Organization of the serotonergic innervation of spinal neurons in rats—I. Neuropeptide coexistence in varicosities innervating some spinothalamic tract neurons but not in those innervating postsynaptic dorsal column neurons

Previous studies have suggested that peptides such as substance P and thyrotropin-releasing hormone coexist with serotonin in the same varicosities in the ventral horn and intermediate gray of the spinal cord in rat. However, coexistence of these peptides with serotonin is rare in fibers in the superficial dorsal horn. Since it has been proposed that serotonergic fibers in the superficial dorsal horn act to modulate nociception, it was hypothesized that the serotonergic neurons that contain neither substance P nor thyrotropin-releasing hormone might constitute a specifically antinociceptive subset of serotonergic neurons. This being the case, it would be expected that different types of serotonergic neurons innervate nociceptive and non-nociceptive spinal neurons. In order to test this hypothesis, a group of cells that include nociceptive neurons (spinothalamic tract neurons) and a group of predominantly non-nociceptive neurons (postsynaptic dorsal column neurons) in the spinal cord of rat were retrogradely labeled. Sections of the spinal cord containing retrogradely labeled spinothalamic tract or postsynaptic dorsal column neurons were stained for serotonin and either substance P or thyrotropin-releasing hormone using two-color immunohistochemistry. A retrogradely labeled cell was classified as “apposed” if there was no discernible distance between an immunohistochemically labeled varicosity and the cell. Eighty per cent of spinothalamic tract and 83% of postsynaptic dorsal column profiles were apposed by serotonin-immunoreactive varicosities in the spinal cord. Thirty-one per cent of the spinothalamic tract profiles that were apposed by serotonergic varicosities were apposed by serotonergic varicosities that were also stained for thyrotropin-releasing hormone. The distribution of the latter spinothalamic neurons was similar to that reported for spinothalamic tract neurons responsive to joint movement. In addition, at least 63% of the spinothalamic tract profiles which were apposed by serotonergic varicosities were apposed by “serotonin-only” varicosities, including most spinothalamic tract neurons in the marginal zone, suggesting that at least some “serotonin-only” neurons are antinociceptive. However, contrary to the hypothesis, at least 94% of the postsynaptic dorsal column profiles apposed by serotonergic varicosities were apposed by “serotonin-only” varicosities.

These findings suggest that there may be a relationship between the sensory modality to which a spinal neuron responds and the type of serotonergic innervation it receives. However, it appears that “serotonin-only” neurons may not constitute a specifically antinociceptive category of serotonergic neurons.     

Fine structure of synapses associated with characterized postsynaptic dorsal column neurons in the cat

Fourteen dorsal horn neurons with axons projecting through the dorsal columns were identified either by electrophysiological methods (and subsequently injected with horseradish peroxidase) or by retrograde labelling with horseradish peroxidase in cats. All neurons were contacted by small (less than 2 micron) boutons containing spherical or elongated agranular vesicles. One neuron with its soma located in lamina III received additional contacts from central elements of glomerular complexes. Neurons with somata located more ventrally (deep lamina IV and V) were also postsynaptic to large (greater than 2 microns) electron lucent profiles which formed multiple synapses with the labelled cells. Some boutons presynaptic to postsynaptic dorsal column neurons were themselves postsynaptic to profiles containing pleiomorphic agranular vesicles at axoaxonic synapses. They also occasionally participated in triadic complexes. It is concluded that the synaptic arrangements formed by boutons in association with postsynaptic dorsal column neurons differ significantly from those associated with spinocervical neurons. Such differences might provide the anatomical substrate for the observed receptive field characteristics of these neurons.     

The differential distribution and relationship of serotoninergic and peptidergic fibers to sympathoadrenal neurons in the intermediolateral cell column of the rat: a combined retrograde axonal transport and immunofluorescence study

The preganglionic sympathetic neurons in the intermediolateral cell column of the thoracic and upper lumbar segments of the spinal cord which innervate the chromaffin cells in the adrenal medulla, sympathoadrenal preganglionic neurons, were identified by the method of retrograde axonal transport of the fluorescent dyes Fast Blue and True Blue. In rats, Fast Blue or True Blue was injected into the medulla of the left adrenal gland. After a survival period of 5 days, the animals were perfusion fixed, the thoracic and lumbar spinal cord sectioned and processed for the immunofluorescent localization of met-enkephalin, neurophysin, oxytocin, serotonin, somatostatin and substance P immunoreactivity. Neuronal perikarya which were retrogradedly-labeled with Fast Blue or True Blue were observed in the intermediolateral cell column from the T1 to the L2 spinal cord segments. The distribution of the sympathoadrenal neurons was determined by counting the number of retrogradedly-labeled neurons per spinal cord segment. In the five animals used for quantifying the sympathoadrenal preganglionic neurons, the majority (72.3%) of the retrogradely-labeled neurons counted per spinal cord were located within the T7-T12 segments. The T9 segment contained the largest average number (20.1%) of retrogradely-labeled cells in a single segment. Met-enkephalin, serotonin and substance P immunoreactive fibers were prominent in the intermediolateral cell column, whereas oxytocin, neurophysin and somatostatin immunoreactive fibers were sparse. The met-enkephalin, serotonin and substance P fibers were seen surrounding both unlabeled and retrogradely-labeled neurons; somatostatin fibers appeared to preferentially contact retrogradely-labeled neurons; whereas, the neurophysin and oxytocin fibers were not found in proximity to retrogradely-labeled neurons. Met-enkephalin, neurophysin, oxytocin, somatostatin and substance P immunoreactivity were depleted in the intermediolateral cell column below the level of a spinal cord transection. Serotonin immunoreactivity was depleted in the intermediolateral cell column below the level of the transection for five to six segments, but sparse networks of immunoreactive fibers were observed in both the intermediolateral cell column and the ventral horn in more caudal segments. Met-enkephalin, serotonin, somatostatin and substance P immunoreactivity were decreased in both the contralateral and ipsilateral intermediolateral cell column below the level of a spinal cord hemisection, suggesting that both crossed and uncrossed descending pathways exist. Neurophysin and oxytocin immunoreactivity were depleted below the level of the hemisection in the ipsilateral intermediolateral cell column without noticeable decrease in the level of immunoreactivity in the contralateral intermediolateral cell column, suggesting that a decussation does not occur at the level of the spinal cord, but may exist above the level of the hemisection...     

Converging and diverging cholinergic inputs from submucosal neurons amplify activity of secretomotor neurons in guinea-pig ileal submucosa.

The organization of synaptic connections between guinea-pig ileal submucosal neurons was examined using intracellular recordings from single or pairs of submucosal neurons. Synaptic inputs were elicited by stimulating cholinergic neurons using pressure-pulse application of 5-hydroxytryptamine (5-HT) in ganglia adjacent to those where intracellular recordings were obtained. In addition, when pairs of intracellular recordings were obtained, one neuron was activated by intracellular stimulation and synaptic responses were recorded in the other neuron. Neurobiotin-filled microelectrodes were employed to characterize cells electrophysiologically and immunohistochemically. Recordings were obtained from 176 (173 S-type and three AH-type) neurons; 81% of cells were classified as vasoactive intestinal peptide (VIP) neurons. No fast excitatory postsynaptic potentials and only rare slow excitatory postsynaptic potentials were recorded following intracellular stimulation of paired S-type neurons. However, when paired intracellular recordings were obtained from neurons within the same ganglion and 5-HT was applied to an adjacent ganglion, this stimulation evoked synchronized fast excitatory postsynaptic potentials in 94% of pairs. In contrast, when cell bodies of VIP-VIP pairs were located in different ganglia, fast synaptic activation evoked by 5-HT stimulation was not synchronized in 87% of pairs. When intracellular recordings were obtained from a single neuron and two separate ganglia were stimulated by 5-HT pressure-pulse activation, fast excitatory postsynaptic potentials originating from both sources were recorded in the same VIP neuron. Morphological study of 34 S-type and three AH-type horseradish peroxidase-labeled neurons was conducted. AH-type neurons had multiple axonal branches with dense arborization of collaterals containing numerous varicosities in three to nine ganglia, whereas axons of S-type neurons exhibited relatively rare collaterals and varicosities within adjacent ganglia.These results demonstrate that cholinergic neurons provide both diverging and converging inputs to VIP neurons, providing a mechanism to enhance activation of VIP secretomotor neurons. The axonal projections of AH-type neurons suggest they are likely candidates to provide diverging inputs to multiple VIP neurons.     

Synaptic connections of an interneuron with axonal arcades in the cat visual cortex

Summary A single, isolated interneuron with axonal arcades in the cat visual cortex was analysed in detail by both light and electron microscopy. The neuron was impregnated by the Golgi-Kopsch method, gold-toned, and processed for electron microscopy using the ethanolic phosphotungstic acid (PTA) staining method of Bloom & Aghajanian (1968). These methods, in combination, resulted in the successful identification of a large number of synaptic boutons arising from the axon of the cell under study. We examined serially at the electron microscope level 210 boutons of the axonal arborization of the cell. Of these, 152 formed identifiable symmetrical synaptic contacts with a variety of postsynaptic elements. The vast majority of the postsynaptic targets were dendritic profiles, which represented 95.7% of all the synaptic contacts identified. Only one example was observed of two labelled boutons making contacts with the same postsynaptic element; the rest were apparently on different elements. This distribution of synapses, characterized by the lack of convergence, is very similar to that reported by other authors for a certain kind of double bouquet cell which, in turn, shares some morphological features with the neurons with axonal arcades. It is suggested that fine details of the geometry of the axonal arborization of a given cell are an important reflection of the distribution of its synapses.     

The differential synaptic organization of the spinal and lemniscal projections to the ventrobasal complex of the rat thalamus. Evidence for convergence of the two systems upon single thalamic neurons

The synaptic organization of terminals originating either from the spinal cord (spinothalamic) or from the dorsal column nuclei (lemniscal) was investigated in the ventrobasal complex of the rat thalamus. Wheatgerm agglutinin conjugated to horseradish peroxidase was used as an anterogradely transported axonal tracer, using benzidine dihydrochloride as a chromogen for the identification by electron microscopy of spinal and lemniscal projections to the ventrobasal thalamus. A double anterograde tract tracing strategy, based labeling by wheatgerm agglutinin conjugated to horseradish peroxidase of spinal terminals and simultaneous visualization of lemniscal terminals identified by Wallerian degeneration induced by lesion of the neurons of origin in the dorsal column nuclei, was used to compare the postsynaptic elements contacted by the two pathways and to look for a possible convergence of the two pathways onto single thalamic neurons. Spinal and lemniscal terminals are large (2-2.5 microns mean average diameter) terminals containing several mitochondria and numerous rounded vesicles. A quantitative analysis of the mean average diameters of the terminals revealed that one could not differentiate between synapses formed by the two pathways on a morphological basis. Terminals of the two pathways make asymmetrical contacts (Gray type I) with dendrites of varying diameter, dendritic protrusions, and cell somata. A quantitative analysis of the least diameter of the postsynaptic elements demonstrates projections of the two systems to different, partially overlapping regions of thalamic neurons. Lemniscal terminals originating from the dorsal column nuclei frequently contact cell somata; axosomatic spinothalamic contacts are uncommon. In addition, lemniscal projections tend to contact more proximal dendrites than do spinal projections, and this differential synaptic organization is statistically significant. From a functional point of view, this differential synaptic organization might indicate that lemniscal inputs have greater influence than spinal inputs in affecting the activity of thalamic neurons. Labeled spinothalamic terminals contact the same dendritic profile as do degenerating lemniscal terminals in about 10% of single sections. Because the present study did not include a complete reconstruction of ventrobasal complex neurons of the thalamus or even regions of dendritic arbors, the degree of convergence is likely to be significantly underestimated. These findings indicate that the anatomical basis exists for an interaction between nociceptive and non-nociceptive somesthetic systems at the level of single ventrobasal neurons of the thalamus of the rat.     

Synaptogenesis and distribution of presynaptic axonal varicosities in low density primary cultures of neocortex: an immunocytochemical study utilizing synaptic vesicle-specific antibodies, and an electrophysiological examination utilizing whole cell recording

Summary Low-density primary cultures of neocortical neurons were utilized to examine: (i) early interactions of growing neurites with morphological characteristics of axons with other neuronal elements, and (ii) the distribution of presynaptic axonal varicosities closely apposed to MAP-2 immunoreactive, putatively postsynaptic, dendrites. At the light microscopical level axonal varicosites, presumably presynaptic terminals, were identified using immunocytochemistry incorporating antibodies specific for the synaptic vesicle antigens synaptophysin and synapsin. The presence of synaptophysin- and synapsin-immunoreactive swellings along axonal processes was first detected at 5 days post-plating and was also apparent in axons growing in isolation. At 5–7 daysin vitro, immunolabelled axonal varicosities in close apposition to putative postsynaptic dendrites (MAP-2 immunoreactive) dendrites were detected. Electrophysiologically active synaptic contacts can also readily be detected at this stage. After 3 weeksin vitro presynaptic contacts do appear to be distributed heterogeneously along postsynaptic dendrites of many neurons in culture. As the culture matures a higher number of presynaptic profiles can be seen along dendrites, with a centrifugal distribution, e.g. a higher density of presynaptic axonal terminals in close apposition to more distal regions of larger dendrites, putatively considered to be apical dendrites of pyramidal-like neurons. In our cultures, the overall increase in the density and the pattern of distribution of presynaptic axon terminals immunoreactive for synaptic vesicle antigens closely apposed to putative post-synaptic structures mimics the general postnatal increase of synaptic density in the neocortexin vivo. Thus, low density primary cultures of neocortical neurons offer a valuable system to explore and manipulate (i) the molecular and cellular basis of neocortical synaptogenesis, and (ii) the pharmacology of neocortical synaptic transmission.     

Synaptic relationships of a type of GABA-immunoreactive neuron (clutch cell), spiny stellate cells and lateral geniculate nucleus afferents in layer IVC of the monkey striate cortex

The precise stimulus specificity of striate cortical neurons is strongly influenced by processes involving gamma-aminobutyric acid (GABA). In the visual cortex of the monkey most afferents from the lateral geniculate nucleus terminate in layer IVC. We identified a type of smooth dendritic neuron (clutch cell) that was immunoreactive for GABA, and whose Golgi-impregnated dendrites and axon were largely restricted to layer IVC beta. The slightly ovoid somata were 8-12 micron by 12-15 micron and the dendritic field was often elongated, extending 80-200 micron in one dimension. The axonal field was 100-150 micron in diameter and densely packed with large bulbous boutons. Although mainly located in IVC beta both the dendritic and axonal processes entered IVC alpha. Fine structural features of GABA-immunoreactive and-impregnated clutch cells and impregnated spiny stellate cells were compared. Clutch cells had more cytoplasm, more densely packed mitochondria and endoplasmic reticulum, and made type II as opposed to type I synapses. A random sample of 159 elements postsynaptic to three clutch cells showed that they mainly terminated on dendritic shafts (43.8-58.5%) and spines (20.8-46.3%), rather than somata (10-17%). The majority of the postsynaptic targets were characteristic of spiny stellate cells. This was directly demonstrated by studying synaptic contacts between an identified GABA positive clutch cell and the dendrites and soma of an identified spiny stellate cell. The termination of clutch cells mainly on dendrites and spines of spiny stellate cells suggests that they interact with other inputs to the same cells. Following an electrolytic lesion in the ipsilateral lateral geniculate nucleus we examined the distribution of degenerating terminals on three identified spiny stellate neurons in layer IVC beta. Out of eight synapses from the lateral geniculate nucleus one was on a dendritic shaft, the rest on spines. Only a small fraction of all synapses on the cells were from degenerating boutons. A clutch cell within the area of dense terminal degeneration was not contacted by terminals from the lateral geniculate nucleus. The results show that layer IVC in the monkey has a specialized GABAergic neuron that terminates on spiny stellate cells monosynaptically innervated from the lateral geniculate nucleus. Possible functions of clutch cells may include inhibitory gating of geniculate input to cortex; maintenance of the antagonistic subregions in the receptive fields; and the creation from single opponent of double colour opponent receptive fields.     

The development of inhibitory synaptic specializations in the mouse deep cerebellar nuclei.

Using confocal laser scanning microscopy and immunohistochemistry, this study shows the complete morphological development of GABAergic synaptic contacts between Purkinje cells and neurons of the deep cerebellar nuclei of the mouse. Firstly, presynaptic varicosities visualized with antibodies against synaptophysin, synapsin or glutamic acid decarboxylase, were detected when the postsynaptic GABA(A) receptors were not yet aggregated in the membrane but had a diffuse cytoplasmic distribution, which indicated a lead in maturation of presynaptic terminals over target cells. Secondly, receptor aggregates developed suddenly after an initial week of diffuse expression and these clusters matured into more numerous and larger synaptic aggregates. During this postsynaptic maturation, the presynaptic varicosities develop into numerous and well-defined spots. As soon as both pre- and postsynaptic clusters were detectable, these sites are always colocalized. We therefore consider the aggregation of postsynaptic receptor during development as a landmark of synapse formation. Our observations are consistent with a developmental model in which the presynaptic neuron differentiates its axon before the target neuron expresses the mature form of its receptors on the membrane. The presynaptic neuron can therefore instruct the target neuron about the distribution and aggregation of the postsynaptic receptors at the synapse.     

An immunogold electron microscopic analysis of FMRFamide-like immunoreactive neurons in the CNS ofHelix pomatia: ultrastructure and synaptic connections

Summary The ultrastructure and synaptic connections of FMRFamide-like immunoreactive neurons were investigated in the CNS of the snailHelix pomatia, following the application of a post-embedding immunogold method. For comparison, first, we analyzed the ultrastructure and granule content of the identified FMRFamide-containing C3 neuron in the cerebral ganglion. Three types of unidentified immunoreactive neuronal perikarya, and five types of varicosities could be distinguished on the basis of granule content. The different granule types revealed a highly selective accumulation of gold particles. One granule type contained by one FMRFamide-like immunoreactive neuron type (N1) and by one varicosity type (T₂) showed similar ultrastructure to that of the granules seen in the C3 cell. In the neuropil, the majority of FMRFamide-like immunoreactive varicosities (four of the five varicosity types) established specialized synaptic contacts with unidentified postsynaptic profiles. In the connective tissue sheath around the ganglia, three types of FMRFamide-like immunoreactive varicosities were found to establish unspecialized contacts with smooth muscle fibres or to be free in the mass of collagen fibres. On the basis of these observations, we suggest (1) an extensive diversity of the localization of FMRFamide (and related substances) at the ultrastructural level; (2) the involvement of FMRFamide-like immunoreactive varicosities in synaptic, modulatory and neurohormonal regulatory processes in theHelix nervous system.     

Immunocytochemical and electron microscopic study of serotonin neuronal organization in the dorsal raphe nucleus of the monkey

Serotonin neurons in the dorsal raphe nucleus were identified using an antibody to a serotonin-bovine serum albumin conjugate and the peroxidase anti-peroxidase method. Nerve cell bodies showing serotonin-like immunoreactivity ranged in size from 15 to 22 micron in diameter; their dendrites were also immunoreactive. Immunostaining was present in the cytoplasmic matrix, outer membranes of mitochondria, rough endoplasmic reticulum, multivesicular bodies and dense-cored vesicles. Heavily immunoreactive axonal varicosities contained small round vesicles (18-35 nm) and larger dense-cored vesicles (50-90 nm). Both unmyelinated (0.2-0.5 micron) and myelinated (0.8-1.1 micron) serotonin-like immunoreactive axons were found, often interspersed within bundles of similar caliber unlabeled axons. Serotonin-like immunoreactive somata and dendrites were postsynaptic to numerous unlabeled terminals that contained either (a) clear round vesicles (18-25 nm) with many small dense-cored vesicles (30-50 nm), (b) clear round vesicles (18-25 nm) with large dense-cored vesicles (90-110 nm) or (c) clear round vesicles (18-25 nm) with or without flat vesicles. In addition pairs of unlabeled terminals formed crest synapses onto serotonin-like immunoreactive dendritic spines. This variety of unlabeled terminals making contact with serotonin-like immunoreactive elements suggests that several neuronal systems with possibly different transmitters may regulate serotonin raphe neurons. We occasionally observed serotonin-like immunoreactive dendrites and terminals in apposition to other serotonin-like immunoreactive dendrites with membrane specializations at the site of contact. This might represent a possible site for the self inhibition of serotoninergic neurons reported in physiological studies of the serotonin system in the dorsal raphe nucleus.     

Synaptic connections of enkephalin-immunoreactive nerve terminals in the neostriatum: a correlated light and electron microscopic study

Summary Two different antisera to leucine-enkephalin were used to study the localization of enkephalin-like immunoreactive material in the neostriatum and globus pallidus of the rat, by means of the unlabelled antibody-enzyme method. Thin immunoreactive varicose fibres are scattered throughout the neostriatum. In the ventral striatum, fibres come together and follow a relatively straight course for several micrometers, forming tube-like structures which can be traced to cell bodies; these cell bodies are completely surrounded by immunoreactive fibres. Occasional immunoreactive varicose fibres are also found close to another type of neuron throughout the whole neostriatum.Examination by electron microscopy of immunoreactive structures that had been identified first in the light microscope, showed that each of the nearly 200 varicosities examined was a vesicle-containing bouton that formed a synaptic contact. Rarely were asymmetrical synaptic contacts found between immunoreactive boutons and dendritic spines. All other synapses formed by enkephalin-immunoreactive boutons were symmetrical. Two types of postsynaptic neuron were identified; the first type was a medium-sized neuron with the ultrastructural features of a typical striatal spiny neuron. The second type had a larger perikaryon surrounded by numerous immunoreactive varicosities that were found to be boutons forming symmetrical synapses. The long dendrites of this second type of neuron likewise received a dense input of immunoreactive boutons forming symmetrical synapses; such ensheathed dendrites were found to be the tube-like structures seen in the light microscope. The ultrastructural features of these neurons, notably a highly indented nucleus, were those of a rare type of striatonigral neuron. In the globus pallidus, all the enkaphalin-immunoreactive boutons studied formed symmetrical synapses with ensheathed dendrites and perikarya that were similar to the latter type of postsynaptic neuron in the neostriatum. Axo-axonic synapses involving immunoreactive boutons were not seen in our material.The results are consistent with the view that enkephalin-like substances may be synaptic transmitters in the neostriatum and that they may have different actions according to the nature of the postsynaptic target. The finding that one type of neostriatal neuron, and a very similar neuron in the globus pallidus, receives multiple enkephalin-immunoreactive boutons all over its perikaryon and along its dendrites indicates a potentially important role of enkephalin in the convergence of information within the neostriatum and pallidum on to output neurons.     

Anatomical distribution and ultrastructural organization of the gabaergic system in the rat spinal cord. An immunocytochemical study using anti-GABA antibodies

γ-Aminobutyric acid (GABA)-containing elements have been studied by light and electron microscopy in the rat spinal cord, using immunocytochemistry with anti-GABA antibodies. Light microscopy showed immunoreactive somata localized principally in laminae I–III, and occasionally in the deeper laminae of the dorsal horn and in the ventral horn. Small somata were also observed around the central canal. Punctate GABA-immunoreactive profiles were particularly concentrated in laminae I–III, and moderately abundant in the deeper laminae and in the ventral horn where they were observed surrounding the unlabelled motoneurons.

At the ultrastructural level, the punctate profiles corresponded to GABA-containing axonal varicosities or small dendrites. GABA-immunoreactive varicosities were presynaptic to labelled or unlabelled dendrites and cell bodies. Some unlabelled terminals presynaptic to unlabelled dendrites received symmetrical synaptic contacts from GABA-immunoreactive terminals.

These results confirm data obtained withl-glutamate decar☐ylase immunocytochemistry, and support the role of GABA in pre- and postsynaptic inhibition in the spinal cord, respectively via axoaxonal and axosomatic or axodendritic synapses.     

Organization of crustacean neuropil. II. Distribution of synaptic contacts on identified motor neurons in lobster stomatogastric ganglion

Summary Identified neurons in the stomatogastric ganglion of the lobster were examined and reconstructed by serial section electron microscopy. Each motor neuron consists of a soma, a primary process leading directly from the soma to the motor axon which leaves the ganglion, and a group of secondary processes which branch from the primary process and ramify within the neuropil. Synapses are found only on small processes in the synaptic neuropil, never on the primary processes or on larger secondary processes in the coarse neuropil. Nearly every secondary process of every neuron examined makes both pre- and postsynaptic contacts. Hence these neurons are not polarized into distinct pre- and postsynaptic regions but have both input and output distributed over each of the secondary processes in the neuropil. The connection between a specific pair of neurons is also distributed over several branches of both the pre- and the postsynaptic neurons.The restriction of synapses to the more distal portions of the secondary processes suggests that no single contact or localized group of contacts can exert an overriding influence on the neuron by virtue of an especially advantageous position. The close proximity of input and output on most secondary processes suggests that synaptic input may be capable of directly influencing output without the intervention of action potentials. The distribution of specific synapses over several branches of both pre- and postsynaptic neurons suggests that each neuron functions as a whole without differentiation into specialized branches.     

Light microscopic and ultrastructural localization of immunoreactive substance P in the dorsal horn of monkey spinal cord

Light- and electron-microscopic localization of substance P in the monkey spinal cord was studied by the peroxidase anti-peroxidase technique with the particular aim of examining types of interactions made by substance P-positive boutons with other neuronal elements in the dorsal horn. By light-microscopy dense labeling for immunoreactive substance P was found in laminae I, II (outer zone) and V (lateral region), consistent with findings in other mammalian species. By electron-microscopy, substance P-positive staining was mostly in unmyelinated and in some thinly myelinated small diameter fibers. Substance P-positive terminals contained both large granular vesicles (80-120 nm diameter), which were filled with reaction product, and clear round vesicles (40-60 nm). Substance P-positive large granular vesicles were sometimes observed near presynaptic sites and in contact with dense projection there. Immunoreactive substance P boutons were small to large in size (1-4 micron), formed synapses with somata and large dendrites and were the central axons of synaptic glomeruli where they were in synaptic contact with numerous small dendrites and spines. Substance P-labeled axons frequently formed synapses with dorsal horn neurons which were also postsynaptic to other types of axons. Substance P-positive profiles participated in numerous puncta adhaerentia with unlabeled cell bodies, dendrites and axons. Only rarely, some suggestive evidence was obtained indicating that axons might synapse onto substance P-containing boutons. Biochemical analysis of monkey spinal cord tissue extracts, undertaken to characterize more precisely the immunoreactive substances, indicated that only substance P and its oxide derivative were detected with the antiserum used in the immunocytochemistry. These morphological findings show that substance P is contained within a class of axon terminals, many of which have been shown previously in the monkey to originate from the dorsal root. The results suggest that modulation of substance P primary afferents terminating in the outer dorsal laminae of the monkey spinal cord occurs in part via axonal inputs onto dorsal horn neurons postsynaptic to the primary afferent.     

Functional properties of fast spiking interneurons and their synaptic connections with pyramidal cells in primate dorsolateral prefrontal cortex

Recent studies suggest that fast-spiking (FS) interneurons of the monkey dorsolateral prefrontal cortex (DLPFC) exhibit task-related firing during working-memory tasks. To gain further understanding of the functional role of FS neurons in monkey DLPFC, we described the in vitro electrophysiological properties of FS interneurons and their synaptic connections with pyramidal cells in layers 2/3 of areas 9 and 46. Extracellular spike duration was found to distinguish FS cells from non-FS interneuron subtypes. However, a substantial overlap in extracellular spike duration between these populations would make classification of individual interneurons difficult. FS neurons could be divided into two main morphological groups, chandelier and basket neurons, with very similar electrophysiological properties but significantly different horizontal spread of the axonal arborization. In paired cell recordings, unitary inhibitory postsynaptic potentials (IPSPs) elicited by FS neurons in pyramidal cells had rapid time course, small amplitude at resting membrane potential, and were mediated by GABA(A) receptors. Repetitive FS neuron stimulation, partially mimicking the sustained firing of interneurons in vivo, produced short-term depression of the unitary IPSPs, present at connections made by both basket and chandelier neurons and due at least in part to presynaptic mechanisms. These results suggest that FS neurons and their synaptic connections with pyramidal cells have homogeneous physiological properties. Thus different functional roles of basket and chandelier neurons in the DLPFC in vivo must arise from the distinct properties of the interneuronal axonal arborization or from a different functional pattern of excitatory and inhibitory connections with other components of the DLPFC neuronal network.     

Axonal sprouting following lesions of the rat substantia nigra

Parkinson's disease is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Symptoms do not appear until most nigral neurons are lost, implying that compensatory mechanisms are present. Sprouting has been proposed as one of these mechanisms. This study quantified the extent of compensatory axonal sprouting following injury of dopaminergic neurons within the substantia nigra pars compacta. Specifically, the extent of the axonal arbour and axonal varicosity morphology was measured after partial destruction (with 6-hydroxydopamine) of the substantia nigra of the adult male rat. Four months later, the substantia nigra was injected with the anterograde neuronal tracer dextran-biotin to trace the full extent of individual axons. An unbiased estimate of neuron number was performed in each animal. This demonstrated nigral neuronal loss ranging from 10 to 90% on the side that received the injection whilst a 7% reduction was observed in the side contralateral to the lesion. Coincident with this loss, some nigral neurons lose tyrosine hydroxylase expression. Vigorous axonal sprouting was observed in the terminal arbours of lesioned animals and was associated with an increased axonal varicosity size. Axonal varicosities and branching points were primarily confined to the dorsal 1.5mm of the caudate-putamen, an area predominantly innervated by nigral neurons. It appears that dopaminergic neurons were responsible for this sprouting because the density of dopamine transporter immunoreactive varicosities in the caudate-putamen was maintained until about a 70% loss of neurons. It was concluded that substantial compensation in the form of sprouting and new dopaminergic synapse formation occurs following lesions of the substantia nigra pars compacta.     

Locus coeruleus terminals in intraocularly transplanted spinal cords as compared with catecholamine terminals in normal spinal cords: Their synaptic densities and functional considerations

Locus coeruleus terminals in intraocularly transplanted spinal cords and catecholamine terminals in defined areas of normal spinal cords were investigated qualitatively and quantitatively by immunoelectron microscopy. Results showed that the morphological features of synapses formed in the grafts closely resembled those of normal spinal cords. The incidences of synapses per varicosities, as observed in single sections, were 30.1, 40.2 and 22.8% for the ventral horn, dorsal horn and grafted spinal cord, respectively. In all three groups, most of the postsynaptic targets were small dendrites, although high frequencies of large dendrites were found in the ventral horn. Spines and axons in the grafts were also postsynaptic targets. Several characteristics of relative immaturity were observed in the grafts. It is suggested that the inhibition of spinal neurons by locus coeruleus terminals may be mediated not only by volume transmission through nonsynaptic contacts, but also by direct contacts with catecholamine terminals, and that the excitation of facilitation observed at those terminals may be explained by the suppression of inhibitory neurons by axoaxonic contacts.