The distribution of inhibitory and excitatory synapses on single, reconstructed jaw-opening motoneurons in the cat

In a previous study, we reported that the distribution of inhibitory input, in contrast to excitatory input, decreased somatofugally along dendrites of cat jaw-closing alpha-motoneurons [J Comp Neurol 414 (1999) 454]. The present study examined the distribution of GABA, glycine, and glutamate immunopositive boutons covering horseradish peroxidase-labeled cat jaw-opening motoneurons. The motoneurons were divided into four compartments: the soma, and primary, intermediate, and distal dendrites. Ninety-seven percent of the total number of studied boutons had immunoreactivity for at least one of the three amino acids. The proportion of boutons immunoreactive for GABA and/or glycine was lower than the proportion of boutons immunoreactive for glutamate. Boutons immunoreactive to glycine alone were more numerous than boutons double-labeled for GABA and glycine, which, in turn, occurred more frequently than boutons immunoreactive to GABA alone. The percentage synaptic covering (proportion of membrane covered by synaptic boutons) of the putatively excitatory (glutamate containing) and putatively inhibitory (GABA and/or glycine containing) boutons decreased somatofugally along the dendrites. Such systematic variations were not seen in the packing density (number of boutons per 100 microm(2)); the packing density showed a distinct drop between the soma and primary dendrites but did not differ significantly among the three dendritic compartments. Overall, the packing density was slightly higher for the putatively excitatory boutons than for the inhibitory ones. When taken together with previous analyses of jaw-closing alpha-motoneurons the present data on jaw-opening alpha-motoneurons indicate that the two types of neuron differ in regard to the nature of synaptic integration in the dendritic tree.     

Changes in synaptology of adult cat spinal α-motoneurons after axotomy

The aim of this electron-microscopic study was to analyze the distribution of synaptic contacts on the cell bodies and dendrites of permanently axotomized adult cat spinal α-motoneurons. Following transection and ligation of the medial gastrocnemius nerve, the synaptic covering of the cell bodies and three different dendritic compartments of homonymous α-motoneurons was analyzed quantitatively at 3, 6, and 12 weeks postoperatively. The synaptic boutons were classified according to their size and the shape of their synaptic vesicles. On the soma, a transient increase in the number of boutons was noted at 3 weeks and 6 weeks postoperatively, while after 12 weeks the bouton number had decreased to half of its normal value. The transient increase was mainly due to an increase in the number of F-type boutons. At 12 weeks postoperatively, the synaptic covering was reduced by 83% on the soma and by 57% on the proximal dendrites. In the distal dendritic regions, the values for synaptic covering remained largely unchanged. In summary, axotomized motoneurons exhibit a reduction in synaptic covering which is maximal on the cell body and becomes less pronounced centrifugally along the dendrites. However, if also taking into account the loss of distal dendritic branches that occurs in axotomized motoneurons, the total loss of boutons is several times larger in the dendrites than on the soma. Received: 18 October 1996 / Accepted: 13 June 1997     

GABA and glycine in synaptic microcircuits associated with physiologically characterized primary afferents of cat trigeminal principal nucleus

Previous studies suggest that sensory information conveyed through trigeminal afferents is more strongly controlled at the level of the first synapse by GABA-mediated presynaptic mechanisms in the trigeminal principal sensory nucleus (Vp) than other sensory nuclei. However, it is unknown if such a mechanism is common to functionally different classes of primary afferent in the same nucleus or across the nuclei. To address these issues, the present study focused on synaptic microcircuits associated with slowly adapting (SA) mechanosensory afferents innervating the periodontal ligaments in the cat Vp and attempted to examine GABA, glycine, and glutamate immunoreactivity in axon terminals involved in the circuits. Afferents were physiologically characterized before injection of horseradish peroxidase (HRP) and preparation for electron microscopy. HRP-labeled afferent boutons were serially sectioned and immunostained with antibodies against GABA, glycine, and glutamate using a postembedding immunogold method. All the afferent boutons examined contacted non-primary dendrites and they were frequently postsynaptic to unlabeled axons (p-endings). Axodendritic and axoaxonic contacts per afferent bouton were 1.3 (46/35) and 2.0 (70/35), respectively. Most p-endings were immunoreactive for GABA (63/70) and also glycine was co-stained in the majority of the p-endings (49/63). Thirty percent of p-endings with the colocalization of GABA and glycine participated in synaptic triads where a p-ending formed a synapse with the same dendrite as the afferent bouton. None of the p-endings was immunoreactive for glutamate. Most afferent boutons were enriched with glutamate but were immunonegative for GABA and glycine. This study provides evidence suggesting that transmission from SA afferents is strongly controlled presynaptically by GABAergic interneurons with colocalized glycine, and that a proportion of these interneurons, involved in synaptic triads, may also have postsynaptic inhibitory actions on target neurons of the SA afferents.     

Colocalization of neurotransmitters in presynaptic boutons of inhibitory synapses in the lamprey spinal cord

Electron-microscopic immunocytochemical studies were performed to detect GABA and glycine immunoreactivity in presynaptic axon terminals in the central gray matter of the spinal cord of the lampreyLampetra fluviatilis. The immunopositive presynaptic terminals contacting identified dendrites of motoneurons and unidentified postsynaptic profiles included terminals immunopositive for GABA only (44%) and glycine only (26%), as well as terminals containing GABA and glycine (30%). Glycine-immunopositive presynaptic terminals contained flattened synaptic vesicles. Large synaptic vesicles with dense cores were present along with classical synaptic vesicles in 74% of GABA-immunopositive boutons. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 85, No. 4, pp. 515–522, April, 1999.     

GABA-, glycine-, and glutamate-immunoreactive bouton profiles in apposition to neurons of the central cervical nucleus in the rat

The neurons of the central cervical nucleus (CCN) convey information about the position and movements of the head, and receive excitatory input from dorsal neck muscles and the labyrinth. Both of these afferent sources form glutamatergic synaptic contacts with CCN neurons. However, these sensory afferent sources can also inhibit CCN neurons. To further elucidate the synaptic organization, we made an electron microscopic investigation, identifying and evaluating the relative frequency of bouton profiles containing the inhibitory transmitters GABA and glycine in apposition to identified CCN neurons. In addition, labeling for glutamate was performed. The identification of the CCN neurons was made possible by injections of retrograde tracer substances into the cerebellum. These substances were made visible by preembedding immunocytochemistry or postembedding immunogold staining. Such staining was also used to detect the three amino acids that were found in boutons apposed to the identified neurons (cf. Ornung et al., J. Comp. Neurol. 1996;365:413-426; Lind? et al., J. Comp. Neurol. 2000;425:10-23). Due to the relatively poor transport of the tracer substances into dendrites of the CCN neurons, the analysis was restricted to the cell body and included bouton profiles in direct apposition to the soma membrane. Data from 10 CCN neurons revealed that about 50% of the apposing bouton profiles were immunoreactive for GABA, and about 34% for glycine. In four neurons, the degree of colocalization of GABA and glycine was determined to be close to 30%. Thus, the vast majority of glycine-labeled profiles also contained GABA, while a considerable fraction of the profiles were immunoreactive for only GABA. The values for glycine immunoreactive bouton profiles presented here may represent somewhat low estimates, depending on the method used. Data from four neurons showed that about 18% of the profiles were labeled for glutamate. The large fraction of purely GABA immunoreactive profiles, or at least a substantial group of them, is suggestive of their derivation from axons descending from the brainstem.     

Enkephalin-, thyrotropin-releasing hormone- and substance P-immunoreactive axonal innervation of the ventrolateral dendritic bundle in the cat sacral spinal cord: An ultrastructural study

The distribution and synaptic arrangement of thyrotropin-releasing hormone-, substance P- and enkephalin-immunoreactive axonal boutons have been studied in the ventrolateral nucleus (Onuf's nucleus) of the upper sacral spinal cord segments in the cat. For this purpose, the peroxidase-antiperoxidase immunohistochemical technique was used. Immunoreactive axonal boutons were traced in complete series of sections in order to reveal synaptic contacts with the bundled dendrites of the ventrolateral nucleus. As judged from the cross-sectional diameter of the postsynaptic dendrites, the distribution of immunoreactive boutons was non-random. Enkephalin-immunoreactive axonal boutons, presumed to be mostly of segmental origin, displayed a rather restricted distribution to mainly (> 80%) medium-to-large dendrites. Thyrotropin-releasing hormone-immunoreactive boutons, that derive from supraspinal levels, were also found to impinge on medium-to-large dendrites (> 80%), indicating a proximal location within the dendritic trees. The skewness toward large postsynaptic dendrites was even more marked for thyrotropin-releasing hormone- than for enkephalin-immunoreactive boutons. Substance P-immunoreactive boutons, that are of either supraspinal or spinal origin, showed a more even distribution throughout the dendritic trees, including both thin distal branches and thick proximal dendrites. In view of the well-known fact that virtually all thyrotropin-releasing hormone-immunoreactive boutons in the ventral horn cocontain substance P (and serotonin) it was assumed that substance P-immunoreactive boutons in synaptic contact with the finest-calibre dendrites as well as most of those with a very proximal juxtasomatic location on the dendritic trees were of segmental origin, while those impinging on medium-to-large dendrites could be of either spinal or supraspinal origin. Fine-calibre dendrites (< 1 μm) represent about 25% of the dendritic branches in the ventrolateral nucleus, but receive, with the exception of substance P (8%), very little (< 3%) peptidergic or GABAergic (Ramírez-León and Ulfhake, 1993) input, although the degree of dendritic membrane covering by bouton profiles in the ventrolateral nucleus does not seem to vary much with the calibre of the postsynaptic dendrite (Ramírez-León and Ulfhake, 1993). Both substance P- and enkephalin-immunoreactive axonal boutons established synaptic contact with more than one dendrite. Furthermore, one and the same bouton could be found in contact with two dendrites that were coupled to each other by a dendro-dendritic contact of desmosomal or puncta adherentia type. This synaptic arrangement was, however, not seen among thyrotropin-releasing hormone-immunoreactive boutons, indicating that these axonal boutons act on a single postsynaptic element, while inputs intrinsic to the spinal cord can show a divergence also at the terminal level.     

GABA-like immunoreactive innervation and dendro-dendritic contacts in the ventrolateral dendritic bundle in the cat S1 spinal cord segment: an electron microscopic study

The motoneurons (MNs) in the ventrolateral nucleus (VLN) of the upper sacral spinal cord segments in the cat supply the external sphincters and the ischiocavernosii muscles. The dendrites of the MNs in the VLN are arranged into rostro-caudally oriented bundles (ventrolateral dendritic bundle, VLB). In this study we describe the distribution and synaptic arrangement of -aminobutyric acid-immunoreactive (GABA-IR) axonal bouton profiles innervating the VLB. This was accomplished using the peroxidase-antiperoxidase technique and a polyclonal antibody raised against glutaraldehydeconjugated GABA.The VLN receives an extensive innervation of GABAIR axonal bouton profiles that surround both cell bodies and dendrites. Twenty-five per cent of the total number of vesicle-containing axonal profiles in the VLN neuropil were estimated to be GABA-IR. On cell bodies in the -motoneuron size-range, the membrane covering of GABA-IR bouton profiles was about 18% and they constituted about 29% of the total membrane covering of axonal bouton profiles. Quantitative analysis of GABAIR bouton profiles on dendrites revealed membrane covering figures rather similar to those on the cell bodies. They were not randomly distributed within the dendritic arborisations. Instead, they were very infrequent (2.5% of the covering) on small calibre dendrites (< 1 m) as compared to larger dendrites (> 1 m, 14–18.5% of the covering), although the total membrane covering of axonal bouton profiles was rather similar for all dendrites (42–52%). The data on membrane covering by GABA-IR boutons presented here may be low estimates due to technical limitations, indicating that the GABAergic input to this region might be even more extensive.A frequent finding was that one and the same GABAIR bouton made synaptic contact with two to three adjacent dendrites. This type of synaptic arrangement among the VLN MNs indicates a divergence of the GABAergic input at the terminal level. In addition, the postsynaptic dendrites involved in such arrangements often disclosed dendro-dendritic contacts. In total, 44% of the bundled dendrites in the VLN disclosed direct dendro-dendritic contact regions. These contacts were most often of the puncta adherentia type, while desmosome-type contacts were less frequent. None of the dendro-dendritic contacts studied had the characteristics of a gap junction.Taken together, the present results indicate that GABA may be a transmitter substance in a large fraction of the synaptic input to the VLN MNs. Furthermore, the location of GABA-IR bouton profiles on the cell bodies and more proximal regions of the dendritic trees implies that they could exert a considerable influence on MN activity pattern.     

Immunocytochemical localization of amino acid neurotransmitter candidates in the ventral horn of the cat spinal cord: a light microscopic study

The distribution of immunoreactivities to six amino acids, possibly related to synaptic function, was investigated in the motor nucleus of the cat L7 spinal cord (laminae VII and IX) using a postembedding peroxidase-antiperoxidase technique. Consecutive 0.5 m transverse sections of plastic-embedded tissue were incubated with antisera raised against protein-glutaraldehyde conjugates of -aminobutyric acid (GABA), glycine, aspartate, glutamate, homocysteate, and taurine. This method allowed localization of the different immunoreactivities in individual cell profiles. The results showed that all these amino acids, except homocysteate, could be clearly detected in either neuronal or glial elements in the ventral horn. In cell bodies of neurons in lamina VII, immunoreactivity was observed for aspartate, glutamate, GABA, and glycine. Adjacent section analysis revealed that combinations of immunoreactivity for glycine/glutamate/aspartate, GABA/glycine/glutamate/aspartate and glutamate/aspartate, respectively, may occur in one and the same cell. In the motor nuclei (lamina IX), immunoreactivity to amino acids was observed in two types of neuron. Large cells, probably representing -motoneurons, were harboring immunoreactivity to both glutamate and aspartate, while a few small neurons in this area displayed a colocalization of glycine, glutamate, and aspartate. Dendrites and axons in the motor nuclei cocontained glycine/glutamate/aspartate, GABA/glycine/glutamate/aspartate, and glutamate/aspartate immunoreactivities. In both laminae VII and IX, taurine-like immunoreactivity was absent in neuronal cell bodies, but highly concentrated in perivascular cells and small cells with a morphology resembling that of glial cells. A punctate immunolabeling, in all probability representing labeling of nerve terminals, could be demonstrated in the ventral horn for GABA, glycine, and glutamate, but not with certainty for aspartate or taurine. A quantitative estimate of the covering of cell bodies of -motoneuron size by immunoreactive puncta revealed that glycine immunoreactive terminal-like structures were most abundant (covering 26–42% of the somatic membrane), while glutamate immunoreactive terminals were seen least frequently (5–9% covering). GABA-immunoreactive terminals covered from 10 to 24% of the soma surface. A colocalization of GABA and glycine immunoreactivities in putative nerve terminals could be shown both in the neuropil and in close relation to cell bodies of motoneurons. These results suggest that among the studied amino acids probably only three, namely GABA, glycine, and glutamate, can be considered to be neurotransmitter candidates in the ventral horn of the cat spinal cord.     

Colocalization of neurotransmitters in the deep cerebellar nuclei

Summary An abundance of glycine and glycine receptor immunoreactivities was found in all three parts of the deep cerebellar nuclei. Glycine immunoreactivity was restricted to small neurons throughout most of the deep cerebellar nuclei except for a few large positive neurons in the ventral part of the fastigial nuclei. In addition, glycine immunoreactivity was found in boutons outlining somata of large glycine negative neurons. Complementary to the glycine positive boutons was an intense glycine receptor immunoreactivity on large deep cerebellar nuclei neurons. Comparisons of immunoreactivities for glycine, GABA and aspartate in consecutive one micron sections revealed that many small neurons colocalized glycine and GABA, while some large neurons in the fastigal region colocalized glycine and aspartate.Ultrastructural investigations revealed glycine receptors on postsynaptic sites of dendrites and somata. Most boutons, which were presynaptic to glycine receptor sites, were filled with small flattened vesicles; however, a small percentage of boutons had round clear or dense core vesicles. Frequently, each bouton apposed multiple active zones on the dendrite or soma. One of these active zones was positive for glycine receptor and another was negative.This study supports: (1) glycine as a neurotransmitter in deep cerebellar nuclei, and (2) glycine and GABA colocalization in the same cell and bouton, but releasing to different receptor sites on the target neuron. Furthermore, the coexistence of glycine with GABA in the same deep cerebellar neuron may play an important role in controlling the conset and duration of signal transmission.     

An ultrastructural study of 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive axonal boutons in the motor nucleus of spinal cord segments L7-S1 in the adult cat

The distribution and fine structure of 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive synaptic boutons and varicosities were studied in the motor nucleus of the spinal cord segments L7-S1 in the cat, using the peroxidase-antiperoxidase immunohistochemical technique and analysis of ultrathin serial sections. The 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive boutons had a similar ultrastructural appearance as judged from serial section analysis. The boutons could be classified into two types on the basis of their vesicular content, with one type containing a large number of small agranular vesicles together with only a few, if any large granular vesicles, while the other type contained a large number of large granular vesicles in addition to small agranular vesicles. The vesicles were spherical or spherical-to-pleomorphic. Postsynaptic dense bodies (Taxi bodies) were occasionally observed in relation to all three types of immunoreactive boutons, which almost invariably formed synaptic junctions with dendrites. Judged by the calibre of the postsynaptic dendrites, the boutons were preferentially distributed to the proximal dendritic domains of motoneurons. In one case, a substance P-immunoreactive bouton formed an axosomatic synaptic contact. In addition to synaptic boutons, 5-hydroxytryptamine-, thyrotropin-releasing hormone- and substance P-immunoreactive axonal varicosities containing a large number of large granular and small agranular vesicles but lacking any form of conventional synaptic contact were observed. Such varicosities were either directly apposing surrounding neuronal elements or separated from the neurons by thin glial processes. The origin of the immunoreactive boutons was not traced, but it was thought likely that the main source of the boutons was neurons with their cell bodies located in the medullary raphe nuclei.     

Recovery of synapses in axotomized adult cat spinal motoneurons after reinnervation into muscle

 Peripheral axotomy of adult cat spinal motoneurons induces a marked loss of synaptic boutons from the cell bodies and dendritic trees. The aim of the present study was to analyze the recovery of synaptic contacts in axotomized motoneurons following reinnervation into muscle. Adult cat spinal motoneurons were first deprived of their muscular contacts for 12 weeks and, then, allowed to reinnervate their target muscle. Two years later, regenerated motoneurons were labeled with horseradish peroxidase to allow quantitative ultrastructural analyses of the synaptic covering of the cell bodies and dendrites. Presynaptic boutons were classified according to their size and the shape of their synaptic vesicles. Results show that a recovery of synaptic covering occurs in the axotomized neurons after muscle reinnervation, but it affects various bouton types to different degrees. The number of S-type boutons synapsing with the soma was 70% higher after reinnervation than at 12 weeks after axotomy, while the number of F-type boutons had increased by only 13%. Compared with the normal situation, the number of S-type boutons synapsing with the proximal dendrites increased from 82% at 12 weeks after axotomy to 180% in the reinnervated state. In conclusion, in adult cat spinal motoneurons, the reestablishment of muscular contact is followed by a normalization of some of the synaptological changes induced by a prolonged state of axotomy. In certain respects restitution is incomplete, but in others it results in overcompensation. Received: 10 December 1997 / Accepted: 30 July 1998     

GABA- and glycine-like immunoreactivity in axonal endings presynaptic to the vibrissa afferents in the cat trigeminal interpolar nucleus

The goal of this study was to analyze the synaptic interaction of primary afferents with GABA- and/or glycine-immunopositive presynaptic endings in the cat trigeminal interpolar nucleus (Vi). Fast adapting vibrissa afferents were labeled by intra-axonal injections of horseradish peroxidase. Postembedding immunogold labeling on serially cut ultrathin sections and quantitative ultrastructural analysis of the labeled boutons and their presynaptic endings (p-endings) in the Vi were performed. The majority of p-endings presynaptic to labeled boutons (83%) were immunopositive for both GABA and glycine and 8% were immunopositive for glycine alone. A small fraction of p-endings were immunopositive for GABA alone (4%) or immunonegative for both GABA and glycine (4%). Ultrastructural parameters related to synaptic release, i.e. bouton volume, mitochondrial volume, and active zone area, were significantly larger in the labeled boutons of primary afferents than in the p-endings. The volume of labeled boutons was positively correlated with the number of the postsynaptic dendrites and p-endings. In addition, fairly large-sized labeled boutons and p-endings were frequently observed in the Vi. These results reveal that large majority of vibrissa afferents in the Vi are presynaptically modulated by interneurons immunopositive for both GABA and glycine, and suggest that the Vi plays a distinct role in the processing of orofacial sensory information, different from that of other trigeminal sensory nuclei.     

Ultrastructure and synaptic connectivity of main and accessory olfactory bulb efferent projections terminating in the rat anterior piriform cortex and medial amygdala

Neurons in the main olfactory bulb relay peripheral odorant signals to the anterior piriform cortex (aPir), whereas neurons of the accessory olfactory bulb relay pheromone signals to the medial amygdala (MeA), suggesting that they belong to two functionally distinct systems. To help understand how odorant and pheromone signals are further processed in the brain, we investigated the synaptic connectivity of identified axon terminals of these neurons in layer Ia of the aPir and posterodorsal part of the MeA, using anterograde tracing with horseradish peroxidase, quantitative ultrastructural analysis of serial thin sections, and immunogold staining. All identified boutons contained round vesicles and some also contained many large dense core vesicles. The number of postsynaptic dendrites per labeled bouton was significantly higher in the aPir than in the MeA, suggesting higher synaptic divergence at a single bouton level. While a large fraction of identified boutons (29 %) in the aPir contacted 2–4 postsynaptic dendrites, only 7 % of the identified boutons in the MeA contacted multiple postsynaptic dendrites. In addition, the majority of the identified boutons in the aPir (95 %) contacted dendritic spines, whereas most identified boutons in the MeA (64 %) contacted dendritic shafts. Identified boutons and many of the postsynaptic dendrites showed glutamate immunoreactivity. These findings suggest that odorant and pheromone signals are processed differently in the brain centers of the main and accessory olfactory systems.     

Ultrastructural subtypes of glutamate-immunoreactive terminals on rat trigeminal motoneurones and their relationships with GABA-immunoreactive terminals

 Electron-microscopic immunolabelling methods were used to study the relationships between glutamate-immunoreactive and γ-aminobutyric acid (GABA)-immunoreactive synapses on trigeminal motoneurones labelled by the retrograde transport of horseradish peroxidase. Serial sections were cut through the motor nucleus, alternate sections were incubated with antibodies to glutamate and GABA, and the immunopositive nerve terminal profiles were recognized using a quantitative, postembedding immunogold method. Boutons exhibiting high levels of glutamate immunoreactivity and GABA-immunoreactive boutons both formed axo-dendritic and axo-somatic synaptic contacts on labelled motoneurones. Boutons strongly immunopositive for glutamate were not immunopositive for GABA, and vice versa. Strongly glutamate immunoreactive boutons received axo-axonic synaptic contacts but did not form such contacts, while GABA-immunoreactive boutons formed axo-axonic synapses but did not receive them. The presynaptic elements at all axo-axonic synapses on to glutamate-immunoreactive boutons sampled were GABA-immunopositive. These data provide ultrastructural evidence in support of the roles of glutamate and GABA as transmitters at synapses on trigeminal motoneurones, and for presynaptic control of transmission at glutamatergic synapses by GABA acting at receptors at axo-axonic synapses. The vast majority (more than 90%) of strongly glutamate immunoreactive boutons contained spherical synaptic vesicles, in contrast to GABA-immunoreactive boutons, which contained pleomorphic vesicles. Most of the glutamate-immunoreactive boutons (67%) formed asymmetrical synaptic active zones, many of which (47% of total) were associated with subsynaptic dense ”Taxi” bodies (T-terminals), while a smaller population of boutons (21%) formed symmetrical synapses, and a few (11%) made synapses associated with subsynaptic cisternae (C-terminals). The heterogeneity of active zone ultrastructure of boutons identified as being glutamatergic on the basis of their high levels of immunolabelling is discussed in relation to possible differences in co-transmitters released, origins of the synaptic input or post-synaptic receptor subtypes activated. Received: 13 May 1996 / Accepted: 9 September 1996     

Ultrastructure and immunocytochemical characteristics of cells in the octopus cell area of the rat cochlear nucleus: comparison with multipolar cells

Cells in the octopus cell area of the rat ventral cochlear nucleus have been connected to the monaural interpretation of spectral patterns of sound such as those derived from speech. This is possible by their fast onset of firing after each octopus cell and its dendrites have been contacted by many auditory fibres carrying different frequencies. The cytological characteristics that make these large cells able to perform such a function have been studied with ultrastructural immunocytochemistry for glycine, GABA and glutamate, and compared to that of other multipolar neurons of other regions of the ventral cochlear nucleus. Cells in the octopus cell area have an ultrastructure similar to large-giant D-multipolar neurons present in other areas of the cochlear nucleus, from which they differ by the presence of a larger excitatory axo-somatic synaptic input and larger mitochondria. Octopus cells are glycine and GABA negative, and glutamate positive with different degree. Large octopus cells receive more axo-somatic boutons than smaller octopus cells. Fusiform octopus cells are found sparsely within the intermediate acoustic striae. These cells are large to giant excitatory neurons (23-35 microm) with 62-85% of their irregular perimeter covered with large axo-somatic synaptic boutons. Most boutons contain round vesicles and are glycine and GABA negative but glutamate positive. The latter excitatory boutons represent about 70% of the input to octopus cells. Glycine positive boutons with flat and pleomorphic vesicles account for 9-10% of the input while GABA-ergic boutons with pleomorphic vesicles represent about 20% of the synaptic input. Other few, multipolar cells within the rat octopus cell area are surrounded by more inhibitory than excitatory terminals which contain flat and pleomorphic vesicles, a feature distinctive from that of true octopus cells. The latter resemble multipolar cells seen outside the octopus cell area that project to the contralateral inferior colliculus and cochlear nucleus. Based on this study, two types of large multipolar cells are present in the octopus cell area: 1) those that receive about 70% of axo-somatic R boutons and stain more intensely for glutamate may correspond to pure onset neurons (Oi); 2) those with less than 33% of R axosomatic boutons, with less immunoreactivity to glutamate and sometimes glycine positive may represent the onset chopper neurons (Oc). In the octopus cell area the first type appears more prevalent. The present study suggests that octopus cells are a special type of excitatory D-multipolar neuron confined to the octopus cell area and mainly innervated by glutamatergic cochlear nerve terminals.     

Ultrastructural observations on beaded α-motoneuron dendrites

Beaded dendrites of 1α-motoneurons intracellularly labelled with horseradish peroxidase (HRP) were studied ultrastructurally in eight adult cats. For comparison, adjacent unlabelled beaded dendrites of unknown origin were also included in the study. Electron microscopy revealed no signs of degeneration or poor fixation according to common criteria. With the exception of the HRP-reaction product no difference in structure was observed between labelled and unlabelled beaded dendrites. Both the beads and their interconnecting segments were postsynaptic to boutons of normal appearance containing spherical (S-type boutons) or flattened vesicles (F-type boutons). The values for synaptic covering and synaptic packing density of the beaded dendritic regions, which usually were located in the periphery of the dendritic trees, were clearly lower than values obtained previously for cell bodies and proximal dendrites of a-motoneurons.     

GABA- and glycine-immunoreactive terminals contacting motoneurons in lamprey spinal cord

Double postembedding GABA- and glycine-immunostaining was performed on the lamprey (Lampetra fluviatilis) spinal cord after previous HRP labeling of motoneurons. Immunopositive boutons contacting motoneurons were counted and distinguished as GABA (39%), glycine (30%) and both GABA+glycine-immunopositive (31%). Densely-packed, flattened synaptic vesicles were only observed in glycine-immunopositive boutons while GABA-immunoreactive and GABA+glycine-immunoreactive boutons contained rounded or oval synaptic vesicles. Dense-core vesicles of different diameters were associated with conventional synaptic vesicles in 74% of GABA-only-immunopositive boutons, 50% of double GABA+glycine-immunopositive boutons, but were only observed in 9% of glycine-only-immunopositive boutons. The presence of terminals immunoreactive to either GABA or glycine contacting the motoneurons suggests that there is a morphological substrate for both GABAergic and glycinergic postsynaptic inhibition of motoneurons in the lamprey spinal cord.     

The dendrites of single brain-stem motoneurons intracellularly labelled with horseradish peroxidase in the cat. An ultrastructural analysis of the synaptic covering and the microenvironment

Two laryngeal motoneurons intracellularly stained with horseradish peroxidase were studied ultrastructurally. The precise position of the ultrastructural observations made along the dendrites was obtained from the computer-reconstruction of the motoneurons in three dimensions. The shape and the size of the synaptic boutons, the percentage of membrane covered by bouton appositions and active zones, the number of boutons per 100 microns2 (packing density) were analysed on the soma and on the labelled dendrites at different distances from the soma up to 1000 microns. The results revealed no important regional differences in the mean length of synaptic apposition. The packing density was in the range of 9.3-14.9 boutons per 100 microns2 and was not correlated with the distance from the soma. The percentage apposition covering was higher on the soma and the proximal part of the dendrites than on the remaining part of the dendritic arborization. Close appositions between labelled dendrite and unlabelled somata and/or dendrites together with dendro-dendritic synapses suggested the possibility that the dendrites may be involved in local cell-to-cell communication. Microdendrites emerging from the soma or the proximal dendrites were contacted by synaptic boutons which may be more efficient as revealed by computation.     

The ultrastructure and synaptic architecture of phrenic motor neurons in the spinal cord of the adult rat

Summary Although light microscopic studies have analysed phrenic motor neurons in several different species, there has never been an ultrastructural investigation of identified phrenic motor neurons. In addition, electrophysiological studies have raised questions relating to the function of phrenic motor neurons which may be answered only by direct electron microscopic investigation. Thus, the present study was carried out to provide a detailed ultrastructural analysis of identified phrenic motor neurons. Phrenic motor neurons in the spinal cord of the rat were labelled by retrogradely transported horseradish peroxidase (HRP) after transecting the phrenic nerve in the neck and applying the enzyme directly to the central stump of the transected nerve. The results showed that the general ultrastructural characteristics of phrenic motor neurons were similar to those previously reported for other spinal motor neurons. However, phrenic primary dendrites appeared to be isolated from all other dendritic profiles in the neuropil. Primary dendrites were not fasciculated. Fasciculation occurred only among the more distal secondary and tertiary phrenic dendritic branches. Direct dendrodendritic or dendrosomatic apposition was rarely seen; gap junctions between directly apposing phrenic neuronal membranes were not observed. The membranes of adjacent phrenic neuronal profiles were most frequently separated by intervening sheaths of astroglial processes. Myelinated phrenic axons and a phrenic axon collateral were identified. The initial portion of the phrenic axon collateral was cone-shaped, lacked myelin, and thus resembled a miniature axon hillock. In one instance, a large accumulation of polyribosomes was observed within the hillock-like structure of a phrenic axon collateral. Eight morphological types of synaptic boutons, M, P, NF_s, S, NF_f, F, G and C were classified according to criteria used by previous investigators. Most of these endings (M, NF_s, NF_f, S and F) made synaptic contact with profiles of labelled phrenic somata and dendrites. F, NF_f, and S boutons also terminated on phrenic axon hillocks. C and G boutons contacted exclusively phrenic somata and small calibre dendrites, respectively. P boutons established axo-axonic synaptic contacts with the M and NF_s bouton. The morphological findings of the present study provide new data that may be related to phrenic synchronized output and presynaptic inhibition of primary afferents terminating on phrenic motor neurons.     

Distinct profiles of refilling of inhibitory neurotransmitters into presynaptic terminals projecting to spinal neurones in immature rats

Corelease of glycine and GABA from the single synaptic terminal (synaptic bouton) is well accepted in immature rat spinal cord and brainstem. However, it raises the question of how glycine and GABA are accumulated in the same synaptic vesicles and coreleased. To address this issue, spontaneous miniature inhibitory postsynaptic currents (mIPSCs) and focally evoked IPSCs (eIPSCs) mediated via a single synapse were recorded from synaptic bouton preparations of the rat immature sacral dorsal commissural nucleus (SDCN) neurones by whole-cell patch recording. Focal stimulation of a single synaptic bouton revealed that three different quantal releases occur from a single synaptic bouton: i.e. pure glycine, pure GABA, and mixed. Prolonged treatment with bafilomycin A1, a vacuolar-type H⁺/ATPase inhibitor, to the SDCN neurone greatly suppressed frequency and amplitude of the mIPSCs. During washing out of bafilomycin A1, complete recovery in the amplitude of glycinergic mIPSCs was observed, while that of GABAergic and mixed mIPSCs was incomplete. These observations indicate that three types of vesicles coexist in single synaptic terminals, and that refilling of glycine into the synaptic vesicle predominantes over GABA after pretreatment with bafilomycin A1 in immature rats. This could be explained by the decrease in the cytosolic concentration of GABA, or by the presence of subtypes of vesicular inhibitory amino acid transporter in the synaptic vesicle membrane.