Synapses formed by olivocochlear axon branches in the mouse cochlear nucleus

Cochlear nucleus branches of thick olivocochlear axons were labeled by injections of horseradish peroxidase into the spiral ganglion of the cochlear basal turn in mice. Six labeled axons were traced by light microscopy, and selected portions of seven branches were sectioned serially for electron microscopic examination. Axonal branches most frequently terminated near certain granule cell regions of the ventral cochlear nucleus. This article describes terminals, synapses, and postsynaptic elements of these olivocochlear branches. The olivocochlear branches had both terminal and en passant boutons that contained round vesicles and made asymmetric synapses with other neuronal processes. About a quarter of the synapses also possessed additional specializations, postsynaptic, or subjunctional bodies. Mossy terminals, a multisynaptic type of terminal commonly found in granule cell regions, were not found arising from any of the labeled branches. No somatic synapses were found, although contacts with cell bodies were occasionally observed. The predominant synaptic target of olivocochlear branches were what appeared to be dendrites of large diameter. At least some of these large dendrites received multiple synapses from a single labeled olivocochlear branch. The morphological characteristics of reconstructed dendrites suggest that multipolar cells might be predominant targets for the medial olivocochlear system in the cochlear nucleus. This was demonstrated in one case in which a large dendrite was followed to its cell body of origin.     

The fine structure of the lateral superior olivary nucleus of the cat

The synaptic organization of the lateral superior olivary nucleus of the cat was analyzed under the electron microscope. The predominant cell type, the fusiform cell, has dendrites that extend from opposite poles of the cell body toward the margins of the nucleus, where they terminate in spinous branches. The fusiform cells are contacted by three types of synaptic terminals that can be distinguished by the size and shape of their synaptic vesicles. The somatic and proximal dendritic surfaces are apposed by synaptic terminals containing small, flat synaptic vesicles. Further from the cell body, the dendrites form numerous synaptic contacts with terminals containing large round vesicles as well as with the terminals containing small, flat vesicles. The most distal dendritic branches and their spiny appendages appear to form synapses almost exclusively with the terminals with large, round vesicles. A relatively rare type of terminal that contains small, round vesicles may form synapses with either the somatic or dendritic surfaces. A few small cells are interspersed among the fusiform cells, but they are more commonly located around the margins of the nucleus. The small cells form few axosomatic contacts. The simplest interpretation of the findings is that the terminals with small, flat vesicles arise in the medial nucleus of the trapezoid body and are inhibitory in function, whereas the terminals with large, round vesicles arise in the anteroventral cochlear nucleus and are excitatory; however, this remains to be demonstrated experimentally. In any case, the differential distribution of these two types of inputs on the somatic and dendritic surfaces must be an important determinant of the physiological response properties of the fusiform cells to binaural acoustic stimuli.     

Post-embedding immunogold labeling of gamma-aminobutyric acid in lamina II of the spinal trigeminal subnucleus pars caudalis: I. A qualitative study

This study examines the normal synaptic organization of the feline spinal trigeminal nucleus pars caudalis (PC). A primary goal of this study is to identify and characterize the synaptic complexes within PC based on their specific neurotransmitter content. Post-embedding immunogold techniques are utilized with electron microscopy to determine the ultrastructural localization of gamma-aminobutyric acid (GABA) immunoreactivity within lamina II of PC. The colloidal gold particles (10 nm) are randomly distributed over immunoreactive (IR) profiles without preference toward membranous or cytoplasmic regions. GABA immunoreactivity occurs on small unmyelinated axons, on terminals which form synaptic contacts, and on some vesicle-containing dendrites. The GABA-IR terminals form symmetric (type II) contacts onto unlabeled somata and dendrites of various sizes, and onto other unlabeled axon terminals. The GABA-IR terminal in axo-axonic complexes is presynaptic to a round vesicle-containing terminal, which itself may form a type I asymmetric contact onto an unlabeled dendrite or soma. A proportion of vesicle-containing dendrites show GABA-immunoreactivity and are postsynaptic to unlabeled terminals with round vesicles. Other, but far fewer, vesicle-containing dendrites are GABA negative and postsynaptic to GABA-IR terminals. In summary, the findings are consistent with the localization of GABA in intrinsic neurons, and may be associated with presynaptic and postsynaptic inhibition within nociceptive related pathways. © 1996 Wiley-Liss, Inc.     

Synaptic organization of the rat parafascicular nucleus,with special reference to its afferents from the superior colliculus and the pedunculopontine tegmental nucleus

The synaptic organization of afferents to the parafascicular nucleus (Pf) of the thalamus was studied in rats. In the Pf, three types of axon terminals were identified: the first type was a small terminal with round synaptic vesicles forming an asymmetric synapse, the second type was a large terminal with round synaptic vesicles forming an asymmetric synapse, and the third type was a terminal with pleomorphic vesicles forming a symmetric synapse. They were named SR, LR and P boutons, respectively. In order to determine the origin of these axon terminals, biotinylated dextran amine (BDA) was injected into the main afferent sources of the Pf, the superior colliculus (SC) and the pedunculopontine tegmental nucleus (PPN). Axon terminals from the SC were both SR and LR boutons which made synaptic contacts with somata and dendrites. PPN afferents were SR boutons, which made synaptic contacts with somata and smaller dendrites. Double-labeled electron microscopic studies, in which a retrograde tracer (wheat germ agglutinin conjugated to horseradish peroxidase: WGA-HRP) was injected into the striatum and an anterograde tracer (BDA) into the SC revealed that SC afferent terminals made synapses directly with Pf neurons that projected to the striatum. Another experiment was performed to find out whether two different afferents converged onto a single Pf neuron. To address this question, two different tracers were injected into the SC and PPN in a rat. Electron microscopically, both afferent terminals from the SC and PPN made synaptic contacts with the same dendrite. Our results prove that a single neuron of the rat Pf received convergent projections from two different sources.     

Ultrastructural localization of phenylethanolamine N-methyltransferase-like immunoreactivity in the rat locus coeruleus

Adrenergic afferents from the rostral ventrolateral medulla are known to modulate the activity of noradrenergic neurons of the locus coeruleus (LC). The light and electron microscopic localization of a polyclonal antiserum directed against the adrenaline synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT) was used to determine the identity and targets of the adrenergic afferents to the LC of the rat brain. By light microscopy, varicose processes showing intense PNMT-like immunoreactivity (LI) were seen throughout the neuropil surrounding neuronal perikarya which in adjacent sections were shown to contain immunoreactivity for the noradrenaline synthesizing enzyme, dopamine-beta-hydroxylase. Electron microscopy confirmed that these labeled varicose processes were primarily axon terminals. Terminals containing PNMT-LI constituted 30% (141 out of 464) of all identifiable terminals within the LC. These terminals were 0.5-1.8 micron in diameter and contained many small, clear and from 2 to 10 larger dense-core vesicles. The targets of the terminals with PNMT-LI were principally unlabeled (i.e. non-PNMT-containing) perikarya and dendrites. The synaptic junctions on perikarya were rare and exclusively symmetric; whereas, those on proximal (large) dendrites were somewhat more numerous and included symmetric as well as asymmetric membrane specializations. However, the vast majority (85% from a total of 141) of the terminals with PNMT-LI formed asymmetric synaptic junctions on unlabeled distal (small) dendrites and dendritic spines. In rare instances, the PNMT-immunoreactive terminals also formed synaptic junctions with other similarly labeled terminals. These findings provide the first ultrastructural evidence that adrenergic terminals in the LC (1) are one of the more prevalent synaptic inputs to the principally noradrenergic neurons; (2) have both symmetric and asymmetric synaptic specializations conventionally associated with inhibition and excitation, respectively; and (3) may modulate other adrenergic terminals through presynaptic mechanisms. In addition to the varicose processes, light microscopy revealed diffuse PNMT-LI throughout the LC. The ultrastructural correlate of this labeling was seen as patches of peroxidase product within the cytoplasm of a few perikarya and dendrites and throughout the cytoplasm of astrocytes identified by their discrete bundles of microfilaments. The detection of PNMT-LI in cells that are not known to synthesize adrenaline is surprising and suggests either a functional diversity for PNMT or amino acid sequence homologies with related enzymes which are enriched in the LC.     

Postnatal development of efferent synapses in the rat cochlea

The development of olivocochlear efferent axons and their contacts in the postnatal cochlea was studied after DiI applications to the olivocochlear bundle in the ipsilateral brainstem of rats from 0 to 10 days of age (P0-10). Light microscopic analyses showed that labeled axons reached the vicinity of inner hair cells by P0 and outer hair cells by P2. Electron microscopic analyses demonstrated that labeled immature efferent axons are present among supporting cells of the greater epithelial ridge as well as inner hair cells at P0. The first efferent contacts that contacted inner hair cells contained a few irregularly sized vesicles and, occasionally, mitochondria. Postsynaptic specializations within inner hair cells apposed to labeled efferent axons included subsynaptic cisterns, irregularly sized vesicles, and synaptic bodies. Similar features were present in unlabeled profiles, presumed to be afferents, indicating that immature efferent axons could not be reliably distinguished from afferents without positive labeling. Efferent axons synapsed with outer hair cells by P4 and had synapse-like contacts at the bases of Deiters' cells at P4 and P6. Contacts between afferents and efferents were observed frequently in the inner spiral bundle from P6. As they matured, efferent axon terminals contacting hair cells contained increasing numbers of synaptic vesicles and were typically apposed by well-defined postsynaptic cisterns, thus acquiring distinctive profiles.     

An electron microscopic study of the afferent connections of the lateral reticular nucleus of the cat

The mode and pattern of termination of the afferents to the lateral reticular nucleus (LRN) of the cat were examined at the cellular level through the ultrastructural localization of induced degeneration. Examination of the LRN following hemicordotomy at the fifth and sixth cervical levels revealed that most of the degenerating terminals were in contact with intermediate and distal dendrites, and that most of these degenerating terminals were small and contained round vesicles. Fewer degenerating terminals were observed on the somata and proximal dendrites after spinal hemisection, and most of these terminals were large and contained round vesicles. Following lesions of the pericruciate cortex, small degenerating terminals were occasionally observed making contact onto intermediate and distal dendrites. Degenerating rubral terminals were observed synapsing on somata, somatic and dendritic spines, proximal dendrites and most commonly on intermediate and distal dendrites following lesioning of the red nucleus. The degenerating axosomatic rubro-LRN terminals belonged to the large, round-vesicle terminal population, while those degenerating terminals contacting intermediate and distal dendrites belonged to the small, round-vesicle population. Small, degenerating terminals were occasionally seen following lesions of the fastigial nucleus, and they made synaptic contact mainly onto intermediate and distal dendrites and dendritic spines. The present ultrastructural observations taken together with the convergence pattern of LRN afferents and the available electrophysiological data on inputs to the LRN suggest an extensive integration of converging impulses from two or more afferent sources to the rostral LRN neurons. The results of this study therefore support the view that the rostral LRN functions as a comparator of command signals from the motor cortex and red nucleus and feedback signals from the spinal cord and cerebellum during ongoing movement.     

Ultrastructure of ascending cholinergic terminals in the anteroventral thalamic nucleus of the rat: a comparison with the mammillothalamic terminals

In this study, to identify the ultrastructure and distribution of ascending cholinergic afferent terminals in the anteroventral thalamic nucleus, we used an anti-vesicular acetylcholine transporter antibody as marker of cholinergic afferents, and characterized the immunoreactive terminals at the ultrastructural level. We then compared the distribution pattern of the cholinergic terminals and that of the mammillothalamic terminals identified by anterograde transport of a tracer injected into the mammillary body. The cholinergic terminals were small, and formed both symmetrical and asymmetrical synaptic contacts throughout the dendritic arborizations, particularly in the distal region. This distribution pattern differed from that of mammillothalamic terminals, that were of LR (large terminal containing round synaptic vesicles) type and were preferentially distributed in the proximal region of dendrites. We also found relatively numerous cholinergic terminals making contact directly with immunonegative excitatory terminals, both LR and SR (small terminal containing round vesicles) terminals, without clear postsynaptic specialization. A few cholinergic terminals even seemed to form a synaptic complex with the LR or SR terminals. These findings suggest that the ascending cholinergic afferents in the anteroventral thalamic nucleus can effectively modulate excitatory inputs from both the mammillothalamic and corticothalamic terminals, in close vicinity to a synaptic site.     

Cochlear-nucleus branches of thick (medial) olivocochlear fibers in the mouse: a cochleotopic projection

Olivocochlear neurons have somata in the superior olivary complex and provide an efferent innervation to the cochlea. One subgroup of olivocochlear neurons, medial olivocochlear neurons, sends fibers to innervate the cochlear outer hair cells. En route to the cochlea, medial olivocochlear fibers give off branches to the ventral cochlear nucleus, the first auditory center of the brain. This study examines the cochlear-nucleus branches of medial olivocochlear fibers, comparing those from fibers that innervate the cochlear base with those from fibers that innervate the cochlear apex. Basal fibers give off dorsal branches to the granule cell lamina and ventral branches to the auditory nerve root. Apical fibers give off few dorsal branches but many ventral branches that terminate rostrally to the nerve root. This cochleotopic mapping of medial olivocochlear branches corresponds in a general way to that of afferent fibers. Unlike afferent fibers, however, the branches terminate primarily along the edges of the cochlear nucleus. In the mouse, the particular edges of termination are (1) the medial border of the ventral cochlear nucleus where it meets the underlying vestibular nerve root, and (2) the border between the ventral cochlear nucleus and the granule cell lamina. Neurons and dendrites of these border regions may thus integrate efferent and afferent information in a frequency-specific manner.     

Relationship of Met-enkephalin-like immunoreactivity to vagal afferents and motor dendrites in the nucleus of the solitary tract: a light and electron microscopic dual labeling study

Methionine (Met⁵)-enkephalin has been implicated in autonomic functions involving vagal reflexes within the nucleus of the solitary tract (NTS). We examined the light and electron microscopic relationships between neurons containing methionine (Met⁵)-enkephalin-like immunoreactivity (MELI) and vagal afferents and motor dendrites in the rat NTS. A polyclonal antibody raised against Met⁵-enkephalin and showing maximal cross-reactivity with this peptide was localized by immunoautoradiography. In the same sections, vagal afferents and motor neurons were identified by histochemical detection of anterogradely and retrogradely transported horseradish peroxidase (HRP). By light microscopy, the MELI was detected in perikarya distributed principally in the dorsomedial, intermediate and parasolitary subdivisions of the NTS. These subnuclei as well as medial and commissural divisions of the NTS also showed: (1) aggregates of silver grains thought to overlie terminals containing MELI, and (2) anterogradely transported HRP in varicose processes. Electron microscopic analysis of the dorsomedial NTS at the level of the area postrema established that MELI was detectable in perikarya, dendrites, and axon terminals. Most of the MELI was associated with large dense core vesicles (dcvs). These opioid terminals formed primarily symmetric synapses on proximal and asymmetric synapses on distal dendrites. Analysis of the dendritic targets of terminals containing MELI revealed that 13/222 were in synaptic contact with dendrites also containing MELI. The remainder of the terminals containing MELI either lacked recognized junctions or formed synapses with unlabeled dendrites. In comparison to the terminals containing MELI in the same series of sections, anterogradely labeled vagal terminals extensively formed asymmetric junctions with distal dendrites and spines. Of the observed anterogradely labeled terminals 6/84 formed synapses with dendrites containing MELI and 3/84 with dendrites containing retrogradely transported HRP. The remainder of the junctions were with dendrites lacking detectable immunoautoradiographic or HRP-labeling. The majority of the recognized synapses on labeled dendrites were at more proximal sites possibly reflecting more limited detection of both MELI and retrogradely transported HRP in smaller dendrites. However, the presence of even a few junctions at proximal sites on dendrites where synaptic transmission is known to be more effective suggests a potentially strong modulation of both opioid and vagal motor neurons by visceral afferents in the NTS. In addition to forming synapses on dendrites, both vagal afferents and terminals containing MELI showed frequent synaptic associations with unlabeled terminals, but not with each other. This finding suggests that the previously demonstrated opiate binding sites on vagal afferents is most likely attributed to other endogenous opiates.     

Selective retrograde transport of nipecotic acid, a GABA analog, labels a subpopulation of gerbil olivocochlear neurons.

Perfusion of the gerbil cochlea with micromolar quantities of 3H-gamma-aminobutyric acid (GABA) results in rapid, selective labeling of 50-60% of the olivocochlear (OC) efferent terminals on afferent dendrites beneath the inner hair cells, and all of the efferent terminals beneath the outer hair cells. In order to identify the neurons from which these GABA-accumulating terminals originate, the cell bodies were localized by using retrograde transport of 3H-nipecotic acid, a metabolically inert GABA analog. With survival times of 6-30 hours after cochlear injection, myelinated OC efferent fibers and cell bodies were well labeled, with the greatest number being labeled at 12-18 hours. All of the labeled neurons belonged to the medial OC system, and no lateral OC neurons were labeled. It is concluded that the GABA-accumulating endings in the gerbil cochlea arise from medial OC neurons, and therefore that medial OC efferent neurons in this species project to both inner and outer hair cell regions.     

Ultrastructure of chemically defined neuron systems in the dorsal horn of the monkey. I. Substance P immunoreactivity

The ultrastructural localization of substance P-like immunoreactivity (SPLI) in lamina I (marginal zone) and lamina II₀ (outer substantia gelatinosa) of the dorsal horn of the macaque monkey was examined by the indirect antibody peroxidase-antiperoxidase method. SPLI was found in small unmyelinated and finely myelinated axons and a variety of terminal types. The majority of SPLI terminals contained a few to many large granular vesicles (mean diameter 90 nm) in addition to a population of small clear round vesicles. A very few terminals contained mainly small round vesicles. SPLI terminals were presynaptic in axosomatic, axodendritic and axospinous contacts forming, in all but the axosomatic junctions, asymmetrical synapses. Some axosomatic junctions were symmetrical. SPLI terminals also formed the center of glomeruli with unlabeled dendrites and dendritic spines; some of the unlabeled dendrites contained a few small scattered vesicles and large dense-core vesicles. In more complex formations 2 to 4 SPLI terminals were associated with one another and linked by desmosomal contacts. The individual terminals in the complexes or ‘congregate terminals’ were simple large granular vesicle containing terminals (LGV), LGV-central terminals of associated glomeruli, or terminals containing mainly small round vesicles. In the apical region of lamina I an unlabeled terminal was found occasionally in contact with an SPLI terminal, which in turn synapsed onto a dendrite. These contacts have some synaptic characteristics and the SPLI terminal was possibly postsynaptic. Most of the types of SPLI terminals resemble closely terminal types shown to be of primary afferent origin. These terminals which make direct contact with dorsal horn dendrites may be the morphological substrate for postsynaptic excitation of dorsal horn neurons by substance P. The contacts of unlabeled terminals with SPLI terminals may represent a morphological substrate by which other neurochemical substances such as enkephalin or serotonin may modulate the substance P effects on dorsal horn neuronal activity.     

Differentiation of spinous synapses in the mouse organ of corti

The inner hair cells, the primary auditory receptors, are perceived only as a means for transfer of sound signals via the auditory nerve to the central nervous system. During initial synaptogenesis, they receive relatively few and mainly somatic synapses. However, around the onset of hearing (10-14 postnatal days in the mouse), a complex network of local spinous synapses differentiates, involving inner hair cells, their afferent dendrites, and lateral olivocochlear terminals. Inner hair cell spines participate in triadic synapses between olivocochlear terminals and afferent dendrites. Triadic synapses have not yet been confirmed in the adult. Synaptic spines of afferent dendrites form axodendritic synapses with olivocochlear terminals and somatodendritic synapses with inner hair cells. The latter are of two types: ribbon-dendritic spines and stout dendritic spines surrounded only by a crown of synaptic vesicles. Formation of spinous afferent synapses results from sprouting of dendritic filopodia that intussuscept inner hair cell cytoplasm. This process continues in the adult, indicating ongoing synaptogenesis. Spinous processes of olivocochlear synaptic terminals contact adjacent afferent dendrites, thus integrating their connectivity. They develop about 14 postnatal days, but their presence in the adult has yet to be confirmed. Differentiation of spinous synapses in the organ of Corti results in a total increase of synaptic contacts and in a complexity of synaptic arrangements and connectivity. We propose that spinous synapses provide the morphological substrate for local processing of initial auditory signals within the cochlea.     

Corticotropin-releasing factor-containing axon terminals synapse onto catecholamine dendrites and may presynaptically modulate other afferents in the rostral pole of the nucleus locus coeruleus in the rat brain

Physiological and immunohistochemical studies have suggested that corticotropin-releasing factor (CRF), the hypophysiotropic peptide that initiates endocrine responses to stress, may serve as a neurotransmitter to activate noradrenergic neurons in the nucleus locus coeruleus (LC). We combined immunoperoxidase labeling for CRF and immunogold-silver localization of the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) in single sections through the rat LC to determine potential substrates for interactions between these two transmitters. Light microscopic analysis indicated that CRF processes are dense and highly varicose in the rostral LC region in the vicinity of noradrenergic dendrites. Electron microscopy of this rostral region revealed that immunoperoxidase labeling for CRF was mainly restricted to axons and axon terminals and was rarely seen in somata or dendrites. Axon terminals containing CRF immunoreactivity varied in size, content of synaptic vesicles, and formation of synaptic specializations. The postsynaptic targets of the CRF-labeled axon terminals consisted of both TH-labeled dendrites and dendrites lacking detectable TH-immunoreactivity. Of 113 CRF-immunoreactive axon terminals, approximately 70% were in direct contact with TH-labeled and unlabeled dendrites. Of the CRF-labeled axon terminals forming synapses with TH-labeled and unlabeled dendrites, they were either of the asymmetric (excitatory type; 19%) or symmetric (inhibitory type; 11%) variety or did not form identifiable contacts in the plane of section analyzed. Unlabeled axon terminals and glial processes were also commonly located adjacent to the plasma membranes of CRF-labeled axon terminals. These results provide the first direct ultrastructural evidence that axon terminals containing CRF-immunoreactivity 1) directly contact catecholamine-containing dendrites within the rostral pole of the LC, 2) may presynaptically modulate other afferents, and 3) are often enveloped by astrocytic processes. © 1996 Wiley-Liss, Inc.     

Patterns of glutamate, glycine, and GABA immunolabeling in four synaptic terminal classes in the lateral superior olive of the guinea pig.

The goal of this study was to correlate synaptic ultrastructure with transmitter specificity and function in the lateral superior olive (LSO), a nucleus that is thought to play a major role in sound localization. This was accomplished by means of postembedding immunogold immunocytochemistry. Four classes of synaptic terminals were identified in the LSO. They were distinguishable from one another both morphologically and on the basis of their different patterns of immunolabeling for glutamate, glycine, and gamma-aminobutyric acid (GABA). The highest level of glutamate immunoreactivity was found in terminals that contained round vesicles (R) and formed synaptic contacts with asymmetric synaptic junctions. Round-vesicle terminals predominated on small caliber dendrites by a ratio of at least 2:1 over the other classes combined. The thinnest dendrites were typically contacted by R terminals only. The ratio of R terminals to the other types decreased as the caliber of the dendritic profiles they apposed increased so that on the soma, R terminals were outnumbered by at least 2:1 by the other types. Terminals containing flattened vesicles (F) exhibited intense immunoreactivity for both glycine and glutamate, although the glutamate immunolabeling was not as high as that in the R terminals. Flattened-vesicle terminals formed symmetric synaptic contacts with their targets and their distribution was the reverse of that described for R terminals; i.e., they were most abundant on LSO perikarya and fewest on small caliber dendrites. Two terminal types, both containing pleomorphic vesicles and forming symmetric synaptic junctions, were found in far fewer numbers. One group contained large pleomorphic vesicles (LP) and was immunoreactive for both glycine and GABA. The other group contained small pleomorphic vesicles (SP) along with a few dense-core vesicles and labeled for GABA only. The LP terminals were preferentially distributed on somata and large-caliber dendrites, while the SP terminals most often contacted smaller dendrites. Previous work suggests that a large percentage of the R terminals arise from spherical cells in the ipsilateral cochlear nucleus and are excitatory in action. This pathway may use glutamate as a transmitter. Many of the F terminals are thought to originate from the ipsilateral medial nucleus of the trapezoid body and appear to be the inhibitory (glycinergic) terminals from a pathway that originates from the contralateral ear. The origins and functions of LP and SP terminals are unknown, but a few possibilities are discussed along with the significance of cocontainment of neuroactive substances in specific terminal types.     

Ultrastructure of synaptic input to medial olivocochlear neurons

Medial olivocochlear (MOC) neurons project from the brain to the cochlea to form the efferent limb of the MOC reflex. To study synaptic inputs to MOC neurons, we retrogradely labeled these neurons using horseradish peroxidase injections into the cochlea. Labeled neurons were identified in the ventral nucleus of the trapezoid body and documented with the light microscope before being studied with serial-section electron microscopy. MOC somata and dendrites were innervated by three different types of synapses, distinguished as either having: 1) large, round synaptic vesicles and forming asymmetric contacts; 2) small, round vesicles plus a few dense core vesicles and forming asymmetric contacts; or 3) pleomorphic vesicles and forming symmetric contacts. The first two types were the most frequent on somata. Acetylcholinesterase-stained material confirmed that the type containing large, round vesicles is most common on dendrites. We kept track of the synaptic terminals in serial sections and compiled them into three-dimensional swellings. Swellings with large, round vesicles formed up to seven synapses per swelling, were largest in size, and sometimes formed complex arrangements engulfing spines of MOC neurons. Swellings with small, round vesicles formed up to four synapses per swelling. The morphology of this type of synapse, and the moderate sizes of the swellings forming it, suggests that it originates from posteroventral cochlear nucleus stellate/multipolar neurons. This input may thus provide the sound-evoked input to MOC neurons that causes their reflexive response to sound.     

Ultrastructural examination of enkephalin and substance P input to cholinergic neurons within the rat neostriatum.

Enkephalin and substance P-containing inputs to cholinergic perikarya were examined in the rat neostriatum using an ultrastructural immunocytochemical double-labeling protocol. Sections of rat neostriatum were double-labeled for either choline acetyltransferase (ChAT) and substance P or ChAT and enkephalin using silver intensified colloidal gold and peroxidase as labels. Regions containing both ChAT-positive neurons and peroxidase reaction product were identified in the light microscope prior to sectioning for electron microscopy. Substance P-containing terminals which contained round synaptic vesicles and made symmetrical synaptic contacts were commonly observed in the neostriatum. Substance P synapses onto ChAT-positive perikarya and dendrites were frequently observed: up to 5 synaptic contacts were observed onto a ChAT-positive dendrite. Enkephalin labeling was also seen in a population of axon terminals containing round synaptic vesicles and exhibiting symmetrical synaptic specializations. In contrast to substance P-containing terminals, relatively few synaptic contacts were observed onto ChAT-positive labeled perikarya and dendrites although enkephalin-labeled terminals were seen in frequent contact with perikarya and dendrites of unlabeled spiny neurons. Since enkephalin and substance P are contained within different populations of striatal spiny neurons, the results of the present study suggest that these two types of neurons differ in their intrinsic striatal connections.     

Ultrastructure of chemically defined neuron systems in the dorsal horn of the monkey. III. Serotonin immunoreactivity

Enkephalinergic axons and terminals were identified by the PAP immunohistochemical method in lamina I (marginal zone) and lamina II₀ (outer substantia gelatinosa) in the dorsal horn of the monkey spinal cord. Synaptic profiles with enkephalin-like immunoreactivity (MELI) contained clear, round, vesicles, sometimes a few large granular vesicles, and usually formed asymmetrical contacts.MELI terminals forming synaptic contacts with various sizes of dendrites and with dendritic spines were the most common type of relationship found; axosomatic contacts were few. Additionally, two types of complexes were observed in which an MELI terminal formed a specialized apposition with an unlabelled terminal. The contact of often resembled a synapse and in most cases the MELI terminal was suspected to be presynaptic. One complex consisted of a MELI terminal apposing the LGV type terminal (containing large granular vesicles), which in turn was presynaptic to a dendrite. (The identify of the LGV terminal could not be determined, but it had some characteristics similar to those described for substance P terminals and for a class of primary afferents in the monkey dorsal horn). The other type of complex consisted of a MELI terminal apposing an R-type terminal (containing small, round, clear vesicles) which was in turn presynaptic to a dendrite. Often, the MELI terminal also formed a synapse onto the same dendrite.The axodendritic, axospinous and axosomatic contacts of MELI terminals in the superficial dorsal horn may produce some of the depressive postsynaptic-like effects of enkephalin iontophoresis onto dorsal horn neurons. In these cases the responses of dorsal horn neurons to both low threshold and nociceptive primary afferents is suppressed. However, the opiate receptor-dependent PAD of C-fibers observed in the dorsal horn may be mediated by the MELI complexes formed with LGV and R terminals found in lamina I.     

Organization and synaptic connections of cholinergic fibers in the cat superior colliculus

The cat superior colliculus (SC) receives a dense cholinergic input from three brainstem nuclei, the pedunculopontine tegmental nucleus, the lateral dorsal tegmental nucleus, and the parabigeminal nucleus (PBG). The tegmental inputs project densely to the intermediate gray layer (IGL) and sparsely to the superficial layers. The PBG input probably projects only to the superficial layers. In the present study, the morphology of choline acetyltransferase (ChAT)-immunoreactive axons and synaptic endings in the superficial and deep layers of the SC was examined by light and electron microscopy to determine whether these cholinergic afferents form different types of synapses in the superifical and deep layers. Two types of fibers were found within the zonal (ZL) and upper superficial gray layers (SGL): small diameter fibers with few varicosities and larger diameter fibers with numerous varicosities. Quantitative analysis demonstrated a bimodal distribution of axon diameters, with one peak at approximately 0.3–0.5 μm and the other at 0.9–1.0 μm. On the other hand, ChAT-immunoreactive fibers in the IGL were almost all small and formed discrete patches within the IGL. Two types of ChAT-immunoreactive synaptic profiles were observed within the ZL and upper SGL using the electron microscope. The first type consisted of small terminals containing predominantly round synaptic vesicles and forming asymmetric synaptic contacts, mostly on dendrites. The second type was comprised of varicose profiles that also contained round synaptic vesicles. Their synaptic contacts were always symmetric in profile. ChAT-immunoreactive terminals in the IGL patches contained round or pleomorphic synaptic vescles, and the postsynaptic densities varied from symmetric to asymmetric, including intermediate forms. However, no large varicose profiles were observed. This study suggests that cholinergic fibers include at least two differnt synaptic morphologies: small terminals with asymmetric thickenings and large varicose profiles with symmetric terminals. The large varicose profile in the superficial layers is absent in the IGL. This result suggests that the cholinergic inputs that innervate the superficial layers and the patches in the IGL of the cat SC differ in their synaptic organization and possibly also in their physiological actions. © 1993 Wiley-Liss, Inc.