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.

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.

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.

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.

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.

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.

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.

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.

Clustering of the group III metabotropic glutamate receptor 4a at parallel fiber synaptic terminals in the rat cerebellar molecular layer.

We report in this study with a pre-embedding immunogold method, the clustering of the group III metabotropic glutamate receptor 4a (mGluR4a) along the presynaptic membrane of parallel fiber synaptic terminals in the cerebellar molecular layer. The mGluR4a clusters were homogeneously distributed and interspaced by about 60 nm. These results suggest a particular arrangement of mGluR4a which might help to a rapid and effective activation of this receptor by glutamate.

Glutamate-like immunoreactivity in ascending spinofugal afferents to the rat periaqueductal grey.

The midbrain periaqueductal gray is a key structure for the mediation of an integrated defence behaviour. Although a prominent role for glutamate in PAG mechanisms is supported by both behavioural and morphological studies, whether PAG afferents conveying somatosensory information constitute a source of glutamatergic input to the PAG remains unknown. Here, we have compared the projection pattern of orthogradely-labelled spinoannular fibres with the distribution of glutamate-like immunoreactivity in the PAG at the light microscopic level. Transaxonal labelling was observed throughout the whole rostrocaudal axis of the PAG except for the dorsolateral regions. Cell-processes and terminal-reminiscent puncta were strongly immunoreactive in all PAG regions, including the dorsolateral areas. To ascertain whether glutamate-immunoreactive puncta observed at light microscopy indeed constituted axon terminals of the spinoannular system, glutamate-like immunoreactivity was assessed in orthogradely-labelled synaptic terminals using a post-embedding immunogold procedure for electron microscopy. Quantitative analysis of gold particle densities revealed over twice as strong an immunoreactivity in anatomically-identified spinoannular axon terminals as in dendrites postsynaptic to them, perikarya and inhibitory Gray II synapses, as well as an over 5-fold heavier immunolabelling than in glial profiles. These findings reveal that glutamate is accumulated in synaptic terminals of the spinoannular system, supporting a neurotransmitter role for this acidic amino acid in spinofugal afferents to the PAG.

Distribution of the metabotropic glutamate receptor subtype mGluR5 in rat midbrain periaqueductal grey and relationship with ascending spinofugal afferents.

The periaqueductal grey matter (PAG) is known to adjust somatic and neurovegetative elements of the defence behaviour. We have used specific polyclonal antibodies to examine the distribution of the metabotropic glutamate receptor subtype 5 (mGluR5) in this region. Immunolabelling for mGluR5 displayed a net preference for dorsolateral areas at rostral and intermediate levels. Electronmicroscopic examination revealed that mGluR5 is expressed in neuronal perikarya and in dendrites receiving synaptic contacts of Gray I type. To investigate the possible relevance of mGluR5 to integration of somatosensory information, spinoannular (SA) neurones were peroxidase-labelled and their relationship with mGluR5-expressing PAG neurones was examined at the ultrastructural level. A number of synaptic terminals of the SA pathway established synaptic contact of asymmetric type onto mGluR5-immunoreactive dendrites. It is suggested that mGluR5 might be involved in the temporal integration of somatosensory inputs to the PAG.

The metabotropic glutamate receptor subtype mGluR 2/3 is located at extrasynaptic loci in rat spinal dorsal horn synapses.

The position of neurotransmitter receptors relative to active neurotransmitter release sites may be a major factor influencing neuronal responses. The location of the metabotropic glutamate receptor subtype mGluR2/3 was investigated in synaptic structures in the rat superficial spinal dorsal horn laminae by using a pre-embedding immunogold technique. Immunostaining for mGluR2/3 occurred in laminae I through III. Gold particles were encountered both in the cytosol and along the plasma membrane. Distinctive plasmalemmal immunodeposits were detected in vesicle-containing profiles, where they were located to membrane compartments distant from active release sites rather than in the close vicinity of synaptic specialisations. No distinct immunolabelling was observed in profiles meeting characteristics of primary afferent terminals. The extrasynaptic occurrence of mGluR2/3 suggests a presynaptic heteroreceptor role for these receptor subtypes in the spinal dorsal horn.

Subtype-specific changes in 5-HT receptor-mediated modulation of C fibre-evoked spinal field potentials are triggered by peripheral nerve injury.

Neurotransmitter serotonin (5-HT) released from descending pain modulation pathways to the dorsal horn is crucial to spinal nociception processing. This study sought to gain insight into the modulatory roles of specific serotonin receptor subtypes in experimentally induced neuropathic pain. In rats subjected to spinal nerve ligation (SNL) surgery, we recorded field potentials evoked in the spinal dorsal horn by C fibre-input, during spinal superfusion with subtype-selective drugs. In neuropathic rats, subtype 5-HT1A agonist 8-OH-DPAT (100 nM) was found to potently depress evoked field potentials, as opposed to 5-HT2A or 5-HT2B subtype agonists TCB-2 (100 nM) or BW 723C86 (1 microM), respectively, which consistently enhanced evoked potentials. All three failed to alter spinal field potentials in sham operated rats. CP 94253 (1 microM), WAY 161503 (1 mM) or SR 57227 (at 1 microM in SNL rats, and 100 microM in sham rats), selective agonists for 5-HT1B, 5-HT2C and 5-HT3 receptors, respectively, significantly depressed evoked field potentials in both animal groups. The 5-HT4 agonist RS 67333 (1 microM) was depressant only in sham operated animals. Only after SNL, spinal superfusion with 5-HT1A- or 5-HT1B receptor-antagonists (S)-WAY 100135 (100 microM) or SB 224289 (100 microM), respectively, disinhibited C fibre-evoked potentials, whereas 5-HT2A or 5-HT2B receptor-antagonists 4F 4PP (100 microM) or SB 204741 (100 microM) depressed evoked potentials, suggesting tonic activity of all four subtypes as a consequence of experimental nerve injury. The present findings reveal profound subtype-specific changes in the functional modulatory activities of spinal serotonin receptors following peripheral nerve injury. In particular, spinal hyperexcitation promoted by receptors 5-HT2A and 5-HT2B is suggested as a novel pathogenic pathway contributing to neuropathic pain.

Neuronal and glial localization of homocysteate-like immunoreactivity in the rat retina.

To study the distribution of L-homocysteate in the rat retina, specific polyclonal and monoclonal anti-homocysteate antibodies have been used in combination with a highly sensitive postembedding method for light microscopic immunocytochemistry. In central and peripheral retina, the most strongly immunoreactive cell bodies lay in the inner nuclear layer. They represented about 17% of the total neuronal cell population of the layer and were identified as bipolar cells (19-20% of cells in the outer half of the inner nuclear layer) and amacrine cells (15% of cells in the inner half of the inner nuclear layer). A third cell type showing heavy homocysteate-like immunoreactivity was identified as Müller glial cells. Characteristically, their descending processes formed three immunoreactive bands in the inner plexiform layer. Furthermore, the outer and inner limiting membranes as well as glia around and between ganglion cell axons and in the vicinity of blood vessels were labelled intensely. Photoreceptors and their terminals, and ganglion cells, were not immunostained. These findings indicate the presence of homocysteate in some bipolar and amacrine cells of the inner nuclear layer and support a role for this sulphur-containing excitatory amino acid as a neurotransmitter candidate in the retina.

Long-term depression of C-fibre-evoked spinal field potentials by stimulation of primary afferent A delta-fibres in the adult rat.

Long-term potentiation (LTP) of spinal C-fibre-evoked field potentials can be induced by brief electrical stimulation of afferent C-fibres, by natural noxious stimulation of skin or by acute nerve injury. Here, we report that in urethane anaesthetized, adult rats prolonged high frequency burst stimulation of the sciatic nerve at Adelta-fibre strength produced long-term depression (LTD) of C-fibre-evoked field potentials, and also depressed the increased amplitudes of C-fibre-evoked field potentials recorded after LTP had been established (depotentiation). Electrical stimulation of Abeta-fibres failed to induce LTD or depotentiation. In spinalized rats, prolonged Adelta-fibre conditioning stimulation induced LTP rather than LTD of C-fibre-evoked field potentials. Thus, tonic descending inhibition may determine the direction of plastic changes in C-fibre-mediated synaptic transmission. Spinal application of the N-methyl-D-aspartic acid receptor antagonist D-APV blocked induction of LTD in intact rats and LTP in spinalized rats. The presently described LTD and the depotentiation of established LTP of C-fibre-evoked field potentials in spinal dorsal horn may underlie some forms of prolonged analgesia induced by peripheral nerve stimulation procedures.

Peripheral nerve insult induces NMDA receptor-mediated, delayed degeneration in spinal neurons.

Injury of a peripheral nerve gives rise to adaptive functional and structural alterations in spinal neurons. We report that the rearrangement of the spinal circuitry in response to sciatic nerve transection in adult rats involves a delayed mode of degeneration of lumbar spinal cord neurons. Nuclear fragmentation was detected by the TUNEL technique 7 days after sciatic neurectomy but not after 3 or 14 days. Dying cells were preferentially located in the ipsilateral superficial dorsal horn and expressed the neuronal cytoskeletal marker SMI-31. Degeneration was prevented by continuous systemic treatment with the NMDA receptor-antagonist MK-801. These data are supportive that apoptosis is induced in spinal neurons in a transsynaptic manner by an early signal from injured afferent fibres via activation of spinal NMDA receptors.

Localization of the mGlu4a metabotropic glutamate receptor in rat cerebellar cortex.

The subcellular localization of the mGlu4a metabotropic glutamate receptor was investigated in rat cerebellum. At the light microscopical level, strong mGlu4a immunoreactivity was found in the molecular layer. A post-embedding immunogold method for electron microscopy revealed gold particles at the presynaptic sites of synapses made by parallel fiber terminals with dendritic spines of Purkinje cells. These observations support electrophysiological evidence indicating an autoreceptor function of mGlu4 receptors at these synapses.

Immunolocalization of the mGluR1b splice variant of the metabotropic glutamate receptor 1 at parallel fiber-Purkinje cell synapses in the rat cerebellar cortex.

Several metabotropic glutamate receptor (mGluR) subtypes have been identified in the cerebellar cortex that are targeted to different compartments in cerebellar cells. In this study, preembedding immunocytochemical methods for electron microscopy were used to investigate the subcellular distribution of the mGluR1b splice variant in the rat cerebellar cortex. Dendritic spines of Purkinje cells receiving parallel fiber synaptic terminals were immunoreactive for mGluR1b. With a preembedding immunogold method, approximately 25% of the mGluR1b immunolabeling was observed perisynaptically within 60 nm from the edge of the postsynaptic densities. Values of extrasynaptic gold particles beyond the first 60 nm were maintained at between 10 and 18% along the whole intracellular surface of the dendritic spine membranes of Purkinje cells. For comparison, the distribution of mGluR1a was studied. A predominant (approximately 37%) perisynaptic localization of mGluR1a was seen in dendritic spines of Purkinje cells, dropping the extrasynaptic labeling to 15% in the 60-120-nm bin from the edge of the postsynaptic specialization. Our results reveal that mGluR1b and mGluR1a are localized to the same subcellular compartments in Purkinje cells but that the densities of the perisynaptic and extrasynaptic pools were different for both isoforms. The compartmentalization of mGluR1b and mGluR1a might serve distinct requirements in cerebellar neurotransmission.