Acute slice preparation for electrophysiology increases spine numbers equivalently in the male and female juvenile hippocampus: a DiI labeling study.

Hippocampal slices are widely used for in vitro electrophysiological experiments to study underlying mechanisms for synaptic transmission and plasticity, and there is a growing appreciation for sex differences in synaptic plasticity. To date, several studies have shown that the process of making slices from male animals can induce synaptogenesis in cornu ammonis area 1 (CA1) pyramidal cells, but there is a paucity of data for females and other brain regions. In the current study we use microcrystals of the lipophilic carbocyanine dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) to stain individual neurons in the CA1 and dentate gyrus (DG) hippocampal subfields of postnatal day 21 male and female rats. We show that the preparation of sections for electrophysiology produces significant increases in spines in sections obtained from females, similar to that observed in males. We also show that the procedures used for in vitro electrophysiology also result in significant spine increases in the DG and CA1 subfields. These results demonstrate the utility of this refined DiI procedure for staining neuronal dendrites and spines. They also show, for the first time, that in vitro electrophysiology slice preparations enhance spine numbers on hippocampal cells equivalently in both juvenile females and males.NEW & NOTEWORTHY This study introduces a new DiI technique that elucidates differences in spine numbers in juvenile female and male hippocampus, and shows that slice preparations for hippocampal electrophysiology in vitro may mask these differences.

Nerve sprouting in innervated adult skeletal muscle induced by exposure to elevated levels of insulin-like growth factors.

Partial denervation or paralysis of adult skeletal muscle is followed by nerve sprouting, probably due to release of diffusible sprout-inducing activity by inactive muscle. Insulin-like growth factors (IGF1 and IFG2) are candidates for muscle-derived sprouting activity, because (a) they induce neurite growth from peripheral neurons in vitro; and (b) their mRNA levels in adult skeletal muscle increase severalfold after denervation or paralysis. We sought to determine whether the presence of elevated levels of IGFs in innervated adult skeletal muscle was sufficient to produce intramuscular nerve growth. Low concentrations of IGFs induced massive neurite growth from enriched embryonic chick motoneurons in vitro. Half-maximal responses required 0.2 nM IGF2 or IGF1, or 20 nM insulin. Similar hormone binding properties of motoneuron processes in vitro were observed. Exposure of adult rat or mouse gluteus muscle in vivo to low quantities of exogenous IGF2 or IGF1 led to intramuscular nerve sprouting. Numbers of sprouts in IGF-exposed muscles were 10-fold higher than in vehicle-exposed or untreated muscles, and 12.2% of the end plates in IGF-exposed muscle (control: 2.7%) had sprouts growing from them. The nerve growth reaction was accompanied by elevated levels of intramuscular nerve-specific growth-associated protein GAP43. Additional properties of IGF-exposed muscle included modest proliferation of interstitial cells and elevated interstitial J1 immunoreactivity. These results suggest that elevated levels of IGFs in denervated or paralyzed muscle might trigger coordinate regenerative reactions, including nerve sprouting and expression of nerve growth-supporting substrate molecules by activated interstitial cells.

Subcellular localization of the voltage-gated potassium channels Kv3.1b and Kv3.3 in the cerebellar dentate nucleus of glutamic acid decarboxylase 67-green fluorescent protein transgenic mice.

Deep cerebellar dentate nuclei are in a key position to control motor planning as a result of an integration of cerebropontine inputs and hemispheric Purkinje neurons signals, and their influence through synaptic outputs onto extracerebellar hubs. GABAergic dentate neurons exhibit broader action potentials and slower afterhyperpolarization than non-GABAergic (presumably glutamatergic) neurons. Specific potassium channels may be involved in these distinct firing profiles, particularly, Kv3.1 and Kv3.3 subunits which rapidly activate at relatively positive potentials to support the generation of fast action potentials. To investigate the subcellular localization of Kv3.1b and Kv3.3 in GAD- and GAD+ dentate neurons of glutamic acid decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice a preembedding immunocytochemical method for electron microscopy was used. Kv3.1b and Kv3.3 were in membranes of cell somata, dendrites, axons and synaptic terminals of both GAD- and GAD+ dentate neurons. The vast majority of GAD- somatodendritic membrane segments domains labeled for Kv3.1b and Kv3.3 (96.1% and 84.7%, respectively) whereas 56.2% and 69.8% of GAD- axonal membrane segments were immunopositive for these subunits. Furthermore, density of Kv3.1b immunoparticles was much higher in GAD- somatodendritic than axonal domains. As to GAD+ neurons, only 70.6% and 50% of somatodendritic membrane segments, and 53.3% and 59.5% of axonal membranes exhibited Kv3.1b and Kv3.3 labeling, respectively. In contrast to GAD- cells, GAD+ cells exhibited a higher density labeling for both Kv3 subunits at their axonal than at their somatodendritic membranes. Taken together, Kv3.1b and Kv3.3 potassium subunits are expressed in both GAD- and GAD+ cells, albeit at different densities and distribution. They likely contribute to the distinct biophysical properties of both GAD- and GAD+ neurons in the dentate nucleus.

Immunoreactivity for the group III receptor subtype mGluR4a in the visual layers of the rat superior colliculus.

Several studies indicate that metabotropic glutamate receptors (mGluRs) participate in the transmission of visual stimuli in optic layers of the superior colliculus (SC). We examined the cellular and subcellular distribution of the group III mGluR4a in superficial layers of the rat SC by means of a specific antiserum and a preembedding immunogold method for electron microscopy. Deposits of mGluR4a immunoparticles were mostly observed on presynaptic membranes of large synaptic terminals, which made asymmetrical synapses and contained abundant spherical, clear synaptic vesicles and numerous electron translucent mitochondria. These characteristic ultrastructural features correspond to retinocollicular synaptic terminals. Also, chains of synaptic retinal terminals along dendrites were labeled for mGluR4a. About 70% of morphologically identified retinal terminals were mGluR4a immunopositive. Furthermore, mGluR4a immunoreactivity in SC greatly disappeared following retinal ablation. About 28% of cortical terminals identified by anterograde tracing showed mGluR4a labeling, whereas only 2% of collicular GABAergic profiles were labeled for mGluR4a. These results reveal that retinal terminals are the major contributors to the mGluR4a immunoreactivity observed in the superior collicular circuitry.

Differential distribution of metabotropic glutamate receptors 1a, 1b, and 5 in the rat spinal cord.

Metabotropic glutamate receptors (mGluRs) modulate somatosensory, autonomic, and motor functions at spinal levels. mGluR postsynaptic actions over spinal neurons display the pharmacologic characteristics of type I mGluRs; however, the spinal distribution of type I mGluR isoforms remains poorly defined. In this study, the authors describe a differential distribution of immunoreactivity to various type I mGluR isoforms (mGluR1a, mGluR5a,b, and mGluR1b) that suggests a correlation between specific isoforms and particular aspects of spinal cord function. Two different antisera raised against mGluR5a,b detected intense immunoreactivity within nociceptive afferent terminal fields (laminae I and II) and also in autonomic regions (parasympathetic and sympathetic). In contrast, two of three anti-mGluR1a antibodies did not immunostain lamina I or II. Laminae I and II immunostaining by a third anti-mGluR1a antibody was competed by a peptide sequence obtained from a homologous region in mGluR5, suggesting possible cross reactivity in fixed tissue. Autonomic neurons did not express mGluR1a immunoreactivity. All anti-mGluR1a antibodies strongly and specifically immunolabeled dendritic and somatic membranes of neurons in the deep dorsal horn (lamina III-V) and the ventral horn (lamina VI-IX). Somatic motoneurons expressed mGluR1a immunoreactivity but little or no mGluR5 immunoreactivity. Phrenic and pudendal motoneurons expressed the highest level of mGluR1a immunoreactivity in the spinal cord. Intense mGluR1b immunoreactivity was restricted to a few scattered neurons and a prominent group of neurons in lamina X. Lamina II neurons expressed low levels of mGluR1b immunoreactivity. Ultrastructurally, type I mGluR immunoreactivity was found mostly at extrasynaptic sites on the plasma membrane, but it was also found perisynaptically, in the body of the postsynaptic regions or in relation to intracytoplasmic structures.

Immunohistochemical localization of the mGluR1beta metabotropic glutamate receptor in the adult rodent forebrain: evidence for a differential distribution of mGluR1 splice variants.

Alternative splicing has been shown to occur at the metabotropic glutamate receptor 1 (mGluR1) gene. Three main isoforms that differ in their carboxy-termini have been described so far and named mGluR1alpha, mGluR1beta and mGluR1c. These variants when expressed in recombinant systems all activate phospholipase C, although the [Ca2+] signals generated have different kinetics. Tissue distribution studies of specific mGluR1 splice variants are limited to the mGluR1alpha isoform. In the present work, we examined the localization of mGluR1beta in the adult rat and mouse forebrain by using a specific antipeptide antibody. Furthermore, the mGluR1beta immunostaining was compared with that obtained with antibodies specific for mGluR1alpha or with a pan-mGluR1 antibody which recognizes all isoforms. mGluR1beta-like immunoreactivity (LI) was found confined to the neuropil and neuronal perikarya and appeared discretely distributed in the rodent forebrain. Differential cellular distribution between mGluR1alpha and mGluR1beta was observed. In the hippocampus, mGluR1alpha-LI was restricted to non-principal neurons in all fields, whereas mGluR1beta-LI was strongest in principal cells of the CA3 field and dentate granule cells but absent in CA1. We have also shown that the vast majority of neurons in the striatum express mGluR1. The predominant form appeared to be mGluR1beta, with a distribution pattern reflecting the patch-matrix organization of the striatum. The specificity of the immunoreactivity described for mGluR1 splice variants was confirmed in mGluR1-deficient mice. The observation of a different cellular and regional distribution of mGluR1 splice variants, in particular in the hippocampus, suggests that they may mediate different roles in synaptic transmission.

Immunocytochemical localization of the mGluR1b metabotropic glutamate receptor in the rat hypothalamus.

The mGluR1 metabotropic glutamate receptor is a G-protein-coupled receptor that exists as different C-terminal splice variants. When expressed in mammalian cells, the mGluR1 splice variants exhibit diverse transduction mechanisms and also slightly differ in their apparent agonist affinities. In the present study, we used an affinity-purified antiserum, specifically reactive to the mGluRlb splice variant, in combination with a highly sensitive preembedding immunocytochemical method for light microscopy to investigate the distribution of this receptor in the rat hypothalamus. An intense immunoreactivity for mGluRlb was observed in distinct hypothalamic nuclei. Thus, neuronal cell bodies and dendrites were stained in the preoptic area, suprachiasmatic nucleus, dorsal hypothalamus, lateral hypothalamus, dorsomedial nucleus, tuberomammilary nucleus, and lateral mammilary body. The ventromedial nucleus exhibited neuropil immunostaining but neuronal cell bodies were not labeled. Strong mGluRlb immunoreactivity was observed in magnocellular neurons of the neuroendocrine supraoptic, paraventricular, and arcuate nuclei. Also, neuronal cell bodies were heavily labeled in the retrochiasmatic nucleus, anterior commissural nucleus, and periventricular nucleus. These immunocytochemical observations, together with previous studies, suggest that mGluRlb is coexpressed with other class I mGluRs in some nuclei throughout the hypothalamus. However, mGluRlb is so far the only receptor of this class strongly expressed in the supraoptic, paraventricular, and arcuate nuclei, which might have relevant implications in the physiological control of the neuroendocrine hypothalamic-pituitary system.

Immunoreactivity for the group III metabotropic glutamate receptor subtype mGluR4a in the superficial laminae of the rat spinal dorsal horn.

Studies indicate that metabotropic glutamate receptors (mGluRs) may play a role in spinal sensory transmission. We examined the cellular and subcellular distribution of the mGluR subtype 4a in spinal tissue by means of a specific antiserum and immunocytochemical techniques for light and electron microscopy. A dense plexus of mGluR4a-immunoreactive elements was seen in the dorsal horn, with an apparent accumulation in lamina II. The immunostaining was composed of sparse immunoreactive fibres and punctate elements. No perikaryal staining was seen. Immunostaining for mGluR4a was detected in small to medium-sized cells but not in large cells in dorsal root ganglia. At the electron microscopic level, superficial dorsal horn laminae demonstrated numerous immunoreactive vesicle-containing profiles. Labelling was present in the cytoplasmic matrix, but accretion of immunoreaction product to presynaptic specialisations was commonly observed. Axolemmal labelling was confirmed by using a preembedding immunogold technique, which revealed distinctive deposits of gold immunoparticles along presynaptic thickenings with an average centre-to-centre distance of 41 nm (41.145 +/- 13.59). Immunoreactive terminals often formed synaptic contacts with dendritic profiles immunonegative for mGluR4a. Immunonegative dendritic profiles were observed in apposition to both mGluR4a-immunoreactive and immunonegative terminals. Diffuse immunoperoxidase reaction product was also detected in dendritic profiles, some of which were contacted by mGluR4a-immunoreactive endings, but only occasionally were they observed to accumulate immunoreaction product along the postsynaptic density. Terminals immunoreactive for mGluR4a also formed axosomatic contacts. The present results reveal that mGluR4a subserves a complex spinal circuitry to which the primary afferent system seems to be a major contributor.

Immunocytochemical localization of the metabotropic glutamate receptor mGluR4a in the piriform cortex of the rat.

This study evaluates the localization of the metabotropic glutamate receptor mGluR4a in the piriform cortex of rats using preembedding immunocytochemical methods. At the light microscopic level, punctate labeling was evident in layers Ia and Ib of the piriform cortex, and immunolabeled fibers were present in layers II and III. Following bilateral destruction of the olfactory bulb, the density of labeled puncta in layer Ia decreased. These results suggest that the receptor is present on the terminals of the lateral olfactory tract (LOT). Electron microscopic evaluation of layers Ia and Ib revealed that mGluR4a was localized in synaptic terminals in layers Ia and Ib. The terminals had clear, round synaptic vesicles and terminated on asymmetric synapses on dendritic spines and shafts. There was also immunolabeling of some dendritic profiles in layers Ia and Ib that were postsynaptic to unlabeled presynaptic terminals. These observations suggest that mGluR4a is present on presynaptic terminals in the layers of the piriform cortex that receive LOT and associational synapses. This is the same area in which previous studies have revealed the presence of mGluR7 and mGluR8, suggesting that all three receptors may be colocalized.

Developmental expression of the group III metabotropic glutamate receptor mGluR4a in the medial nucleus of the trapezoid body of the rat.

A preembedding immunocytochemical method for light microscopy was used to study the postnatal development of expression of the group III metabotropic glutamate receptor mGluR4a in the medial nucleus of the trapezoid body (MNTB) of the rat. Immunoreactivity for mGluR4a was localized in axonal endings wrapping the principal globular neurons in MNTB, known as calyces of Held. The percentage of calyces of Held immunoreactive for mGluR4a increased progressively from postnatal day 3 (PND3), showing the highest density of labeled calyces by PND9. From this postnatal age on, a gradual reduction in the number of mGluR4a-immunopositive calyces of Held was observed, reaching the lowest level of labeled profiles in adult tissue. The developmental expression of mGluR4a in calyces of Held correlates well with previous studies in young animals showing a modulation of synaptic neurotransmission by group III mGluRs in these giant excitatory synapses made on MNTB principal neurons. All these observations together suggest that the expression of mGluR4a mainly between PND7 and PND12 might be relevant to the maturation and modulation of synaptic transmission at the calyces of Held.

Effect of perforant path lesion on pattern of glutamate-like immunoreactivity in rat dentate gyrus.

To investigate the relation between perforant path and the pattern of glutamate-like immunoreactivity in its target regions in the rat hippocampal formation, unilateral lesions of various size and location were placed to interrupt certain contingents of these afferent fibers. Postembedding immunohistochemistry at the level of light microscopy yielded the same pattern of immunoreactivity in the hippocampal formation contralateral to the lesion as in untreated animals. On the ipsilateral side, however, extensive transections of the perforant path led to a drastic loss of glutamate-immunoreactive terminal-like elements in the outer part of the dentate molecular layer. More restricted lesions induced a loss of punctate glutamate-like immunoreactivity in narrower bands within this zone. The width and the location of the affected bands appeared to depend on the extent of the transections and their topographical relation to the perforant path fiber system. These results and those obtained using a postembedding immunogold method at the level of electron microscopy strongly indicate that perforant path terminals in the dentate molecular layer of the rat contain high levels of glutamate and, thus, provide further support for an already well-documented role of this excitatory amino acid as neurotransmitter in this system.

Changes in the pattern of glutamate-like immunoreactivity in rat superior colliculus following retinal and visual cortical lesions.

We have investigated the pattern of glutamate-like immunoreactivity in the superficial layers of the rat superior colliculus by means of postembedding immunocytochemical methods for light and electron microscopy. At the light microscopic level, labelling was faintly to moderately intense in most perikarya of the stratum zonale, stratum griseum superficiale and stratum opticum. Furthermore, strong glutamate-immunoreactive terminal-like elements were accumulated most densely in stratum zonale, stratum griseum superficiale and stratum opticum. At the electron microscopic level, a postembedding immunogold method revealed that the vast majority of those labelled elements corresponded to retinal and visual cortical terminals. These profiles were about twice as heavily labelled as their postsynaptic partners. To determine the contribution of retinal and cortical afferents to the pattern of glutamate-like immunoreactivity, rats were subjected to right retinal ablation, left cortical ablation or combined right retinal and left cortical ablations. After retinal ablation, strongly labelled perikarya were observed in the retinorecipient layers. Furthermore, a prominent loss of glutamate-immunoreactive terminal-like elements occurred in stratum zonale and stratum griseum superficiale. Ipsilateral superior colliculus to cortical ablation exhibited subtle changes characterized by a moderate increase in perikaryal immunostaining in stratum zonale, stratum griseum superficiale and stratum opticum and by an apparent discrete reduction of labelled dots in stratum griseum superficiale and stratum opticum. In cases with combined lesions, strongly immunoreactive cell bodies and dendrites were accompanied by a massive disappearance of labelled terminal-like elements in stratum zonale, stratum griseum superficiale and stratum opticum. The effect of retinal and visual cortical ablations on the pattern of glutamate-like immunoreactivity suggests that these afferents are the major sources for glutamate-immunoreactive terminals in the rat superior colliculus. In addition, these findings provide further evidence for glutamate as neurotransmitter in the visual pathways studied.

Group II metabotropic glutamate receptors are differentially expressed in the medial nucleus of the trapezoid body in the developing and adult rat.

The existence of a neuronal-glial signalling through the activation of neurotransmitter receptors expressed in glia is well-documented. In excitatory synapses, glutamate released from presynaptic terminals activates not only postsynaptic receptors, but also ionotropic and metabotropic glutamate receptors localized in the glia ensheathing the synapses. The medial nucleus of the trapezoid body of the auditory system is involved in the localization of sounds in the space. In this nucleus, the large excitatory synaptic terminals formed by the calyces of Held on the principal globular cell bodies are wrapped by astrocytic processes. Since these synapses are functional from early postnatal days, glia receiving excitatory synaptic signals from the calyces may participate in modulating the maturation and development of the system. Groups I and II of metabotropic glutamate receptors (mGluRs) have been localized in glial cells in different brain regions. To investigate whether group II mGluRs are present in the medial nucleus of the trapezoid body, we have studied the pattern of expression of mGluR2/3 in the developing and mature nucleus by means of immunocytochemichal methods. The most remarkable finding was the switch in the occurrence of mGluR2/3 from glial to neuronal compartments. Thus, a preferential localization of mGluR2/3 immunoreactivity was observed in astrocytic processes surrounding the calyces of Held during the early postnatal development. In contrast, the main feature in adult rats was the presence of the group II mGluRs in presynaptic calyces of Held and postsynaptic principal globular cells.From these observations we suggest a role for group II mGluRs in neuronal-glial signalling in the calyx of Held-principal globular neuron synapses. Activation of these receptors might be relevant to the maturation and modulation of synaptic transmission in the medial nucleus of the trapezoid body.

Subcellular localization of the voltage-dependent potassium channel Kv3.1b in postnatal and adult rat medial nucleus of the trapezoid body.

A pre-embedding immunocytochemical method was used to study the subcellular distribution of the voltage-dependent potassium channel Kv3.1b in the medial nucleus of the trapezoid body (MNTB) in developing and adult rat. The main finding was the localization of the channel in specific membrane compartments of the calyces of Held and principal globular neurons. Thus, at postnatal day (P) 9 immunoparticles were densely localized in plasma membranes of globular cell bodies and their main dendrites. At P16, a strong Kv3.1b labeling was still observed in these globular cell compartments, but the most remarkable feature was the presence of immunoparticles in synaptic terminal membranes of the calyces of Held. However, the presynaptic and postsynaptic specializations of the calyx of Held-globular cell synapses were virtually devoid of immunoparticles. This same subcellular distribution of Kv3.1b was seen in adult, with membranes of calycine terminals more uniformly labeled. The developmental profile of Kv3.1b expression in MNTB coincides with the functional maturation of the calyx of Held-principal globular neuron synapse. The presence of the channel in this system is crucial for the high-frequency synaptic transmission of auditory signals.

Localization and release of homocysteic acid, an excitatory sulfur-containing amino acid.

In addition to the excitatory role played by the amino acid transmitters glutamate and aspartate in the central nervous system, their sulfur-containing analogues homocysteic acid (HCA) and cysteine sulfinic acid (CSA) may also play a similar role. HCA is released and taken up by rat CNS tissue; it excites neurons predominantly via NMDA receptors whenever present, and is neurotoxic. The pattern of HCA-like immunoreactivity in the rat indicates a localization of HCA mostly in glial elements, although its presence in nerve terminals and neuronal perikarya cannot be excluded. In the cerebellum of newborn and adult animals, the Bergmann glial cells and the astrocyte endfeet are immunoreactive, either in the presence or in the absence of climbing fibers. In the cortex, hippocampus, and retina, labeling is seen in both glial and neuronal elements. Excitatory signaling involving glial elements is discussed.

Glutamate-like immunoreactivity in synaptic terminals of the posterior cingulopontine pathway: a light and electron microscopic study in the rabbit.

A postembedding immunoperoxidase method for light microscopy was used to localize glutamate-like immunoreactivity in the rabbit basilar pontine nuclei. Labelled fibre bundles, neuronal cell bodies and numerous puncta of diverse size were heavily glutamate immunoreactive throughout all subdivisions of the pontine nuclei. To determine whether some of the glutamate-immunoreactive puncta were synaptic terminals of posterior cingulate cortical neurons, a double-labelling technique involving an anterograde tract-tracing method and a postembedding immunogold procedure for electron microscopy was used. A quantitative evaluation of gold particle densities revealed that anterogradely labelled cingulopontine synaptic terminals were about twice as immunoreactive as their postsynaptic dendrites, perikaryal and glial profiles and about three times more than symmetric synaptic terminals. The present results indicate that the posterior cingulopontine projection contains high levels of glutamate at its synaptic terminals. This observation provides further support to the role for glutamate as a neurotransmitter in the corticopontine pathway.

Retinal afferents on Golgi-identified vertical neurons in the superior colliculus of the rabbit. A Golgi-EM, degenerative and autoradiographic study.

The characteristics and distribution pattern of retinal afferent terminals making synaptic contacts on narrow field vertical neurons in the stratum griseum superficiale of the rabbit superior colliculus were studied using the Golgi-gold substitution technique in combination with either autoradiographic or degenerative methods. At the level of light microscopy, identified gold-toned vertical neurons showed similar features to those previously described by others. Although their axons were frequently seen rising from the basal dendritic tuft, they could also emerge from an apical dendrite, but rarely from the cell body. The electron microscopic study revealed that these neurons received radiolabelled or degenerated profiles with typical features of retinal terminals, while more proximal parts seemed to receive fewer terminals but of larger size. Axo-somatic synaptic junctions were very rare and, interestingly, retinal terminals were not seen forming synapses on the basal dendritic arborization. The present results indicate that the narrow field vertical cells are targets for retinotectal projection.

Differential cellular localization of three splice variants of the mGluR1 metabotropic glutamate receptor in rat cerebellum.

Antibodies were raised against C-terminal peptides of the splice variants a, b and c of the rat mGluR1 metabotropic glutamate receptor. Affinity purified antibodies each specifically reacting with mGluR1a, mGluR1b and mGluR1c were used to study the cellular localization of these receptors in rat cerebellum. The mGluR1a antibody strongly labelled Purkinje cells at their cells bodies, portions of their dendritic trees and numerous small punctate elements reminiscent of dendritic spines. Also labelled were some stellate, basket, Golgi and Lugaro cells. Granule cells were devoid of staining. The mGluR1b antibody strongly labelled Purkinje cell bodies and their dendrites at levels which varied within the same lobule of the vermis or the hemispheres. No significant labelling was observed at stellate, basket, Golgi and granule cells, while occasionally a fraction of basket cells and cerebellar glomeruli was moderately immunoreactive. The mGluR1c antibody strongly labelled cell bodies and thick principal dendrites of Purkinje cells but not dendritic spines. Immunonegative Purkinje cells were intermingled with strongly labelled ones in lobules 4-10, while in lobules 1, 2 and 3, no stained Purkinje cells were detected. The mGluR1c antibody also labelled stellate, basket, some Golgi and some Lugaro cells as well as granule cells.

Immunohistochemical localiztion of homocysteate in human primary visual cortex.

Polyclonal and monoclonal anti-homocysteate antibodies were used with a postembedding immunohistochemical method for light microscopy to localize homocysteate-like immunoreactivity in human primary visual cortex. Densely accumulated dots of diverse size resembling astrocytic processes were labelled in supragranular layers, mainly in layers I and II. Some glial elements intermingled with fibre bundles in the white matter, and astrocytic endfeet in the vicinity of capillaries were also stained. In addition, very few round or elongated neuronal cell bodies in layer IVc were intensely homocysteate immunoreactive. These observations extend to human primary visual cortex previous studies on the preferential localization of L-homocysteate in glia.

Light microscopic comparison of the patterns of glutamate-like and homocysteate-like immunoreactivities in rat dorsal lateral geniculate after combined visual cortical and retinal ablations.

To study the contribution of retinal and cortical afferents to the patterns of glutamate- and homocysteate-like immunoreactivities in dorsal lateral geniculate, combined retinal and cortical ablations were performed in rats. In controls, glutamate immunoreactivity was in terminal-like dots and neurons. Homocysteate immunoreactivity was in small puncta. In lesioned animals, most glutamate-immunoreactive dots disappeared. In contrast, abundant puncta resembling parts of glial cells were immunoreactive for homocysteate.