Ultrastructural and immunocytochemical characterization of terminals of postsynaptic ascending dorsal column fibers in the rat cuneate nucleus

The morphology, synaptic contacts, and neurotransmitter enrichment of postsynaptic dorsal column terminals in the cuneate nucleus of rats were investigated and compared with those of identified primary afferents. For this purpose, anterograde transport of horseradish peroxidase–based tracers injected in the spinal cord was combined with postembedding immunogold labeling for glutamate and gamma-aminobutyric acid (GABA). Anterogradely labeled postsynaptic dorsal column terminals were morphologically homogeneous: they were small (mean area = 1. 37 μM2) and dome-shaped, contacted single dendritic shafts or cell bodies, and were not involved in axoaxonic synapses. The majority of them were not enriched in glutamate or GABA immunoreactivity compared with other tissue components. Their morphology, size, and neurotransmitter content thus differed from that of primary afferents. These differences are consistent with distinct functional roles for the two main afferent systems ascending to the cuneate nucleus. © 1995 Wiley-Liss, Inc.     

Identification of signal substances in synapses made between primary afferents and their associated axon terminals in the rat trigeminal sensory nuclei

The relationships between primary afferent terminals (PATs) and their associated presynaptic terminals in the rat trigeminal sensory nuclear complex (TSNC) were examined with special reference to amino acid transmitters glutamate (Glu) and gamma-aminobutyric acid (GABA). Primary afferent terminals anterogradely labeled from the trigeminal ganglion with the B subunit of cholera toxin conjugated to horseradish peroxidase (CTB-HRP) were sectioned for electron microscopy. Serial sections from the principal nucleus (Vp), dorsomedial parts of the oral and interpolar nuclei (Vdm), and lamina III/IV of caudal nucleus (Vc) were immunostained for Glu and GABA by using a postembedding immunogold technique. The tracer, CTB-HRP to the trigeminal ganglion, preferentially labeled myelinated primary afferents. Sections immunostained with Glu antiserum showed that most labeled PATs were enriched with immunoreactivity (IR) for Glu. The Glu-IR PATs contained clear, round, synaptic vesicles and formed asymmetric synaptic contacts with somata or dendrites. They were frequently postsynaptic to, unlabeled axon terminals filled with a mixture of clear, round, oval, and flattened vesicles (p-endings), with symmetric synaptic junctions. The frequency of synapses onto somata or primary dendrites per Glu-IR PAT was higher in the Vdm than in either the Vp or Vc lamina III/IV. The frequency of contacts of the p-endings per Glu-IR PAT was higher in the Vp than in the Vdm and Vc lamina III/IV. Sections immunostained with GABA antiserum showed that most axon terminals presynaptic to PATs were enriched with GABA in the three nuclei. The GABA-IR axon terminals and their postsynaptic PATs had a similar ultrastructural character to p-endings and their postsynaptic Glu-IR PATs, respectively. The present study suggests that primary afferent neurons with large-caliber fibers use glutamate as a neurotransmitter and are subject to presynaptic modulation by GABAergic fibers.     

Afferent synaptic contacts on glycine-immunoreactive neurons in the rat cuneate nucleus

This study was aimed to clarify whether the primary afferent terminals (PATs), GABAergic terminals, and glutamatergic terminals made direct synaptic contacts with glycine-IR neurons in the cuneate nucleus of rats. In this connection, injection of the anterograde tracer WGA-HRP into brachial plexus, antiglycine preembedding immunoperoxidase, and anti-GABA, along with antiglutamate postembedding immunogold labeling, were used to identify the PATs, glycine-IR neurons, GABA-IR terminals, and glutamate-IR terminals, respectively. The present results showed that HRP-labeled PATs, immunoperoxidase-labeled glycine-IR terminals, immunogold-labeled GABA-IR, and glutamate-IR terminals made axodendritic synaptic contacts with immunoperoxidase-labeled glycine-IR neurons. The latter three presynaptic elements also formed axosomatic synapses with glycine-IR neurons. Statistical analysis has shown that the minimum diameter of the glycine-IR dendrites postsynaptic to the above-mentioned four presynaptic elements did not differ significantly. In addition, the synaptic ratio of the glutamate-IR terminals on the glycine-IR dendrites was higher than that of GABA-IR terminals. The synaptic ratio of the GABA-IR terminals on glycine-IR dendrite was in turn higher than that of the PATs and glycine-IR terminals. It is suggested that the PATs and glutamate-IR terminals on the glycine-IR neurons may be involved in subsequent postsynaptic inhibition for spatial precision of lateral inhibition. On the other hand, the GABA-IR and glycine-IR terminals which make synaptic contacts with the dendrites of glycine-IR neurons may provide a putative means for disinhibition or facilitation to maintain the baseline neuronal activity in the rat cuneate nucleus. The results of quantitative analysis suggest that glutamate act as the primary excitatory neurotransmitter, while GABA, when compared with glycine, may serve as a more powerful inhibitory neurotransmitter on glycine-IR neurons in the rat cuneate nucleus.     

Retinal synapses of the cat medial interlaminar nucleus and ventral lateral geniculate nucleus differ in size and synaptic organization

The retinal terminals of the medial interlaminar nucleus (MIN) and ventral lateral geniculate nucleus (VLG) have been examined quantitatively to determine if there are morphological differences in their synaptic ultrastructure which reflect their distinctive physiologies. The cross-sectional area and density (number per unit area) of synaptic contact zones with conventional and presynaptic dendrites (F2 profiles) were measured for each retinal terminal. The densities of F2 presynaptic dendrites and F1 flattened vesicle axon terminals were also measured. Retinal terminals in MIN were often large (mean size= 2.7 μm² area) and had a high density of synaptic contacts (0.14 per μm surface area) with conventional dendrites, presynaptic dendrites, and dendritic spines. A high density of F2 presynaptic dendrites (0.08 per μm² area) was found in MIN. F1 axon terminals were also found frequently (0.04 per μm²). MIN retinal terminals were often organized in glomeruli like those of the dorsal lateral geniculate nucleus. The retinal terminals in VLG were almost always small (mean size= 0.94 μm² area), although they also had a high density of synaptic contacts (0.17 per μm surface area). They frequently synapsed on small dendrites and dendritic spines and less frequently on large dendrites. Unlike MIN, retinal terminals in VLG rarely contacted F2 presynaptic dendrites which were much less frequent in VLG (0.01 per μm² area). Like MIN, VLG contained numerous F1 axon terminals (0.06 per μm² area). No typical retinal glomeruli were found in VLG. These results show that MIN, which contains many Y cells, has a population of large retinal terminals and many F2 presynaptic dendrites. VLG, which apparently has only W cells, contains only small retinal terminals and has fewer F2 presynaptic dendrites. Both have a high density of F1 flat vesicle axon terminals.     

Postsynaptic dorsal column pathway of the rat. III. Distribution of ascending afferent fibers

The distribution in the dorsal column nuclei (DCn) of post-synaptic dorsal column (PSDC) fibers was examined in rats following injections of Phaseolus vulgaris leucoagglutinin (PHA-L) in the spinal cord. Lemniscal neurons in the DCn were retrogradely labeled in the same animals by injecting the thalamus with Fluoro-Gold. In some experiments, primary afferent fibers were also labeled by injecting dorsal root ganglia with choleragenoid-conjugated HRP. Injections of PHA-L into the cervical enlargement labeled many fibers and varicosities throughout most of the ipsilateral cuneate nucleus. Labeled fibers were also present in the external cuneate and internal basilar nuclei. Injections of PHA-L into thoracic cord labeled fibers and varicosities in the medial cuneate and lateral gracile nuclei, as well as the external cuneate nucleus. Injections into the lumbar enlargement labeled fibers and varicosities throughout most of the gracile nucleus. Injections in sacral cord labeled fibers in the most medial part of the gracile nucleus. Dense labeling of PSDC fibers was found in areas with high densities of retrogradely labeled lemniscal neurons and areas with high densities of primary afferent fibers. In all regions of the DCn and in the external cuneate nucleus, fibers and varicosities labeled for PHA-L were seen in apposition to retrogradely labeled lemniscal cells. The distribution of postsynaptic afferent fibers in the DCn of the rat and its relationship to lemniscal neurons and primary afferent fibers contrast sharply with these features in cats.     

Synaptic relationship of the neurons containing a metabotropic glutamate receptor, MGluR5, with nociceptive primary afferent and GABAergic terminals in rat spinal superficial laminae

Recent pharmacological evidence showed that metabotropic glutamate receptors (mGluRs), particularly mGluRs1/5, had a potential role in spinal nociceptive processing. However, previous morphological studies on mGluRs have been limited mainly to their distribution in the spinal cord. In the present study, electron microscopic immunocytochemistry was employed to identify the synaptic relationship of the neurons containing mGluR5, with nociceptive primary afferent and gamma-aminobutyric acid-ergic (GABAergic) terminals in the superficial dorsal horn of the spinal cord. Nociceptive C- and A(delta)-primary afferent terminals selectively labeled with horseradish peroxidase conjugated to wheat-germ agglutinin were in asymmetric synaptic contacts with or in direct apposition to mGluR5 positive dendritic profiles. The double-labeling studies revealed that mGluR5 immunoreactive dendrites also received symmetric synaptic contacts from axon terminals labeled with immunogold particles indicating GABA. The present demonstration of mGluR5 neurons receiving inputs from both nociceptive primary afferents and GABAergic terminals of presumed interneurons further supports the involvement of mGluR5 in the transmission and modulation of nociceptive information in the spinal cord.     

Pallidal afferent territory of the Macaca mulatta thalamus: Neuronal and synaptic organization of the VAdc

Ventral anterior thalamic nucleus pars densicellularis (VAdc) as delineated earlier (Ilinsky and Kultas-Ilinsky [1987] J. Comp. Neurol. 262:331–364) was analyzed by using qualitative and quantitative neuroanatomical techniques. Projection neurons (PN), retrogradely labeled with wheat germ agglutinin conjugated horseradish peroxidase from the cortex, were small to medium in size (mean area, 312 μm²) with numerous primary dendrites displaying a tufted branching pattern. Local circuit neurons (LCN), immunoreactive for gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase, were small (mean area, 110 μm²), and gave off few dendrites. Two subpopulations of GABA positive boutons (F1 type) were distinguished: large (mean area, 2.6 μm²) terminals with symmetric synapses containing few pleomorphic vesicles and numerous mitochondria densely covered proximal PN sites; smaller F1 boutons with a slightly different morphology contacted mostly distal PN dendrites. Two subpopulations of terminals containing round vesicles and forming asymmetric synapses were distinguished by bouton size (mean areas, 0.4 μm² and 1.6 μm², respectively). These targeted mainly distal PN dendrites, but some synapsed proximally next to large F1 boutons. On distal dendrites, representatives of both types were labeled from the cortex. The density of boutons with symmetric and asymmetric synapses (the number of boutons per 100 μm of PN membrane length) was 3.3:0.2 on primary, 2.5:1.2 on secondary, and 0.8:12 on distal dendrites. The numerical density of synapses formed by presynaptic LCN dendrites on all PN levels was 20 to 40 times less than that of axon terminals at the same sites. Afferent input to LCN from boutons of all types, including that from 50% of labeled cortical boutons, mainly targeted distal dendrites. Overall, the findings suggest that PN in VAdc receive massive inhibitory input proximally intermingled with some presumably excitatory input, and that LCN contribution to PN inhibition is modest. J. Comp. Neurol. 386:573–600, 1997. © 1997 Wiley-Liss, Inc.     

Ultrastructural analysis of glutamate‐, GABA‐, and glycine‐immunopositive boutons from supratrigeminal premotoneurons in the rat trigeminal motor nucleus

The supratrigeminal region (Vsup) is important for coordination of smooth jaw movement. However, little is known about the synaptic connections of the Vsup premotoneurons with the trigeminal motor neurons. In the present study, we examined axon terminals of Vsup premotoneurons in the contralateral trigeminal motor nucleus (Vmo) by a combination of anterograde tracing with cholera toxin B–horseradish peroxidase (CTB‐HRP), postembedding immunohistochemistry for the amino acid transmitters glutamate, GABA, and glycine, and electron microscopy. Tracer injections resulted in anterograde labeling of axon terminals of the Vsup premotoneurons in the motor trigeminal nucleus (Vmo). The labeled boutons in Vmo exhibited immunoreactivity for glutamate, GABA, or glycine: glutamate‐immunopositive boutons (69%) were more frequently observed than GABA‐ or glycine‐immunopositive boutons (19% and 12%, respectively). Although most labeled boutons (97%) made synaptic contacts with a single postsynaptic dendrite, a few glutamate‐immunopositive boutons (3%) showed synaptic contact with two dendrites. No labeled boutons participated in axoaxonic synaptic contacts. Most labeled boutons (78%) were presynaptic to dendritic shafts, and the remaining 22% were presynaptic to somata or primary dendrites. A large proportion of GABA‐ or glycine‐immunopositive boutons (40%) were presynaptic to somata or primary dendrites, whereas most glutamate‐immunopositive boutons (86%) were presynaptic to dendritic shafts. These results indicate that axon terminals of Vsup premotoneurons show simple synaptic connection with Vmo neurons. This may provide the anatomical basis for the neural information processing responsible for jaw movement control. © 2008 Wiley‐Liss, Inc.     

Brainstem projection of the vestibular nerve in the guinea pig: An HRP (horseradish peroxidase) and WGA-HRP (wheat germ agglutinin-HRP) study

We explored the course and termination of primary vestibular afferent fibers within the brainstem of the guinea pig by means of anterograde axonal transport of horseradish peroxidase (WGA-HRP). Primary vestibular afferent fibers distribute within the entire vestibular nuclear complex, with the exception of the dorsal part of the lateral vestibular nucleus. The superior vestibular nucleus is characterized by the concentration of terminals within its central part. Although terminal labeling is weaker within the periphery, no area completely lacks primary input. The lateral vestibular nucleus can be divided into a ventral and a dorsal part; within the ventral part small and giant cells receive primary afferent fibers, whereas no significant terminal labeling occurs in the dorsal part. The medial vestibular nucleus shows the most uniform labeling, although the lateral part of its rostral third has a few more terminals than the medial half. Primary projection to the descending vestibular nucleus is widespread, although in its rostrodorsal part it is less impressive. Of the small cell groups commonly associated with the vestibular nuclear complex, only group y receives abundant primary input. Whereas group z completely lacks labeled fibers as well as terminals, single primary axons can be observed passing groups x and f. However, no terminals can be found within the borders of these two cell groups. Scanty projections can be detected within the prepositus hypoglossi nucleus, as well as within the external cuneate nucleus, the cochlear nucleus, the abducent nucleus, and parts of the reticular formation.     

GABA and glycine-like immunoreactivity at axoaxonic synapses on 1a muscle afferent terminals in the spinal cord of the rat

The object of this study was to analyze the synaptic interactions of identified muscle spindle afferent axon terminals in the spinal cord of the rat. Group 1a muscle afferents supplying the gastrocnemius muscle were impaled with microelectrodes in the dorsal white matter of the spinal cord and stained by intracellular injection with Neurobiotin. Postembedding immunogold techniques were used to reveal GABA- and glycine-like immunoreactivity in boutons presynaptic to afferent terminals in the ventral horn and the deep layers of the dorsal horn. Serial-section reconstruction was used to reveal the distribution of synaptic contacts of different types on the afferent terminals. The majority of afferent boutons received axoaxonic and made axodendritic or axosomatic synaptic contacts. In the ventral horn, 91% of boutons presynaptic to the afferent terminals were immunoreactive for GABA alone and 9% were immunoreactive for both GABA and glycine. The mean number of axo-axonic contacts received per terminal was 2.7, and the mean number of synaptic contacts at which the terminal was the presynaptic element was 1.4. In the deep layers of the dorsal horn, 58% of boutons presynaptic to afferent terminals were immunoreactive for GABA alone, 31% were immunoreactive for GABA and glycine, and 11% for glycine alone. The mean number of axoaxonic contacts received per afferent terminal in this region was 1.6 and the mean number of synaptic contacts at which the terminal was the presynaptic element was 0.86. This clearly establishes the principle that activity in 1a afferents is modulated by several neurochemically distinct populations of presynaptic neuron.     

Quantitative analysis of immunogold labeling indicates low levels and non-vesicular localization of L-aspartate in rat primary afferent terminals

The role of L-aspartate as an excitatory neurotransmitter in primary afferent synapses in the spinal cord dorsal horn is disputed. To further investigate this issue, we examined the presence of aspartate-like immunoreactivity in primary afferent nerve terminals and other tissue components of the dorsal horn. We also examined the relationship between aspartate and glutamate immunogold labeling density and the density of synaptic vesicles in primary afferent terminals and presumed inhibitory terminals forming symmetric synapses. Weak aspartate immunosignals, similar to or lower than those displayed by presumed inhibitory terminals, were detected in both C-fiber primary afferent terminals in lamina II (dense sinusoid axon terminals, identified by morphological criteria) and in A-fiber primary afferent terminals in laminae III-IV (identified with anterograde transport of choleragenoid-horseradish peroxidase conjugate). The aspartate immunogold signal in primary afferent terminals was only about one-fourth of that in deep dorsal horn neuronal cell bodies. Further, whereas significant positive correlations were evident between synaptic vesicle density and glutamate immunogold labeling density in both A- and C-fiber primary afferent terminals, none of the examined terminal populations displayed a significant correlation between synaptic vesicle density and aspartate immunogold labeling density. Thus, our results indicate relatively low levels and a non-vesicular localization of aspartate in primary afferent terminals. It is therefore suggested that aspartate, rather than being a primary afferent neurotransmitter, serves a role in the intermediary metabolism in primary afferent terminals.     

The time course of functional alterations in degenerating dorsal column afferents to lateral cuneate nucleus

Unilateral dorsal funiculotomies at the C5 level were done in 13 adult cats. At 24-hr intervals from 24 to 120 hr after funiculotomy the function of the dorsal column afferents to the lateral cuneate nucleus was studied. Synaptic function was compromised by 24 hr and essentially absent by 48 hr. Cortically induced primary afferent depolarization in the dorsal column terminals in lateral cuneate nucleus was present at 24 hr but could not be detected at 48 hr or later. Axonal conduction persisted for 48 hr but was not detected at 72 hr. These results are compared to the development of deafferentation hyperactivity in lateral cuneate nucleus neurons and to the sequence of fine structural alterations in the lateral cuneate nucleus following dorsal rhizotomy.     

Glutamatergic inputs from the pedunculopontine nucleus to midbrain dopaminergic neurons in primates: Phaseolus vulgaris-leucoagglutinin anterograde labeling combined with postembedding glutamate and GABA immunohistochemistry

To verify the possibility that the pedunculopontine nucleus is a source of glutamatergic terminals in contact with midbrain dopaminergic neurons in the squirrel monkey, we used the anterograde transport of Phaseolus vulgaris-leucoagglutinin in combination with preembedding immunohistochemistry for tyrosine hydroxylase and for calbindin D-28k and postembedding immunocytochemistry for glutamate and for γ-aminobutyric acid. Following tracer injections in the pedunculopontine nucleus, numerous anterogradely labeled fibers emerged from the injection sites to innervate densely the pars compacta of the substantia nigra and ventral tegmental area. The major type of labeled fibers were thin with multiple collaterals and varicosities that established intimate contacts with midbrain dopaminergic neurons. At the electron microscopic level, the anterogradely labeled boutons were medium sized (maximum diameter between 0.9 μm and 2.5 μm) and contained numerous round vesicles and mitochondria. Postembedding immunocytochemistry revealed that 40–60% of anterogradely labeled terminals were enriched in glutamate and formed asymmetric synapses with dendritic shafts of substantia nigra and ventral tegmental area neurons. In triple-immunostained sections, some of the postsynaptic targets to these terminals were found to be dopaminergic. In addition, 30–40% of the anterogradely labeled terminals in both regions displayed immunoreactivity for γ-aminobutyric acid and, in some cases, formed symmetric synapses with dendritic shafts. In conclusion, our results provide the first ultrastructural evidence for the existence of synaptic contacts between glutamate-enriched terminals from the pedunculopontine nucleus and midbrain dopaminergic neurons in primates. Our results also show that the pedunculopontine nucleus is a potential source of γ-aminobutyric acid input to this region. These findings suggest that the pedunculopontine nucleus may play an important role in the modulation of the activity of midbrain dopaminergic cells by releasing glutamate or γ-aminobutyric acid as neurotransmitter. © 1996 Wiley-Liss, Inc.     

The release of amino acids with proposed neurotransmitter function from the cuneate and gracile nuclei of the rat in vivo

The release of both exogenous and endogenous amino acid neurotransmitter-suspects from the superfused rat dorsal column nuclei preparation was investigated. Stimulation of dorsal column tract fibres produced a significant increase in release of [¹⁴C]glutamate and [³H]GABA, when compared with spontaneous efflux and which was calcium-dependent. Corresponding increases in the release of endogenous glutamate and GABA occured during this type of stimulation. Stimulation of the medial lemniscus, which produces inhibition of cuneothalamic relay cells, resulted in a significant increase in release of both GABA and glycine. Superfusion with40mM K⁺ resulted in an increased release of all amino acids tested. The results are discussed in relation to current knowledge of amino acid transmitter release and the neuropharmacology of cuneate synaptic transmission.     

Ultrastructural quantitative analysis of glutamatergic and GABAergic synaptic terminals in the phrenic nucleus after spinal cord injury

Quantitative analysis of electron microscopic postembedding immunochemically stained material indicates that 48% of all terminals in the rat phrenic nucleus are glutamatergic and 33% are γ-aminobutyric acid (GABA) ergic. Three distinct types of glutamatergic terminals were observed in the rat phrenic nucleus: terminals characterized by large, loosely arranged spherical synaptic vesicles (SI) or small, compact spherical synaptic vesicles (Ss) and elongated terminals containing spherical synaptic vesicles with neurofilaments (NFs). All three types of glutamatergic terminals display asymmetrical synaptic membrane densities with postsynaptic dense bodies being present in some of the S-type terminals. The GABAergic immunoreactive terminals in the phrenic nucleus most closely resemble F-type terminals. They are characterized by flattened or pleomorphic synaptic vesicles and symmetric synaptic membrane densities. Among the 48% glutamatergic terminals, 27% are SI, 65% are Ss, and 8% are NFs, respectively. Significantly fewer glutamate, GABA, and unlabeled terminals per unit area are present in the phrenic nucleus 30 days after a C2 spinal cord hemisection as compared to nonhemisected controls. The average number of active zones per terminal, however, is greater in the hemisection group (1.45 ± 0.03) than in the control group (1.34 ± 0.03), with the active zones in the glutamate terminals mainly accounting for this difference. Moreover, the length of the active zones in the glutamate terminals was significantly longer in the hemisection group (0.37 ± 0.013 μm) as compared to the controls (0.24 ± 0.008 μm). In addition, the mean length of synaptic active zones in GABAergic terminals was also found to be longer in the hemisection group (0.36 ± 0.022 μm) as compared to controls (0.28 ± 0.014 μm). Finally, there is also a significantly higher ratio of synaptic active zones to the total number of glutamate-labeled terminals after injury (1.73 ± 0.08) as compared to controls (1.41 ± 0.04). The number of double/multiple synapses, the percentages of Sl, Ss, and NFs-type terminals, and the percentages of synaptic active zones contacting either distal dendrites or proximal dendrites/somata do not change significantly 30 days after injury. These results are important for a more complete understanding of the synaptic plasticity that occurs in the phrenic nucleus after spinal cord injury and to show how the plasticity may relate to the unmasking of latent bulbospinal respiratory connections which restore function to the hemidiaphragm paralyzed by an ipsilateral spinal cord hemisection. © 1996 Wiley-Liss, Inc.     

Primary afferent terminal sprouting after a cervical dorsal rootlet section in the macaque monkey

We examined the role of primary afferent neurons in the somatosensory cortical "reactivation" that occurs after a localized cervical dorsal root lesion (Darian-Smith and Brown [2000] Nat. Neurosci. 3:476-481). After section of the dorsal rootlets that enervate the macaque's thumb and index finger (segments C6-C8), the cortical representation of these digits was initially silenced but then re-emerged for these same digits over 2-4 postlesion months. Cortical reactivation was accompanied by the emergence of physiologically detectable input from these same digits within dorsal rootlets bordering the lesion site. We investigated whether central axonal sprouting of primary afferents spared by the rhizotomy could mediate this cortical reactivation. The cortical representation of the hand was mapped electrophysiologically 15-25 weeks after the dorsal rootlet section to define this reactivation. Cholera toxin subunit B conjugated to horseradish peroxidase was then injected into the thumb and index finger pads bilaterally to label the central terminals of any neurons that innervated these digits. Primary afferent terminal proliferation was assessed in the spinal dorsal horn and cuneate nucleus at 7 days and 15-25 postlesion weeks. Labeled terminal bouton distributions were reconstructed and the "lesion" and control sides compared within each monkey. Distributions were significantly larger on the side of the lesion in the dorsal horn and cuneate nucleus at 15-25 weeks after the dorsal rootlet section, than those mapped only 7 days postlesion. Our results provide direct evidence for localized sprouting of spared (uninjured) primary afferent terminals in the dorsal horn and cuneate nucleus after a restricted dorsal root injury.     

Electron microscopy of immunoreactivity patterns for glutamate and γ-aminobutyric acid in synaptic glomeruli of the feline spinal trigeminal nucleus (subnucleus caudalis)

We studied the ultrastructure of the synaptic organization in the feline spinal trigeminal nucleus, emphasizing specific neurotransmitter patterns within lamina II of the pars caudalis/medullary dorsal horn. Normal adults were perfused, and Vibratome sections from pars caudalis were processed for electron microscopy. Ultrathin sections were reacted with antibodies for the excitatory neurotransmitter glutamate (Glu) and for the inhibitory neurotransmitter γ-aminobutyric acid (GABA) by using postembedding immunogold techniques. Both single- and double-labeled preparations were examined. Results with single labeling show that Glu-immunoreactive terminals have round synaptic vesicles and form asymmetric synaptic contacts onto dendrites. GABA-immunoreactive axon terminals and vesicle-containing dendrites have pleomorphic vesicles, and the axon terminals form symmetric contacts onto dendrites and other axons. Double labeling on a single section shows glomeruli with central Glu-immunoreactive terminals that are presynaptic to dendrites, including GABA⁺ vesicle-containing dendrites. These Glu⁺ terminals are also postsynaptic to GABA⁺ axon terminals, and these GABA-immunoreactive terminals may also be presynaptic to the GABA⁺ vesicle-containing dendrites. Quantitative analyses confirm the specificity of the Glu and GABA immunoreactivities seen in the various glomerular profiles. The results suggest that a subpopulation of Glu-immunoreactive primary afferents (excitatory) may be under the direct synaptic influence of a GABA-immunoreactive intrinsic pathway (inhibitory) by both presynaptic and postsynaptic mechanisms. © 1996 Wiley-Liss, Inc.     

Fine structure, synaptology and immunocytochemistry of large neurons in the rat dorsal cochlear nucleus connected to the inferior colliculus

Neurons in the rat dorsal cochlear nucleus that project to the inferior colliculus (pyramidal and giant) were retrograde labelled with wheat germ agglutinin conjugated to horseradish peroxydase. Both cell types showed a similar ultrastructural feature, particularly the rough endoplasmic reticulum was well developed and sometimes surrounded the nucleus. The synaptological profile was similar in pyramidal and giant cells. Axo-somatic terminals covered 40-70% of the perimeter of pyramidal cells and 35-60% of the perimeter of giant neurons. Giant neurons featured bipolar or multipolar shape and different orientation but they possessed a similar synaptic profile. Most axo-somatic terminals contained flat and pleomorphic synaptic vesicles, some pleomorphic vesicles. Few terminals contained round vesicles. These cells were consistently immuno-negative for both glycine and GABA and variably positive for glutamate. The immunoelectron microcopic study of thin sections showed that glycine immunoreactivity was constantly present in terminals enriched with flat vesicles, which often did not show GABA immunoreactivity. Few anterograde labelled boutons containing flat vesicles were in contact with the proximal dendrites and the cell bodies of pyramidal and giant neurons. The origin of these terminals is discussed. No other cells of the dorsal cochlear nucleus, in particular cartwheel and tuberculo-ventral neurons, were in contact with labelled boutons. The present results suggest that descending inhibitory collicular projections are essentially directed to the large excitatory neurons of the dorsal cochlear nucleus.     

Glutamate-like Immunoreactivity in Retinal Terminals of the Mouse Suprachiasmatic Nucleus

With a view to identifying the neurotransmitter content of retinal terminals within the mouse suprachiasmatic nucleus, a highly specific antiserum to glutaraldehyde-coupled glutamate was used in a postembedding immunogold procedure at the ultrastructural level. Retinal terminals were identified by cholera toxin–horseradish peroxidase transported anterogradely from the retina and reacted with tetramethyl benzidine/tungstate/H₂O₂, or by their characteristically pale and distended mitochondria with irregular cristae. Controls included model ultrathin sections containing high concentrations of various amino acids. Alternate serial sections were labelled with anti-glutamate and anti-γ-aminobutyric acid (GABA). Data were analysed by computer-assisted image analysis. Density of glutamate labelling (gold particles per μm²) on whole retinal terminals was > 3 times higher than that on postsynaptic dendrites, and > 5 times higher than that on miscellaneous non-retinal non-glutamatergic terminals in the suprachiasmatic nucleus. The overall density of gold particles over retinal terminals was ～ 3 times higher than that over GABAergic terminals, in which glutamate-like immunoreactivity was mainly mitochondrial. Labelling of vesicles in retinal terminals was almost 5 times greater than the apparent labelling of vesicles in GABAergic terminals, underscoring the location of transmitter glutamate within synaptic vesicles in retinal terminals. In the retino-recipient region of the suprachiasmatic nucleus there was also a small population of non-retinal glutamatergic terminals. Their overall immunoreactivity was similar to or exceeded that of retinal terminals, but morphological features clearly distinguished between these two types of glutamate-containing terminals. The present results indicate that the vast majority of retinal terminals may use glutamate as a transmitter, in keeping with electrophysiological and neuropharmacological data from other sources. The possibility of cotransmitters within retinal terminals, suggested by the presence of dense-core vesicles among the glutamate-containing synaptic vesicles, has still to be addressed.     

Electron microscopic analysis of lesion-induced changes in synaptic structure and immunogold labeling of neurotransmitters within the feline trigeminal nucleus

This report uses lesion and postembedding immunogold protocols to examine the ultrastructural details of lesion-induced synaptic and neurotransmitter changes in the feline trigeminal nucleus. Electron microscopic (EM) analysis concentrated on lamina II (substantia gelatinosa) of the subnucleus pars caudalis (PC) which is one relay site of trigeminal fibers involved in nociception. Special attention was directed to analysis of reoccupation of synaptic sites vacated by primary afferent degeneration. Primary afferents were caused to degenerate by performing unilateral retrogasserian rhizotomy. After survival times of 1, 2, 6, and 7 days, sections of PC were processed for postembedding immunogold labeling with antibodies to the neurotransmitters gamma aminobutyric acid (GABA) and glutamate (Glu). The results show: (1) degenerating primary afferent terminals were easily identified in various stages of degeneration; (2) Glu immunoreactivity was observed in early forms of degenerated primary afferent terminals with clumped vesicles as well as in the highly distorted, electron dense terminals of later degeneration; and (3) some GABA immunoreactive terminals formed atypical synapses which exhibited both asymmetric (excitatory) and symmetric (inhibitory) synaptic densities. A possible model is presented of the progression of events following trigeminal nerve lesion which results in atypical synapse formation. Such altered synaptic relationships seen in PC following trigeminal rhizotomy may be related to hyperactivity that is seen in animals and to the atypical facial pain following nerve lesions in humans. © 1996 Wiley-Liss, Inc.