GABA(C) receptors whereas the majority of tectothalamic calbindin neurons and intrinsically projecting parvalbumin neurons do not.

Visual experience regulates Kv3.1b and Kv3.2 expression in developing rat visual cortex.

Among the GABAergic neocortical interneurons, parvalbumin-containing fast-spiking (FS) basket cells are essential mediators of feed-forward inhibition, network synchrony and oscillations, and timing of the critical period for sensory plasticity. The FS phenotype matures after birth. It depends on the expression of the voltage-gated potassium channels Kv3.1b/3.2 which mediate the fast membrane repolarization necessary for firing fast action potentials at high frequencies. We have now tested in rat visual cortex if visual deprivation affects the Kv3 expression. During normal development, Kv3.1b/3.2 mRNA and protein expression increased in rat visual cortex reaching adult levels around P20. Dark rearing from birth neither prevented nor delayed the upregulation. Rather unexpectedly, the expression of Kv3.1b protein and Kv3.2 mRNA and protein increased to higher levels from the third postnatal week onwards. Triple-labeling revealed that in dark-reared visual cortex Kv3.2 was upregulated in parvalbuminergic interneurons in supragranular layers which in normal animals rarely display Kv3.2 expression. Recovery from dark rearing normalized Kv3.2 expression. This showed that visual experience influences the Kv3 expression. The results suggest that an altered expression of Kv3 channels affects the functional properties of FS neurons, and may contribute to the deficits in inhibition observed in the sensory-deprived cortex.

Neuronal activity and TrkB ligands influence Kv3.1b and Kv3.2 expression in developing cortical interneurons.

Among the GABAergic neocortical interneurons, fast-spiking (FS) basket and chandelier cells are essential mediators for feed-forward inhibition, network synchrony and oscillations. The FS properties are in part mediated by the voltage-gated potassium channels Kv3.1b/3.2 which allow the fast repolarization of the membrane necessary for firing non-adapting action potentials at high frequencies. It has been recently reported that the FS phenotype fails to mature in BDNF knockout mice suggesting a role for neurotrophins. We now describe the role of neuronal activity and neurotrophins for Kv3.1b/3.2 expression using organotypic cultures of rat visual cortex as model system. Chronic activity deprivation from 2 days in vitro (DIV) prevented the postnatal developmental increase of Kv3.2, but not Kv3.1b mRNA expression. However, chronic activity deprivation failed to alter Kv3.1b and marginally delayed Kv3.2 protein expression. Activity deprivation by glutamate receptor blockade from 10 to 20 DIV reduced both mRNAs, whereas deprivation with tetrodotoxin (TTX) reduced both mRNAs and the Kv3.2 protein. Thalamic and cortical afferents in cocultures failed to alter the expression. BDNF and NT4 supplemented from 2 DIV onwards increased the expression of Kv3.1b, but not Kv3.2 mRNA in young cultures. Only NT4 increased the expression of both mRNAs later in development. Kv3 protein levels were not changed by exogenous tropomyosin-related kinase B (TrkB) ligands, but the levels decreased upon inhibiting the MAPK signaling suggesting a role for endogenous factors and in particular MEK2 signaling for translation. The results show that Kv3.1b/3.2 expression is differentially controlled by neuronal activity and neurotrophic factors.

Characterization of pretectal-nuclear-complex afferents to the pulvinar in the cat.

We investigated anatomical and physiological properties of the projection from the pretectal nuclear complex (PNC) to the ipsilateral lateral posterior-pulvinar complex in the cat. After Phaseolus vulgaris leucoagglutinin injections into the PNC, the majority (70%) of anterogradely labeled terminals was localized in the pulvinar proper, the remaining 30% were scattered in the lateral and medial portions of the LP. No PNC neuron retrogradely labeled from the pulvinar was found to also express glutamic acid decarboxylase (GAD) mRNA, although a large number of neurons carrying the GAD label were found in close vicinity. In contrast, 69% of retrogradely labeled PNC cells also displayed glutamate-like immunoreactivity. Twenty-six out of 96 (27%) visually responsive pulvinar neurons were orthodromically activated by electrical stimulation of the ipsilateral PNC at latencies between 1 and 10 ms (median 1.9 ms). All orthodromically activated neurons responded well to the onset and offset of large visual stimuli and to sudden stimulus shifts. Whenever a saccadic eye movement was executed, these neurons were also activated, except during saccades in darkness. The comparison of saccade-evoked response with responses to visual stimuli that elicit similar retinal image shifts revealed that pretectorecipient pulvinar neurons also seem to receive a saccade-related non-visual input. All response properties correspond to those of a specific class of pulvinar neurons that have been termed "SV" neurons because they respond to visual stimulation as well as during saccades. They also closely resemble response properties of PNC neurons that project to the ipsilateral pulvinar. The results support the proposal that PNC cells not only directly activate their postsynaptic target neurons in the pulvinar, but that they also provide a visual input to these neurons that greatly contributes to their response characteristics.

Specification of neuropeptide Y phenotype in visual cortical neurons by leukemia inhibitory factor.

Building the complex mammalian neocortex requires appropriate numbers of neurochemically specified neurons. It is not clear how the highly diverse cortical interneurons acquire their distinctive phenotypes. The lack of genetic determination implicates environmental factors in this selection and specification process. We analysed, in organotypic visual cortex cultures, the specification of neurons expressing neuropeptide Y (NPY), a potent anticonvulsant. Endogenous brain-derived neurotrophic factor and neurotrophin 4/5 play no role in early NPY phenotype specification. Rather, the decision to express NPY is made during a period of molecular plasticity during which differentiating neurons with the potential to express NPY compete for the cytokine leukemia inhibitory factor which is produced in the cortex, but is negatively regulated by thalamic afferences. The neurons that fail in this competition are parvalbuminergic basket and chandelier neurons, which express NPY transiently, but will not acquire a permanent NPY expression. They switch into a facultative NPY expression mode, and remain responsive to the neurotrophins which modulate NPY expression later in development.

Transgenic activation of Ras in neurons promotes hypertrophy and protects from lesion-induced degeneration.

Ras is a universal eukaryotic intracellular protein integrating extracellular signals from multiple receptor types. To investigate its role in the adult central nervous system, constitutively activated V12-Ha-Ras was expressed selectively in neurons of transgenic mice via a synapsin promoter. Ras-transgene protein expression increased postnatally, reaching a four- to fivefold elevation at day 40 and persisting at this level, thereafter. Neuronal Ras was constitutively active and a corresponding activating phosphorylation of mitogen-activated kinase was observed, but there were no changes in the activity of phosphoinositide 3-kinase, the phosphorylation of its target kinase Akt/PKB, or expression of the anti-apoptotic proteins Bcl-2 or Bcl-X(L). Neuronal Ras activation did not alter the total number of neurons, but induced cell soma hypertrophy, which resulted in a 14.5% increase of total brain volume. Choline acetyltransferase and tyrosine hydroxylase activities were increased, as well as neuropeptide Y expression. Degeneration of motorneurons was completely prevented after facial nerve lesion in Ras-transgenic mice. Furthermore, neurotoxin-induced degeneration of dopaminergic substantia nigra neurons and their striatal projections was greatly attenuated. Thus, the Ras signaling pathway mimics neurotrophic effects and triggers neuroprotective mechanisms in adult mice. Neuronal Ras activation might become a tool to stabilize donor neurons for neural transplantation and to protect neuronal populations in neurodegenerative diseases.

Development of neuronal activity and activity-dependent expression of brain-derived neurotrophic factor mRNA in organotypic cultures of rat visual cortex.

We have analyzed in organotypic rat visual cortex cultures the way in which expression of brain-derived neurotrophic factor (BDNF) mRNA depends on synaptically generated spontaneous bioelectric activity (SBA) as monitored by recordings of pyramidal cells. SBA was initially low, but from the fourth week onwards 83% of the neurons fired action potentials at 0.2-1.2 impulses/s in a well-balanced state of excitation and inhibition. BDNF mRNA expression increased during the second week to a level surprisingly similar to the adult visual cortex in vivo, despite the fact that activity rates in vitro were approximately 10-fold lower than rates reported in vivo. Thus, SBA generated by a cortical neuronal network in the absence of sensory input is sufficient to elicit and maintain BDNF expression. The transient BDNF peak occurring after eye opening in vivo did not occur in vitro. A blockade of SBA seems not to alter the expression of neurotrophin (NT)-3 and -4/5, and tyrosine kinase receptor C and B mRNA. However, BDNF expression remained extremely low. A recovery of SBA after a period of blockade concurred with a transient hyperexcitability. BDNF immediately increased, driven by calcium influx through voltage-gated channels in synergy with NMDA receptors. Expression transiently reached high levels in neurons of supragranular layers. Infragranular neurons, although firing action potentials, recovered BDNF expression much slower. After 5 days in vitro recovery, the network had de novo established a balanced state of excitation and inhibition. Distribution and expression level of BDNF mRNA had returned to control. Even in 'adult' cultures an acute blockade of SBA downregulated BDNF, and a subsequent recovery of SBA restored BDNF expression. We conclude that BDNF mRNA expression depends on and responds with a fast kinetic to changes of the SBA. Steady-state levels do not depend on the absolute levels of activity, but more likely on the balance between excitation and inhibition, suggesting a role for BDNF in activity homeostasis.

The paleocortical ventricle is the origin of reelin-expressing neurons in the marginal zone of the foetal human neocortex.

The subpial granular layer (SGL) is a transient cell layer in the cortical marginal zone during the period of neuronal migration into the cortical plate. The origin of the SGL has been studied by immunocytochemistry for calretinin (CR) and reelin in human foetuses from 11 to 40 gestational weeks (GW). At 11 GW, the paleocortical ventricle, a rostral dilatation of the lateral ventricle, gives rise to two fountainheads: a medial fountainhead provides neurons for the marginal zone (MZ) of the rostral cortex and rostral hippocampal rudiment, while multiple cell streams migrate from a lateral fountainhead into the MZ of the paleocortex and insula. The latero-medial gradient of neuronal packing density in the neocortical MZ indicates that migration extends farther into the neocortex. Neurons express CR already in the retrobulbar ventricular zone; they express reelin only as they approach the MZ of the paleocortex and rostral archicortex. At 16/17 GW, large numbers of CR-immunoreactive granule cells originate from the same fountainheads, and then direct medially, toward the surface of the anterior perforated substance, and laterally, into the paleocortical MZ, from where they continue into the neocortical SGL following a ventrolateral to dorsomedial gradient. From 13 to 18 GW, reelin is expressed by a subpopulation of granule cells and by Cajal-Retzius-like neurons. By 22 GW, the paleocortical ventricle undergoes regression and no longer supplies the SGL. Our results show that the paleocortical ventricle gives rise to a stream of neurons which extends over the cortical MZ as the subpial granular layer. The fact that SGL derivatives express reelin suggests that this transient cell layer may play a significant role in the establishment of the complex cytoarchitecture of the cerebral cortex.

Patterns of spontaneous activity and morphology of interneuron types in organotypic cortex and thalamus-cortex cultures.

The physiological and morphological properties of interneurons in infragranular layers of rat visual cortex have been studied in organotypic cortex monocultures and thalamus-cortex co-cultures using intracellular recordings and biocytin injections. Cultures were prepared at the day of birth and maintained for up to 20 weeks. Twenty-nine interneurons of different types were characterized, in addition to 170 pyramidal neurons. The cultures developed a considerable degree of synaptically driven "spontaneous" bioelectric activity without epileptiform activity. Interneurons in cortex monocultures and thalamus-cortex co-cultures had the same physiological and morphological properties, and also pyramidal cell properties were not different in the two culture conditions. All interneurons and the majority of pyramidal cells displayed synaptically driven action potentials. The physiological group of fast-spiking interneurons included large basket cells, columnar basket cells (two cells with an arcade axon) and horizontally bitufted cells. The physiological group of slow-spiking interneurons included Martinotti cells and a "long-axon" cell. Analyses of the temporal patterns of activity revealed that fast-spiking interneurons have higher rates of spontaneous activity than slow-spiking interneurons and pyramidal cells. Furthermore, fast-spiking interneurons fired spontaneous bursts of action potentials in the gamma frequency range. We conclude from these findings that physiological and morphological properties of interneurons in organotypic mono- and co-cultures match those of interneurons characterized in vivo or in acute slice preparations, and they maintain in long-term cultures a well-balanced state of excitation and inhibition. This suggests that cortex-intrinsic or cell-autonomous mechanisms are sufficient for the expression of cell type-specific electrophysiological properties in the absence of afferents or sensory input.

Sensory impairments and delayed regeneration of sensory axons in interleukin-6-deficient mice.

Interleukin-6 (IL-6) is a multifunctional cytokine mediating inflammatory or immune reactions. Here we investigated the possible role of IL-6 in the intact or lesioned peripheral nervous system using adult IL-6 gene knockout (IL-6(-/-)) mice. Various sensory functions were tested by applying electrophysiological, morphological, biochemical, and behavioral methods. There was a 60% reduction of the compound action potential of the sensory branch of IL-6(-/-) mice as compared with the motor branch in the intact sciatic nerve. Cross sections of L5 DRG of IL-6(-/-) mice showed a shift in the relative size distribution of the neurons. The temperature sensitivity of IL-6(-/-) mice was also significantly reduced. After crush lesion of the sciatic nerve, its functional recovery was delayed in IL-6(-/-) mice as analyzed from a behavioral footprint assay. Measurements of compound action potentials 20 d after crush lesion showed that there was a very low level of recovery of the sensory but not of the motor branch of IL-6(-/-) mice. Similar results of sensory impairments were obtained with mice showing slow Wallerian degeneration (Wlds) and a delayed lesion-induced recruitment of macrophages. However, in contrast to WldS mice, in IL-6(-/-) mice we observed the characteristic lesion-induced invasion of macrophages and the upregulation of low-affinity neurotrophin receptor p75 (p75LNTR) mRNA levels identical to those of IL-6(+/+) mice. Thus, the mechanisms leading to the common sensory deficiencies were different between IL-6(-/-) and WldS mice. Altogether, the results suggest that interleukin-6 is essential to modulate sensory functions in vivo.

Expression of TrkB and TrkC but not BDNF mRNA in neurochemically identified interneurons in rat visual cortex in vivo and in organotypic cultures.

The mammalian visual cortex contains morphologically diverse populations of interneurons whose neurochemical properties are believed to be regulated by neurotrophic factors. This requires the expression of neurotrophin receptors. We have analysed whether brain-derived neurotrophic factor (BDNF), its receptor trkB and the NT-3 receptor trkC are expressed in interneurons of rat visual cortex in vivo, and in organotypic visual cortex cultures, paying particular attention to the subsets of neuropeptidergic neurons. In situ hybridization in combination with immunofluorescence for calcium-binding proteins and neuropeptides revealed that BDNF is not expressed in interneurons in vivo or in vitro. For the neurotrophin receptors we found in vivo at postnatal day 70 (P70) that approximately 80% of the parvalbumin-immunoreactive (-ir), but only 50% of the intensely calbindin-ir, and only 20% of the calretinin-ir neurons express trkB. Double labelling with neuropeptides revealed that approximately 50% of the neuropeptide Y-ir and approximately 50% of the somatostatin-ir neurons express trkB in a laminar-specific way. Only 25% of the vasoactive intestinal polypeptide (VIP)-ir neurons coexpress trkB. The coexpression of neuropeptide Y with trkB, but not with BDNF or trkC, was confirmed with a double in situ hybridization. In contrast, the percentages differed in the immature cortex; at P14 70% of the NPY-ir neurons and 46% of the calretinin-ir neurons revealed trkB expression, while the ratio for calbindin-ir cells was fairly constant (59%). From the interneuron populations studied, only 12% of the parvalbumin-ir neurons expressed trkC. A triple labelling revealed that some neurons coexpressed both trk mRNAs, while others had only trkC. The analysis of interneurons in organotypic cultures yielded very similar results. The results indicate that trkB ligands synthesized by pyramidal neurons influence neuropeptide or calcium-binding protein expression in a paracrine or transsynaptic manner. However, in contrast to current belief, in the adult only about half of all interneurons appear responsive to trkB ligands. Although the proportion is higher in the immature cortex, not all of the interneurons appear neurotrophin-receptive. With regard to the presence or absence of neurotrophin receptors, the molecular heterogeneity of GABAergic interneurons in the visual cortex is higher than currently assumed, and the responsiveness to neurotrophins changes with development in a cell type-specific way.

GABAergic inhibition in the rat pontine nuclei is exclusively extrinsic: evidence from an in situ hybridization study for GAD67 mRNA.

As clearly indicated by our electrophysiological work, GABAergic inhibition plays a powerful role in the pontine nuclei (PN), the major link between cerebral cortex and the cerebellum. Using the technique of in situ hybridization for the mRNA encoding for the gamma-aminobutyric acid (GABA)-synthesizing isoenzyme glutamic acid decarboxylase67 (GAD67), we demonstrate here the total absence of potentially GABAergic neurons from the rat PN. This negative finding supports the notion that GABAergic inhibition in the PN of rats, unlike that of higher mammals, is exclusively based on extrapontine GABAergic afferents.

Cellular reactivity to mechanical axonal injury in an organotypic in vitro model of neurotrauma.

An in vitro model of traumatic brain injury is described that is based on organotypic cocultures (OTCs) of rat neocortex and thalamus connected by reciprocal axonal projections. Localized mechanical compression of this projection was inflicted with a mechanical device, and the effects on cell viability, axonal morphology, and protein expression levels were analyzed. Within 24 h after insult, major cell damage occurred in infragranular cortical layers containing the corticothalamic projection neurons and in thalamic regions adjacent to the mechanical impact as was assessed through the use of the vital stain Syto 21, and propidium iodide labeling. A small, but significant number of calretinin-positive interneurons in cortical and thalamic areas displayed symptoms of injury. Axonal elements, as revealed by neurofilament (NF-H/M) immunohistochemistry, in the corticothalamic transition zone displayed pathomorphological changes, such as axonal bulbs and swellings, already 4 h after insult. Densitometric analysis revealed that MAP-2a,b expression was not significantly changed within 4 h after injury. A significant reduction in MAP-2a,b amount was evident at 20 h after injury in thalamus (by 31.6%) and cortex (by 30%) maintained for 12 days in vitro (DIV), but not in OTCs aged 20 DIV. The axonally localized form MAP-2c significantly increased in cortex of 12-DIV OTCs at 4 and 20 h after insult (65.6% and 33.4%, respectively). MAP-2c levels in cortex of 20 DIV initially increased by 47.7% and declined below control values 20 h after injury. Thalamic areas revealed a delay in MAP-2c reactivity, in that expression was significantly elevated only at 20 h after injury (by 84.4% in 12-DIV and by 39.6% in 20-DIV OTCs, respectively). These data may reflect the regenerative ability of juvenile, but not of older neurons in response to mechanical axonal injury.

Epigenetic factors regulate the NPY expression in rat cortical neurons.

The NPY phenotype expressed in a subset of rat neocortical neurons is influenced by a variety of epigenetic factors. In the present study, we analyzed the role of synaptically driven spontaneous bioelectric (action potential) activity (SBA) and neurotrophic factors. Our model systems are organotypic monocultures of visual cortex which either grow as spontaneously active cultures or as activity-blocked cultures to which neurotrophic factors can be applied via the medium. NPY mRNA expressing neurons are detected by in situ hybridization and are quantified as a percentage of all neurons. In spontaneously active cultures, about 7% of all neurons express NPY mRNA. This expression is regulated by SBA, because expression is reduced to about 2% by different activity blockade paradigms. When putative NPY neurons differentiate under activity blockade, they are unable to restitute the NPY expression during a subsequent period of SBA. A restitution of the NPY phenotype in 6-7% of the neurons after a transient blockade of activity is only possible when neurons were initially allowed to differentiate in the presence of SBA. We then analyzed whether neurotrophic factors known to promote NPY expression can do so in the absence of SBA. Neurotrophin-4/5 and leukemia inhibitory factor, but not brain-derived neurotrophic factor and neurotrophin-3, stimulate the NPY phenotype in the absence of SBA. In situ hybridization in combination with immuno-fluorescence reveals that NPY-ir neurons express the receptors trkB or LIFRbeta, but not trkC. This coexpression pattern explains why neurotrophin-4/5 and leukemia inhibitory factor are efficient regulators of the NPY-expression. Our results suggest that the NPY expression in neocortical neurons depends on epigenetic factors: spontaneous activity and neurotrophic factors modulate the expression and are thus involved in shaping the neurochemical architecture of the cerebral cortex.

Visual activity is required to maintain the phenotype of supragranular NPY neurons in rat area 17.

Visual activity governs the functional maturation of the mammalian visual cortex. We report here, that visual experience is required for stabilizing the phenotype of a subset of cortical interneurons. Neurons expressing neuropeptide Y mRNA (NPY neurons) display a transiently higher expression in the early postnatal visual areas 18a and 17 that is followed by a phenotype restriction during the second postnatal month: about 50% of the NPY neurons in supragranular and infragranular layers of area 18a, and in infragranular layers of area 17 gradually stop the NPY expression. In contrast, the expression remains unchanged in supragranular layers of area 17. Dark rearing rats from birth to up to 100 days does neither prevent the developmental onset of NPY mRNA expression, nor does it prevent the phenotype restriction from occurring. In contrast, in dark reared animals NPY neurons in supragranular layers of area 17 now also undergo a phenotype restriction. Returning animals to light after variable periods of darkness results in an upregulation of NPY mRNA expression selectively in neurons in supragranular layers of area 17. These neurons acquire a constitutive expression during the second postnatal month. This suggests that the phenotypic specification of a distinct subset of cortical interneurons is regulated by visual experience which thus influences on the maturation of the neurochemical architecture of area 17.

NT-4/5 and LIF, but not NT-3 and BDNF, promote NPY mRNA expression in cortical neurons in the absence of spontaneous bioelectrical activity.

Epigenetic factors are known to influence the differentiation of neocortical neurons. The present study analyses the role of spontaneous bioelectrical activity (SBA) and neurotrophic factors on the expression of neuropeptide Y (NPY) in rat visual cortical neurons using organotypic monocultures prepared from newborn animals and in situ hybridization to detect the NPY messenger ribonucleic acid (mRNA). Spontaneously active cortex cultures display NPY mRNA expression in about 7% of all cortical neurons from 10 days in vitro (DIV) on. Blocking the SBA by chronic application of 10 mM Mg2+ for 3-30 DIV reduces the percentage of NPY neurons to about 2%. Allowing an initial phase of SBA (1-20 DIV) followed by an SBA blockade (for 21-50 DIV) results in 2% labelled neurons, indicating a dramatic reduction of NPY mRNA expression in the absence of SBA. Surprisingly, the reverse experiment (a period of SBA blockade for 1-20 DIV followed by a period of SBA recovery for 21-40 DIV) does not cause an upregulation of NPY mRNA expression. However, allowing cultures to differentiate as spontaneously active cultures, then applying a transient period of SBA blockade which is followed by a second period of SBA, does rescue the NPY mRNA expression in 7% of the cortical neurons. We conclude that SBA is a main trigger for NPY mRNA expression and it is particularly important during an early postnatal period of differentiation. We then analysed whether neurotrophic factors known to modulate cortical neuropeptide expression are able to do so in the absence of SBA. Supplementing chronically blocked cultures with the neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5) and the cytokine, leukaemia inhibitory factor (LIF), reveals that BDNF and NT-3 are unable to increase the percentage of NPY neurons. In contrast, LIF and NT-4/5 increase the percentage of NPY neurons to 4 and 6-7%, respectively. Moreover, neurons treated with NT-4/5 display a very high level of NPY mRNA expression in somata and in the dendritic trees. The data suggest a complex interplay and a hierarchy of epigenetic factors in regulating the neurochemical architecture of the developing neocortex.

Characterization of a directional selective inhibitory input from the medial terminal nucleus to the pretectal nuclear complex in the rat.

The receptive field properties of neurons in the medial terminal nucleus of the accessory optic system (MTN) that project to the ipsilateral nucleus of the optic tract (NOT) and dorsal terminal nucleus (DTN), as identified by antidromic electrical activation, were analysed in the anaesthetized rat. The great majority (88%) of MTN neurons that were antidromically activated from NOT and DTN preferred downward directed movement of large visual stimuli while the remaining cells preferred upward directed stimulus movement. Distinct retrograde tracer injections into the NOT/DTN and the ipsilateral inferior olive (IO) revealed that no MTN neurons project to both targets. MTN neurons projecting to the ipsilateral NOT/DTN were predominantly found in the ventral part of the MTN, whereas those projecting to the IO were found in the dorsal part of the MTN. In situ hybridization for glutamic acid decarboxylase (GAD) mRNA was used as a marker for GABAergic neurons. Up to 98% of MTN neurons retrogradely labelled from the ipsilateral NOT/DTN also expressed GAD mRNA. Earlier studies have shown that MTN neurons that prefer upward directed stimulus movements are segregated from MTN neurons that prefer downward directed stimulus movements. It also has been demonstrated that directionally selective neurons in the NOT/DTN prefer horizontal stimulus movements and receive an inhibitory input from ipsilateral MTN. Our results indicate that this input is mediated by GABAergic cells in the ventral part of MTN, which to a large extent prefer downward directed stimulus movements, and that the great majority of MTN neurons that prefer upward directed stimulus movements project to other targets one of which possibly is the IO.

Postnatal expression pattern of calcium-binding proteins in organotypic thalamic cultures and in the dorsal thalamus in vivo.

The present study describes the postnatal expression of calbindin, calretinin and parvalbumin and glutamic acid decarboxylase (GAD) and microtubule-associated protein 2 (MAP2) in organotypic monocultures of rat dorsal thalamus compared to the thalamus in vivo. Cultures were maintained for up to 7 weeks. Cortex-conditioned medium improved the survival of thalamic cultures. MAP2-immunoreactive material was present in somata and dendrites of small and large-sized neurons throughout the cultures. Parvalbumin immunoreactivity was present in larger multipolar or bitufted neurons along the edge of a culture. These neurons also displayed strong parvalbumin mRNA and GAD mRNA expression, and GABA immunoreactivity. They likely corresponded to cells of the nucleus reticularis thalami. Parvalbumin mRNA, but neither parvalbumin protein nor GAD mRNA, was expressed in neurons with large somata within the explant. They likely represented relay cells. GAD mRNA, but not parvalbumin mRNA, was expressed in small neurons within the explants. Small neurons also displayed calbindin- and calretinin-immunoreactivity. The small neurons likely represented local circuit neurons. The time course of expression of the calcium-binding proteins revealed that all were present at birth with the predicted molecular weights. A low, but constant parvalbumin expression was observed in vitro without the developmental increase seen in vivo, which most likely represented parvalbumin from afferent sources. In contrast, the explantation transiently downregulated the calretinin and calbindin expression, but the neurons recovered the expression after 14 and 21 days, respectively. In conclusion, thalamic monocultures older than three weeks represent a stable neuronal network containing well differentiated neurons of the nucleus reticularis thalami, relay cells and local circuit neurons.

Biochemical and histological analysis of two Müller cell antibodies in developing and adult cat and rat central nervous system.

We investigated the binding characteristics of two monoclonal antibodies, 4F3 and 3F8, which in the retina specifically stain Müller cells, both with protein blots and immunohistochemically in sections of various regions of the central nervous system of neonatal and adult cats and rats. Clear differences emerged between the two antibodies. In addition, some species-specific as well as developmental differences within the staining pattern of each individual antibody were evident. The epitopes recognized by 4F3 lay mainly in the 57-63 kDa range. Histologically, 4F3 labelled mainly glia cells: oligodendrocytes and astrocytes in optic nerve, astrocytes in neocortex and cerebellum, Bergmann glia in the cerebellum and radial glia in neonatal animals. This was confirmed by double-immunofluorescence with the astrocyte marker GFAP. By contrast, 3F8 epitopes lay mainly in the 47-49 kDa range. Histologically, 3F8 labelled oligodendrocytes in the optic nerve, but only neurons in cerebellum and neocortex as confirmed by double-labelling with neuronal markers. Neither 4F3 nor 3F8 recognized GFAP or vimentin. These results clearly indicate (1) that the two antibodies identify new epitopes/molecules, (2) that the antigens are not retina-specific, and (3) that Müller cells share epitopes with other glial cells as well as with neurons outside the retina.