Fine structural morphology of identified X- and Y-cells in the cat's lateral geniculate nucleus

Four physiologically identified neurons in the A laminae of the cat's dorsal lateral geniculate nucleus were filled with horseradish peroxidase and studied using the electron microscope. Two were X-cells and two were Y-cells. Each had electrophysiological properties appropriate for its X- Or Y-cell class, and each also had an axon that projected into the optic radiation, indicative of a geniculocortical relay cell. Representative samples from about 10% of each neuron's entire dendritic arbor (proximal and distal) were taken to obtain an estimate of the types and distributions of synapses contacting these arbors. One X-cell had a cytoplasmic laminar body, but there were no other significant cytological differences seen among the neurons. Common to each of the neurons were the following synaptic features: (i) retinal terminals (r.l.p.) were mostly or entirely restricted to proximal dendrites or dendritic appendages (less than 100 microns from the soma). These terminals constituted about 15-25% of the synapses on the proximal dendrites. (ii) Terminals with flattened or pleomorphic synaptic vesicles (f. terminals) were predominant on the proximal dendrites (30-55% of the total synapses for that region) and were mainly located near the retinal terminals. A smaller percentage (10-20%) were also distributed onto the distal dendrites. (iii) Small terminals with round synaptic vesicles (r.s.d.), many presumably having a cortical origin, predominated (60-80%) on distal dendrites (greater than 100 microns), but also formed a large proportion (40-70%) of the synapses on the intermediate (50-150 microns) dendrites. Total synaptic contacts for one X-cell and one Y-cell were estimated at about 4000 and 5000, respectively. The major fine structural differences observed between X- and Y-cells were almost entirely related to the retinal afferents. First, the retinal synapses for X-cells were mostly made on to dendritic appendages (spines, etc.), whereas Y-cells had most of their retinal synapses onto the shafts of primary and proximal secondary dendrites (that is, near branch points. Second, the retinal terminals that contacted X-cell dendrites nearly always formed triadic arrangements that included nearby f. terminals, but those on Y-cells rarely did so. Finally, the main type of f. terminals associated with X-cells were morphologically different from most of those associated with the Y-cells, and this also related directly to the triadic arrangements; that is, f. terminals in the triadic arrangements were morphologically distinguishable from f. terminals that did not participate in triadic arrangements.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synaptic patterns in the dorsal lateral geniculate nucleus of the monkey

Summary Synaptic junctions are found in all parts of the nucleus, being almost as densely distributed between cell laminae as within these laminae.In addition to the six classical cell laminae, two thin intercalated laminae have been found which lie on each side of lamina 1. These laminae contain small neurons embedded in a zone of small neural processes and many axo-axonal synapses occur there.Three types of axon form synapses in all cell laminae and have been called RLP, RSD and F axons. RLP axons have large terminals which contain loosely packed round synaptic vesicles, RSD axons have small terminals which contain closely packed round vesicles and F axons have terminals intermediate in size containing many flattened vesicles.RLP axons are identified as retinogeniculate fibers. Their terminals are confined to the cell laminae, where they form filamentous contacts upon large dendrites and asymmetrical regular synaptic contacts (with a thin postsynaptic opacity) upon large dendrites and F axons. RSD axons terminate within the cellular laminae and also between them. They form asymmetrical regular synaptic contacts on small dendrites and on F axons. F axons, which also occur throughout the nucleus, form symmetrical regular contacts upon all portions of the geniculate neurons and with other F axons. At axo-axonal junctions the F axon is always postsynaptic.Supported by Grant R 01 NB 06662 from the USPHS and by funds of the Neurological Sciences Group of the Medical Research Council of Canada. Most of the observations were made while R. W. Guillery was a visiting professor in the Department of Physiology at the University of Montreal. We thank the Department of Physiology for their support and Mr. K. Watkins, Mrs. E. Langer and Mrs. B. Yelk for their skillful technical assistance.     

A major role for intracortical circuits in the strength and tuning of odor-evoked excitation in olfactory cortex

In primary sensory cortices, there are two main sources of excitation: afferent sensory input relayed from the periphery and recurrent intracortical input. Untangling the functional roles of these two excitatory pathways is fundamental for understanding how cortical neurons process sensory stimuli. Odor representations in the primary olfactory (piriform) cortex depend on excitatory sensory afferents from the olfactory bulb. However, piriform cortex pyramidal cells also receive dense intracortical excitatory connections, and the relative contribution of these two pathways to odor responses is unclear. Using a combination of in vivo whole-cell voltage-clamp recording and selective synaptic silencing, we show that the recruitment of intracortical input, rather than olfactory bulb input, largely determines the strength of odor-evoked excitatory synaptic transmission in rat piriform cortical neurons. Furthermore, we find that intracortical synapses dominate odor-evoked excitatory transmission in broadly tuned neurons, whereas bulbar synapses dominate excitatory synaptic responses in more narrowly tuned neurons.     

Experience-Driven Axon Retraction in the Pharmacologically Inactivated Visual Cortex Does Not Require Synaptic Transmission

Background

Experience during early postnatal development plays an important role in the refinement of specific neural connections in the brain. In the mammalian visual system, altered visual experiences induce plastic adaptation of visual cortical responses and guide rearrangements of afferent axons from the lateral geniculate nucleus. Previous studies using visual deprivation demonstrated that the afferents serving an open eye significantly retract when cortical neurons are pharmacologically inhibited by applying a γ-aminobutyric acid type A receptor agonist, muscimol, whereas those serving a deprived eye are rescued from retraction, suggesting that presynaptic activity can lead to the retraction of geniculocortical axons in the absence of postsynaptic activity. Because muscimol application suppresses the spike activity of cortical neurons leaving transmitter release intact at geniculocortical synapses, local synaptic interaction may underlie the retraction of active axons in the inhibited cortex.

Method and Findings

New studies reported here determined whether experience-driven axon retraction can occur in the visual cortex inactivated by blocking synaptic inputs. We inactivated the primary visual cortex of kittens by suppressing synaptic transmission with cortical injections of botulinum neurotoxin type E, which cleaves a synaptic protein, SNAP-25, and blocks transmitter release, and examined the geniculocortical axon morphology in the animals with normal vision and those deprived of vision binocularly. We found that afferent axons in the animals with normal vision showed a significant retraction in the inactivated cortex, as similarly observed in the muscimol-treated cortex, whereas the axons in the binocularly deprived animals were preserved.

Conclusions

Therefore, the experience-driven axon retraction in the inactivated cortex can proceed in the absence of synaptic transmission. These results suggest that presynaptic mechanisms play an important role in the experience-driven refinement of geniculocortical axons.     

双眼和单眼视觉剥夺猫外膝体细胞的图形适应

为测定丘脑外膝体细胞的图形适应是否依赖于早期视觉经验,在细胞外记录了双眼和单眼缝合的猫外膝体中断细胞对手工时间运动光栅刺激的反应。在双眼剥夺猫,占６８％的记录到的细胞在３０ｓ内反应下降到稳定值,其平均反应值下降３３％,适应程度较正常猫显著。在单眼剥夺猫,记录到的剥夺眼驱动的和非剥夺眼驱动的细胞中,分别有占５３％和４４％的细胞显示图形适应,两者差别不大。研究表明,早期视剥夺能增强或保持图形适应,提示     

Preserved Excitatory-Inhibitory Balance of Cortical Synaptic Inputs following Deprived Eye Stimulation after a Saturating Period of Monocular Deprivation in Rats

Monocular deprivation (MD) during development leads to a dramatic loss of responsiveness through the deprived eye in primary visual cortical neurons, and to degraded spatial vision (amblyopia) in all species tested so far, including rodents. Such loss of responsiveness is accompanied since the beginning by a decreased excitatory drive from the thalamo-cortical inputs. However, in the thalamorecipient layer 4, inhibitory interneurons are initially unaffected by MD and their synapses onto pyramidal cells potentiate. It remains controversial whether ocular dominance plasticity similarly or differentially affects the excitatory and inhibitory synaptic conductances driven by visual stimulation of the deprived eye and impinging onto visual cortical pyramids, after a saturating period of MD. To address this issue, we isolated visually-driven excitatory and inhibitory conductances by in vivo whole-cell recordings from layer 4 regular-spiking neurons in the primary visual cortex (V1) of juvenile rats. We found that a saturating period of MD comparably reduced visually–driven excitatory and inhibitory conductances driven by visual stimulation of the deprived eye. Also, the excitatory and inhibitory conductances underlying the synaptic responses driven by the ipsilateral, left open eye were similarly potentiated compared to controls. Multiunit recordings in layer 4 followed by spike sorting indicated that the suprathreshold loss of responsiveness and the MD-driven ocular preference shifts were similar for narrow spiking, putative inhibitory neurons and broad spiking, putative excitatory neurons. Thus, by the time the plastic response has reached a plateau, inhibitory circuits adjust to preserve the normal balance between excitation and inhibition in the cortical network of the main thalamorecipient layer.     

Neuronal connections of ocular dominance columns in the cortex of monocularly deprived cats

We have investigated the developmental changes of intrahemispheric neuronal connections of the areas 17 & 18 ocular dominance columns in monocularly deprived cats. Single cortical columns were microiontophoretically injected with horseradish peroxidase and 3D reconstruction of retrogradely labelled cells' region was done. Ocular dominance of injected columns and their coordinates in the visual field map were determined. In area 17 it was shown that for non-deprived eye the connections of columns that are driven via the crossed pathways were longer than connections of columns driven via uncrossed ones, and in both cases they were longer than connections in intact cats. The connections of deprived eye columns are significantly reduced. We have observed some changes in the spatial organization of long-range connections in area 17 for columns driven by the non-deprived eye (more rounded shape of regions of labeled cells, non-uniform distribution of cells within it). Maximal length of such connections did not exceed the length of connections in strabismic cats. We speculate that the length of cell axons providing for the horizontal connections of cortical columns has some intrinsic limit that does not depend on visual stimulation during the critical period of development.     

The development of the retinogeniculate pathways in normal and albino ferrets

The retinogeniculate pathways of normal and albino ferrets have been studied with horseradish peroxidase and tritiated proline used as axonal markers. The uncrossed retinogeniculate projection of adult albino ferrets is abnormally small and occupies only a fraction of the geniculate area normally occupied by uncrossed afferents. The crossed pathway is correspondingly expanded, occupying almost the entire nucleus. The geniculate laminae in the albino ferret are abnormal, showing abnormal fusions between layers receiving crossed input and abnormal discontinuities next to the small cell islands receiving uncrossed afferents. In early development, retinofugal fibres can be labelled within the optic tracts on the 28th intrauterine day and a few crossed fibres can be traced into the lateral geniculate nucleus. At this stage, the uncrossed component is extremely small in normal and albino animals and cannot be traced beyond the tract. By day 32 retinal fibres are invading the lateral geniculate nucleus bilaterally, the invasion by the crossed component being significantly more advanced than that by the uncrossed component. The uncrossed pathway of the albinos is already abnormal in terms of its size, in terms of the position it occupies in the optic tract, and in terms of its limited invasion of the lateral geniculate nucleus. The abnormally reduced size of the uncrossed component appears earlier than the abnormal segregation of the retinogeniculate terminals, suggesting that the primary action of the albino gene upon central visual pathways is prechiasmatic. At postnatal stages (41 days after conception and older) the normal, gradual withdrawal of the uncrossed fibres from the monocular segment, and the separation of crossed from uncrossed retinogeniculate terminal arbors is significantly delayed in the albinos. The uncrossed retinogeniculate terminals are abnormally sparse initially and become distributed in an abnormal, interrupted pattern as development proceeds. The abnormal pattern of geniculate lamination appears to be secondary to the abnormal distribution of retinogeniculate afferents.     

Connection of the auditory cortex secondary zones with the auditory subcortical cerebral centers in the cat

As it has been demonstrated microscopically, the corticofugal fibers in the AII and Ep zones of the auditory cortex in all the auditory subcortical centers (medial geniculate body, posterior colliculi of the tectum mesencephali and the superior olive nuclei) terminate by means of single axodendritic synapses, having an asymmetrically active zone, and mixed (by their form) synaptic vesicles. Small and middle dendrites make their postsynaptic part. A comparison has been carried out on distribution and form of synapses, completing the projection fibers from the zone of the primary acoustic responses (AI) and of the primary acoustic zone (AIV). Basing on the morphological data, concerning distribution and form of the synaptic terminals, a suggestion is made that physiological influence of each acoustic cortex zone is different for the medial geniculate body and posterior colliculi of the tectum mesencephali, but it is unitypical for the superior olive level.     

Synaptic connections of fibers of the rubrospinal tract in the cat spinal cord

The ultrastructure of the lateral part of laminae VI and VII of the spinal gray matter (the location of most of the terminal branches of the rubrospinal tract) was investigated in cats under normal conditions and at various times after destruction of the red nucleus. The neuron population of this region is formed by cells fairly homogeneous in size (25–40µ). The structure of the dendritic profiles is simple and they carry only infrequent and small membranous appendages. Most synapses are axo-dendritic. The axon terminals are divided into three groups depending on the size and shape of the synaptic vesicles and the presence of post-synaptic specialization. A few glomerular axon terminals contacting with various structures are found. Small axon terminals located chiefly on dendrites and their appendages show degenerative changes 1–8 days after destruction of the red nucleus. As a rule the degenerating terminals contain round synaptic vesicles. The glomerular terminals do not degenerate.A. A. Bogomol'ets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 6, No. 6, pp. 610–618, November–December, 1974.     

Modulation of sensory input to the spinal cord by presynaptic ionotropic glutamate receptors

Sensory input from peripheral nerves to the dorsal horn of the spinal cord is mediated by a variety of agents released by the central terminals of dorsal root ganglion (DRG) neurons. These include, but are not limited to, amino acids, especially glutamate, peptides and purines. The unraveling of the mechanisms of synaptic transmission by central terminals of DRG neurons has to take into account various ways in which the message from the periphery can be modulated at the level of the first central synapse. These include postsynaptic and presynaptic mechanisms. Homomeric and heteromeric complexes of receptor subunits for the different transmitters released by DRG neurons and interneurons, clustered at the postsynaptic site of central synapses, can be expressed in different combinations and their rate of insertion into the postsynaptic membrane is activity-regulated. Inhibitory mechanisms are an important part of central modulation, especially via presynaptic inhibition, currently believed to involve GABA released by inhibitory intrinsic neurons. Recent work has established the occurrence of another way by which sensory input can be modulated, i.e. the expression of presynaptic ionotropic and metabotropic receptors in central terminals of DRG neurons. Microscopic evidence for the expression, in these terminals, of various subunits of ionotropic glutamate receptors documents the selective expression of glutamate receptors in functionally different DRG afferents. Electrophysiological and pharmacological data suggest that activation of presynaptic ionotropic glutamate receptors in central terminals of DRG neurons may result in inhibition of release of glutamate by the same terminals. Glutamate activating presynaptic receptors may spill over from the same or adjacent synapses, or may be released by processes of astroglial cells surrounding synaptic terminals. The wide expression of presynaptic ionotropic glutamate receptors, especially in superficial laminae of the dorsal horn, where Adelta- and C fibers terminate, provides an additional or alternative mechanism, besides GABA-mediated presynaptic inhibition, for the modulation of glutamate release by these fibers. Since, however, presynaptic ionotropic glutamate receptors are also expressed in terminals of GABAergic intrinsic interneurons, a decrease of GABA release resulting from activation of these receptors in the same laminae, may also play a role in central sensitization and hyperalgesia.     

Formation of lamina-specific synaptic connections

In many parts of the vertebrate central nervous system, inputs of distinct types confine their synapses to individual laminae. Such laminar specificity is a major determinant of synaptic specificity. Recent studies of several laminated structures have begun to identify some of the cells (such as guidepost neurons in hippocampus), molecules (such as N-cadherin in optic tectum, semaphorin/collapsin in spinal cord, and ephrins in cerebral cortex), and mechanisms (such as activity-dependent refinement in lateral geniculate) that combine to generate laminar specificity.     

Electron microscope study of sources in the cat primary auditory cortex (AI) projecting to the medial geniculate body

An electron microscope study of retrogradely labeled pyramidal neurons in layer VI of the primary auditory cortex (AI) after injecting horseradish peroxidase (HP) into the medial geniculate body was carried out in cats. Not less than 57.8±1.9% on average of the perimeter of perikaryon profiles of corticogeniculate neurons labeled with HP were found to be covered with astroglia processes. Between three and eight synapses occupying an average of 10.8±1.0% of the perimeter length were found on the perikaryon profiles of these neurons. Nearly all synapses (a total of 98.7%) at the soma of corticogeniculate neurons had symmetrical active zones, being made up of axonal terminals with flattened synaptic vesicles. Anterogradely HP-labeled axonal terminals of geniculocortical fibers were also found in the neuropil of layer VI in area AI, in addition to retrogradely labeled neurons. They contained large round synaptic vesicles and formed asymmetrical synapses. The potential role of axosomatic synapses in the shaping of corticogeniculate neuronal activity is discussed.A. A. Bogomolets Institute, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 2, pp. 171–178, March–April, 1990.     

Eye-specific segregation of optic afferents in mammals,fish, and frogs: The role of activity

Eye-specific patches or stripes normally develop in the visual cortex and superior colliculus of many (but not all) mammals and are also formed, after surgically produced binocular innervation, in the optic tectum of fish and frogs. The segregation of ocular dominance patches or columns has been studied using a variety of anatomical pathway-tracing techniques, by electrophysiological recording of postsynaptic units or field potentials, and by the 2-deoxyglucose method following visual stimulation of only one eye. In the tectum of both fish and frogs and in the cortex and colliculus of mammals, eye-specific patches develop from initially diffuse, overlapping projections. Of the various mechanisms that might cause such segregation, the evidence favors an activity-dependent process that stabilizes synapses from the same eye because of their correlated activity. First, several environmental manipulations affect the segregation of afferents in visual cortex: strabismus and alternate monocular exposure apparently enhance segregation, whereas dark rearing slows the segregation process, and monocular deprivation causes the experienced eye to form larger patches at the expense of those of the deprived eye. Second, blocking activity in both eyes is effective in preventing the segregation both in the tectum of fish and frog and in the visual cortex of cat. With the eyes blocked, alternate stimulation of the optic nerves permits the segregation of ocular dominance, at least onto single cells in the cat visual cortex. These findings are discussed in terms of an activity-dependent stabilization of those synapses having correlated activity (those from neighboring ganglion cells within one eye) but not of those lacking correlated activity (those from left and right eyes). We suggest that the eye-specific patches represent a compromise between total segregation of the projections from the two eyes and the formation of a single continuous retinotopic map across the surface of the cortex or tectum.     

Neuronal and synaptic organization of the lateral geniculate nucleus of the tree shrew,Tupaia glis

Summary The ultrastructural study of the lateral geniculate nucleus (LGN) of the tree shrew (Tupaia glis) revealed two types of neurons: (1) a large thalamocortical relay cell (TCR), which may bear cilia, and (2) a small Golgi type-II interneuron (IN) with an invaginated nucleus. The narrow rim of pale cytoplasm of the IN contains fewer lysosomes and fewer Nissl bodies than the cytoplasm of the TCR. The IN perikarya, which in some cases establish somatosomatic contacts, frequently contain flattened or pleomorphic synaptic vesicles. The ratio of TCR to IN is 31.Three types of axon terminals were observed in the LGN. Two of them contain round synaptic vesicles but differ in size. The large RL boutons undergo dark degeneration after enucleation; they are the terminals of retino-geniculate fibers. The smaller RS boutons show dark degeneration after ablation of the visual cortex; they are the terminals of the cortico-geniculate fibers. The third type of bouton (F₁ does not degenerate after either intervention. The boutons of this type are filled with flattened vesicles and are believed to be intrageniculate terminals. F₂-profiles were interpreted as presynaptic dendrites of the IN. The characteristic synaptic glomeruli found in the LGN contain in their center an optic terminal. These optic terminals establish synaptic contacts with dendrites or spine-like dendritic protrusions of TCRs as well as with presynaptic dendrites. Synaptic triads were also seen. The distribution of the individual types of synaptic contacts in layers 3 and 4 was determined. Layer 4 contains only one third of the retino-geniculate synapses and of the synaptic contacts of F₁-terminals.     

Experimental morphological study of primary afferent terminals at the base of the dorsal horn of the cat spinal cord

The distribution and ultrastructure of primary afferent terminals in the gray matter of the cervical and lumbar regions of the cat spinal cord were studied by the experimental degeneration method of Fink and Heimer. Most preterminals of primary afferents were shown to be concentrated in the region of the intermediate nucleus of Cajal (central part of Rexed's laminae VI–VII), in the substantial gelatinosa (laminae II–III), and in the nucleus proprius of the dorsal horn (central and medial parts of lamina IV). Fewer are found in the region of the motor nuclei. The number of degenerating axon terminals in the lateral parts of laminae IV and V differed: 31.5 and 0.4% respectively of all axon terminals. Many terminals of primary afferents in lamina IV contribute to the formation of glomerular structures in which they exist as terminals of S-type forming axo-axonal connections with other terminals. These results are in agreement with electrophysiological data to show that interneurons in different parts of the base of the dorsal horn differ significantly in the relative numbers of synaptic inputs formed by peripheral afferents and descending systems.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 5, No. 4, pp. 406–414, July–August, 1973.     

Distribution and ultrastructure of thalamic afferents in the cat auditory cortex

Ultrastructural features of thalamic afferent fibers were studied in the cat auditory cortex in the early stages (on the 4th day) of experimental degeneration produced by destruction of the medial geniculate body. A coordinate grid was used in conjunction with an electron micro-scope to study the topography of the degenerating elements over wide areas of sections, so that the density of degeneration could be determined quantitatively in different layers of the cortex. Degenerating axons were found in all layers. Most of the large (5–7 µ) degenerating axons are located in layer VI; their diameters were smaller in the upper layers of the cortex. Degenerative changes affecting synaptic terminals of thalamo-cortical afferents were of the "dark" type. Fibers of the geniculo-cortical tract were shown to terminate mainly in cortical layer IV. A few degenerating synapses were found in the molecular layer. Terminals with sperical synaptic vesicles are found mainly on the spines of dendrites where they form "asymmetrical" contacts. A few degenerating axo-somatic synapses were observed on stellate neurons in layer IV. The results are discussed in connection with electrophysiological investigations of the cat auditory cortex during stimulation of specific afferent fibers.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 4, No. 6, pp. 612–620, November–December, 1972.     

Novel processes invaginate the pre-synaptic terminal of retinal bipolar cells

Mixed-rod cone bipolar (Mb) cells of goldfish retina have large synaptic terminals (10 mum in diameter) that make 60-90 ribbon synapses mostly onto amacrine cells and rarely onto ganglion cells and, in return, receive 300-400 synapses from gamma-aminobutyric acid (GABA)-ergic amacrine cells. Tissue viewed by electron microscopy revealed the presence of double-membrane-bound processes deep within Mb terminals. No membrane specializations were apparent on these invaginating processes, although rare vesicular fusion was observed. These invaginating dendrites were termed "InDents". Mb bipolar cells were identified by their immunoreactivity for protein kinase C. Double-label immunofluorescence with other cell-type-specific labels eliminated Müller cells, efferent fibers, other Mb bipolar cells, dopaminergic interplexiform cells, and somatostatin amacrine cells as a source of the InDents. Confocal analysis of double-labeled tissue clearly showed dendrites of GABA amacrine cells, backfilled ganglion cells, and dendrites containing PanNa immunoreactivity extending into and passing through Mb terminals. Nearly all Mb terminals showed evidence for the presence of InDents, indicating their common presence in goldfish retina. No PanNa immunoreactivity was found on GABA or ganglion cell InDents, suggesting that a subtype of glycine amacrine cell contained voltage-gated Na channels. Thus, potassium and calcium voltage-gated channels might be present on the InDents and on the Mb terminal membrane opposed to the InDents. In addition to synaptic signaling at ribbon and conventional synapses, Mb bipolar cells may exchange information with InDents by an alternative signaling mechanism.     

Ultrastructural immunocytochemical localization of excitatory amino acids in the somatosensory system.

We studied the ultrastructure and the synaptic arrangement of glutamate-immunoreactive terminals in rats, in the superficial laminae of the spinal cord, the brainstem cuneate nucleus, and the thalamic ventroposterolateral nucleus, where a role for glutamate as neurotransmitter has been suggested by biochemical, physiological and pharmacological approaches. The antiserum employed was raised against glutaramate conjugated to keyhole limpet hemocyanin with glutaraldehyde, and was used for pre-embedding staining with an avidin-biotin-peroxidase method and for post-embedding staining with an immunogold procedure. Both methods yielded similar results, consisting of labeling of selected terminals in all the areas examined. Double immunogold labeling on the same thin section using antisera against gamma-amino-butyric acid (GABA) or substance P (SP), in combination with the anti-glutamate serum, showed that staining for glutamate and GABA was present in different terminals in all the regions examined; glutamate and SP were co-localized in a few terminals only in the superficial laminae of the spinal cord. By performing immunogold staining in combination with anterograde tracing, glutamate immunoreactivity could be localized in identified primary afferents to the dorsal spinal cord and cuneate nucleus, and in lemniscal afferents to the thalamus.     

Synaptic connections of the neurokinin 1 receptor-like immunoreactive neurons in the rat medullary dorsal horn

The synaptic connections between neurokinin 1 (NK1) receptor-like immunoreactive (LI) neurons and γ-aminobutyric acid (GABA)-, glycine (Gly)-, serotonin (5-HT)- or dopamine-β-hydroxylase (DBH, a specific marker for norepinephrinergic neuronal structures)-LI axon terminals in the rat medullary dorsal horn (MDH) were examined under electron microscope by using a pre-embedding immunohistochemical double-staining technique. NK1 receptor-LI neurons were observed principally in laminae I and III, only a few of them were found in lamina II of the MDH. GABA-, Gly-, 5-HT-, or DBH-LI axon terminals were densely encountered in laminae I and II, and sparsely in lamina III of the MDH. Some of these GABA-, Gly-, 5-HT-, or DBH-LI axon terminals were observed to make principally symmetric synapses with NK1 receptor-LI neuronal cell bodies and dendritic processes in laminae I, II and III of the MDH. The present results suggest that neurons expressing NK1 receptor within the MDH might be modulated by GABAergic and glycinergic inhibitory intrinsic neurons located in the MDH and 5-HT- or norepinephrine (NE)-containing descending fibers originated from structures in the brainstem.