The arrangement of glutamate receptors in excitatory synapses.

Electron microscopic immunogold analyses have revealed a highly differentiated arrangement of glutamate receptors at excitatory synapses in the central nervous system. Studies focused on the hippocampus and cerebellum have shown that the postsynaptic specialization is the preferential site of NMDA and AMPA receptor expression, and that the delta 2 receptor is similarly concentrated at this site. In cases of colocalization (AMPA and NMDA, or AMPA and delta 2) the two receptor types appear to be intermingled rather than segregated to separate parts of the membrane. The different groups of metabotropic receptor exhibit distinct distributions at the synapse: group I receptors occur in membrane domains lateral to the postsynaptic specialization; group II receptors are expressed in preterminal membranes or extra-synaptically; whereas group III receptors are found in, or close to, the presynaptic active zone consistent with their roles as autoreceptors. The differentiated distribution of glutamate receptors reflects their functional heterogeneity and explains why some receptors are activated only at high firing frequencies.

Ultrastructural localization of the alpha4-subunit of the neuronal acetylcholine nicotinic receptor in the rat substantia nigra.

The distribution of the alpha4-subunit of the neuronal nicotinic acetylcholine receptor (nAChR) in the rat brain was examined at light and electron microscopy levels using immunohistochemical staining. In the present study we demonstrate the specificity, in both tissue homogenates and brain sections, of a polyclonal antibody raised against the rat nAChR alpha4-subunit. The characterization of this antibody involved: (1) Western blot analysis of rat brain homogenates and membrane extracts from cells previously transfected with diverse combinations of neuronal nAChR subunits, and (2) immunohistochemistry using transfected cells and rat brain tissue. At the light microscope level, the alpha4-subunit-like-immunoreactivity (LI) was widely distributed in the rat brain and matched the distribution of the alpha4-subunit transcripts observed previously by in situ hybridization. Strong immunohistochemical labeling was detected in the mesencephalic dopaminergic nuclei. The nAChRs in this region are thought to be responsible for the modulation of dopaminergic transmission. The neurotransmitter identity of alpha4-immunolabeled neurons in the substantia nigra pars compacta (SNpc) and the ventral tegmental area was thus assessed by investigating the possible colocalization of the nAChR alpha4-subunit with tyrosine hydroxylase using confocal microscopy. The double labeling experiments unambiguously indicated that the alpha4-subunit-LI is present in dopaminergic neurons. At the electron microscope level, the neurons in the SNpc exhibited alpha4-subunit-LI in association with a minority of postsynaptic densities, suggesting that the alpha4-subunit may be a component of functional nAChRs mediating synaptic transmission between midbrain cholinergic neurons and mesencephalic dopaminergic neurons.

Different modes of expression of AMPA and NMDA receptors in hippocampal synapses.

Postembedding immunogold labeling was used to determine the relationship between AMPA and NMDA receptor density and size of Schaffer collateral-commissural (SCC) synapses of the adult rat. All SCC synapses expressed NMDA receptors. AMPA and NMDA receptors were colocalized in at least 75% of SCC synapses; the ratio of AMPA to NMDA receptors was a linear function of postsynaptic density (PSD) diameter, with AMPA receptor number dropping to zero at a PSD diameter of approximately 180 nm. These findings indicate that 'silent' SCC synapses are smaller than the majority of SCC synapses at which AMPA and NMDA receptors are colocalized. Thus synapse size may determine important properties of SCC synapses.

Terminals of subthalamonigral fibres are enriched with glutamate-like immunoreactivity: an electron microscopic, immunogold analysis in the cat.

Wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) histochemistry was combined with post-embedding immunogold cytochemistry in order to establish whether the subthalamic nucleus (STN) gives origin to glutamate (Glu)-enriched nerve terminals in substantia nigra, pars reticulata (SNr). Two adult cats served as normal controls and in two other animals crystalline WGA-HRP had been implanted bilaterally in STN. In all four animals ultrathin sections from SN were subjected to an immunogold procedure using antiserum raised against either Glu or gamma-aminobutyric acid (GABA). In some experiments the sections were subjected to consecutive incubations with both GABA and Glu antisera. These two antisera label two morphologically distinct types of boutons in SNr. The GABA antiserum labels boutons with pleomorphic vesicles, and they establish symmetrical synaptic contacts, mainly with dendritic shafts and spines, and occasionally with cell bodies. The Glu antiserum labels boutons with vesicles which are smaller and more uniform with regard to size and shape than those seen in the GABA-labelled boutons. The Glu-labelled boutons are engaged in asymmetrical synaptic contacts mainly with dendritic shafts and more rarely with cell bodies. The number of GABA-labelled boutons in SNr greatly exceeds the number of Glu-labelled ones. In the experimental material a considerable number of boutons in SNr are labelled with WGA-HRP reaction product. Several of these boutons are enriched in Glu-like immunoreactivity (Glu-LI), but not in GABA-LI. It is concluded that the subthalamonigral projection in the cat is likely to use Glu as a transmitter. The findings are briefly discussed with respect to the role played by STN in movement disorders and the involvement of excitatory amino acids in SN for the propagation of epileptic seizures and development of neurotoxicity.

Demonstration of a reciprocal connection between the periaqueductal gray matter and the reticular nucleus of the thalamus.

Lectin-conjugated horseradish peroxidase was either injected or implanted in crystalline form in various parts of the periaqueductal gray substance in the cat. After survival times ranging between 24 and 48 h the animals were fixed, and the mesencephalon and thalamus were sectioned and processed for peroxidase histochemistry, using tetramethylbenzidine as the chromogen. Light microscopic examination of the sections revealed that there exists a prominent reciprocal connection between the ventral and lateral parts of the periaqueductal gray matter on one hand, and the reticular nucleus of the thalamus on the other. The connections are mainly ipsilateral, and involve the entire rostrocaudal extent of the thalamic reticular nucleus, but mainly the ventrolateral sector of its caudal two thirds. There is a differential labelling within the thalamic reticular nucleus.

GABA, glycine, aspartate, glutamate and taurine in the vestibular nuclei: an immunocytochemical investigation in the cat.

The distributions of five amino acids with well-established neuroexcitatory or neuroinhibitory properties were investigated in the feline vestibular complex. Consecutive semithin sections of plastic-embedded tissue were incubated with antisera raised against protein-glutaraldehyde conjugates of GABA, glycine, aspartate, glutamate and taurine. This approach allowed us to study the relative densities of the different immunoreactivities at the level of individual cell profiles. The results indicate that in the vestibular nuclei, neuronal colocalization of two or more neuroactive amino acids is the rule rather than an exception. Colocalization was found of immunoreactivities for GABA and glycine; glycine, aspartate and glutamate; glycine and aspartate, and glutamate and aspartate. GABA immunoreactive neurons were generally small and were found scattered throughout the vestibular complex. Glycine immunoreactive neurons were similarly distributed, except in the superior nucleus where the latter type of neuron could not be detected. Neuronal profiles colocalizing immunoreactivities for GABA and glycine occurred in all nuclei, but were most numerous in the lateral nucleus. The vast majority of the neurons showed noteworthy staining for glutamate and aspartate, although the level of immunoreactivities varied (e.g., the large neurons in the lateral and descending nuclei were more intensely aspartate immunoreactive than the smaller ones). Taurine-like immunoreactivity did not occur in neuronal cell bodies but appeared in Purkinje cell axons and in glial cell profiles. The functional significance of the complex pattern of amino acid colocalization remains to be clarified. In particular it needs to be distinguished between metabolic and transmitter pools of the different amino acids. The present results call for caution when attempts are made to conclude about transmitter identity on the basis of amino acid contents alone.

GABA, glycine, glutamate, aspartate and taurine in the perihypoglossal nuclei: an immunocytochemical investigation in the cat with particular reference to the issue of amino acid colocalization.

The differential distribution of glutamate (Glu), aspartate (Asp), glycine (Gly), gamma-aminobutyric acid (GABA) and taurine (Tau) was investigated in the cat's perihypoglossal nuclei. Serial semi-thin (0.5 micron) sections through the perihypoglossal nuclei were incubated with antisera raised against the mentioned amino acids with the aim of studying possible co-localization. In each experiment different measures were undertaken in order to screen for possible cross-reactivities, and all sections were processed together with test conjugates in order to ascertain the specificity of the antisera used. A very high proportion of the neurons in the perihypoglossal nuclei (about 90%) shows strong immunostaining for Asp and also displays distinct immunoreactivity for Glu in neighbouring sections. About 25% of the cells in the perihypoglossal nuclei are intensely immunostained for Gly, but very few cells show immunoreactivity for GABA. Only glial cells appear to be immunostained for Tau. Neurons that are Gly(+) also display Glu and Asp immunoreactivities. The neuropil of the perihypoglossal nuclei shows a high density of GABA(+), Gly(+) and Glu(+) puncta mainly representing stained axons and terminals. Fewer Asp(+) puncta and very few Tau(+) nerve terminal-like puncta are seen. Details of the regional distribution of immunopositive neurons and puncta within the perihypoglossal nuclei are described. The findings are discussed with particular reference to the possible role of the mentioned amino acids as transmitter substances in the known synaptic circuitry of the perihypoglossal nuclei.

Gamma-aminobutyrate-like immunoreactivity in the thalamus of the cat.

Serial sections of the cat's thalamus were incubated with a purified antiserum raised against gamma-aminobutyric acid conjugated to bovine serum albumin by distilled glutaraldehyde. This serum has been extensively characterized and appears to react selectively with fixed gamma-aminobutyric acid in brain tissue treated with glutaraldehyde. Adjoining sections were stained with thionin and served as invaluable guides for a correct evaluation of the immunolabelling pattern. In the neuropil the intensity of the immunostaining varies considerably between thalamic nuclei and even between nuclear subdivisions. The neuropil staining appears particularly dense in the nuclei parataenialis, periventricularis, centralis medialis, reuniens, rhomboideus, habenularis lateralis, centrum medianum, parafascicularis, subparafascicularis, submedius, dorsal and ventral parts of the lateral geniculate body, the dorsal part of the medial geniculate body, the posterior complex, suprageniculate nucleus, pulvinar and parts of the lateral posterior nucleus. The pulvinar/lateralis posterior complex shows a particularly well-differentiated staining pattern which closely matches Updyke's [Updyke (1983) J. comp. Neurol. 219, 143-181] parcellation of this region. In several thalamic nuclei or subareas--and notably in those relay nuclei which are known to project upon non-primary sensory cortical areas--the immunostained neuropil is characterized by many puncta encircling an unstained profile. With few exceptions all thalamic nuclei displayed immunoreactive nerve cell bodies. Several examples were found of a mismatch between the number of such cells and the staining intensity of the neuropil. Thus the nuclei periventricularis, parafascicularis, subparafascicularis, parataenialis, limitans and centrum medianum although being very rich in neuropil staining have practically no immunostained perikarya. Rough estimates were made of the size and the proportion of gamma-aminobutyric acid labelled neurons in all major--and some minor--thalamic nuclei and their subdivisions. In some thalamic nuclei, notably the nuclei reticularis, anterior dorsalis, lateralis dorsalis, centralis lateralis, ventralis posterior and the dorsal lateral geniculate body, the population of immunoreactive neurons is distinctly heterogeneous with regard to soma size. The findings are discussed with regard to previous immunocytochemical studies of the distribution of gamma-aminobutyric acid and its synthesizing enzyme in the thalamus. Particular emphasis is put on the great species differences which appear to exist in this respect.

Thalamic commissural connections in the cat.

In order to identify possible connections between the two thalami injections of wheat germ agglutinin-coupled horseradish peroxidase (WGA-HRP) were made unilaterally in the thalamus of adult cats. Except for the ventral lateral geniculate body, the nucleus reticularis thalami (R) is the only thalamic structure which shows labeled cells bilaterally following multiple unilateral thalamic injections of WGA-HRP. Furthermore, it appears that the nucleus ventralis medialis and adjoining ventralis lateralis is one main - and possibly sole - recipient area of a bilateral input from R.

Ultrastructural demonstration of a projection from the flocculus to the nucleus prepositus hypoglossi in the cat.

Small electrolytic lesions were made in the flocculus of two adult cats by means of a stereotactic approach avoiding any damage to the cerebellar nuclei. After a survival time of 3 days the animals were killed and the brains fixed and prepared according to standard procedures for ultrastructural studies. The brains of two unoperated cats were similarly treated and served as normal controls. In the experimental animals a large number of boutons in the rostral part of the nucleus prepositus hypoglossi (Ph) ipsilateral to the floccular lesion showed degenerative changes. These were characterized by hypertrophy, a prominent aggregation of densely packed parallel tubules or concentric arrays of cisternae and a filamentous hyperplasia. Only very rarely were such abnormal boutons seen in the caudal half of the ipsilateral Ph, or on the contralateral side or in the unoperated animals. The degenerating boutons contain clusters of pleomorphic vesicles and they establish symmetrical synaptic contacts with somata, dendritic shafts and dendritic spines. Some of the degenerating boutons appear to be of the en passant type. These findings thus affirm the existence of a direct flocculo-prepositus projection in the cat. It is suggested that this pathway could be responsible for mediating information about eye position and velocity to Ph neurons.

Branched projections from the nucleus prepositus hypoglossi to the oculomotor nucleus and the cerebellum. A retrograde fluorescent double-labeling study in the cat.

The retrograde transport of fluorescent substances was used in order to investigate divergent axon collaterals of neurons in the nucleus prepositus hypoglossi (Ph). Fast blue (FB) was injected into the flocculus, paraflocculus and/or the vermis, while nuclear yellow (NY) was injected into the oculomotor nucleus alone or combined with injections in the nucleus of Darkschewitsch, the interstitial nucleus of Cajal and the medial longitudinal fascicle. Within optimal survival time, separate populations of single-labeled neurons of both dyes were found in Ph in all cases. Double-labeled neurons were seen in the rostral Ph following FB injections into the flocculus and the paraflocculus and NY injections restricted to the oculomotor nucleus. The present findings demonstrate that many neurons in the rostral Ph give collateral branches to the cerebellum and to the oculomotor nucleus.

The vestibulothalamic projections in the cat studied by retrograde axonal transport of horseradish peroxidase.

Horseradish peroxidase (HRP) was injected or iontophoretically ejected in various thalamic nuclei in 63 adult cats. In 11 other animals HRP was deposited outside the thalamic territory. The number and distribution of labelled cells within the vestibular nuclear complex (VC) were mapped in each case. To a varying degree all subgroups of VC appear to contribute to the vestibulothalamic projections. Such fibres are distributed to several thalamic areas. From the present investigation it appears that generally speaking, there exist three distinct vestibulothalamic pathways with regard to origin as well as to site of termination of the fibres. One projection appears to originate mainly in caudal parts of the medial (M) and descending (D) vestibular nuclei and in cell group z. This pathway terminates chiefly in the contralateral medial part of the posterior nucleus of the thalamus (POm) including the magnocellular part of the medial geniculate body (Mgmc), the ventrobasal complex (VB) and the area of the ventral lateral nucleus (VL) bordering on VB. A second projection originates mainly in the superior vestibular nucleus (S) and in cell group y and terminates mainly in the contralateral nucleus centralis lateralis (CL) and the adjoining nucleus paracentralis (Pc). A third, more modest, pathway originates chiefly in the middle M and D, with a minor contribution from S and cell group y, and terminates in the contralateral ventral nucleus of the lateral geniculate body (GLV). There is some degree of overlap between the origin of these three vestibulothalamic pathways.

Mesencephalic and diencephalic afferents to the superior colliculus and periaqueductal gray substance demonstrated by retrograde axonal transport of horseradish peroxidase in the cat.

The mesencephalic and diencephalic afferent connections to the superior colliculus and the central gray substance in the cat were examined by means of the retrograde transport of horseradish peroxidase (HRP). After deep collicular injections numerous labeled cells were consistently found in the parabigeminal nucleus, the mesencephalic reticular formation, substantia nigra pars reticulata, the nucleus of posterior commissure, the pretectal area, zona incerta, and the ventral nucleus of the lateral geniculate body. A smaller number of cells was found in the inferior colluculus, the nucleus of the lateral lemniscus, the central gray substance, nucleus reticularis thalami, the anterior hypothalamic area, and, in some cases, in the contralateral superior colliculus, Forel's field, and the ventromedial hypothalamic nucleus. Only the parabigeminal nucleus and the pretectal area showed labeled cells following injections in the superficial layers of the superior colliculus. In the cats submitted to injections in the central gray substance, labeled cells were consistently found in the contralateral superior colliculus, the mesencephalic reticular formation, substantia nigra parts reticulata, zona incerta and various hypothalamic areas, especially the ventromedial nucleus. In some cases, HRP-positive cells were seen in the nucleus of posterior commissure, the pretectal area, Forel's field, and nucleus reticularis thalami. A large injection in the mediodorsal part of the caudal mesencephalic reticular formation, which included the superior colliculus and the central gray substance, resulted in numerous labeled cells in nucleus reticularis thalami. The findings are discussed with respect to the suggested functional division of the superior colliculus into deep and superficial layers. Furthermore, the possible implications of labeled cells in zona incerta and the reticular thalamic nucleus are briefly discussed.

Studies on the cerebellar projections from the main and external cuneate nuclei in the cat by means of retrograde axonal transport of horseradish peroxidase.

The cerebellar projections from the main and external cuneate nuclei in the cat have been studied by means of retrograde axonal transport of horseradish peroxidase. The main projection from the external cuneate nucleus (ECN) is to the intermediate and, possibly, the small lateral part of lobule V and to the paramedian lobule on the ipsilateral side. The projection from the ECN to the cerebellar regions mentioned is topographically organized. Cells in the caudal part of the ECN send their axons to the caudal parts of lobule V and to the rostral part of the paramedian lobule. Cells in the rostral part of the ECN project to the rostralmost part of lobule V and to the folia in the caudal part of the paramedian lobule. The experimental study also shows that cells in the main cuneate nucleus (MCN) send their axons to the cerebellum. These axons, like those from the ECN, terminate in the intermediate part of lobule V of the anterior lobe and in the paramedian lobule. However, the axons of the cells in the MCN terminate only in the superficial parts of the folia, whereas those from the ECN terminate in the depth of the folia in these two cerebellar areas. The present study also gives evidence that cells in the ventral part of the gracile nucleus send their axons to lobules I and II of the anterior lobe vermis. The observations referred to here are to our knowledge the first anatomical findings demonstrating a projection from the main cuneate and gracile nuclei onto the cerebellar cortex. The observations confirm previous physiological studies.