Projections from the spinal trigeminal nucleus to the cochlear nucleus in the rat

The integration of information across sensory modalities enables sound to be processed in the context of position, movement, and object identity. Inputs to the granule cell domain (GCD) of the cochlear nucleus have been shown to arise from somatosensory brain stem structures, but the nature of the projection from the spinal trigeminal nucleus is unknown. In the present study, we labeled spinal trigeminal neurons projecting to the cochlear nucleus using the retrograde tracer, Fast Blue, and mapped their distribution. In a second set of experiments, we injected the anterograde tracer biotinylated dextran amine into the spinal trigeminal nucleus and studied the resulting anterograde projections with light and electron microscopy. Spinal trigeminal neurons were distributed primarily in pars caudalis and interpolaris and provided inputs to the cochlear nucleus. Their axons gave rise to small (1-3 microm in diameter) en passant swellings and terminal boutons in the GCD and deep layers of the dorsal cochlear nucleus. Less frequently, larger (3-15 microm in diameter) lobulated endings known as mossy fibers were distributed within the GCD. Ventrally placed injections had an additional projection into the anteroventral cochlear nucleus, whereas dorsally placed injections had an additional projection into the posteroventral cochlear nucleus. All endings were filled with round synaptic vesicles and formed asymmetric specializations with postsynaptic targets, implying that they are excitatory in nature. The postsynaptic targets of these terminals included dendrites of granule cells. These projections provide a structural substrate for somatosensory information to influence auditory processing at the earliest level of the central auditory pathways.     

Projections from auditory cortex to the cochlear nucleus in rats: Synapses on granule cell dendrites

Previous work has demonstrated that layer V pyramidal cells of primary auditory cortex project directly to the cochlear nucleus. The postsynaptic targets of these centrifugal projections, however, are not known. For the present study, biotinylated dextran amine, an anterograde tracer, was injected into the auditory cortex of rats, and labeled terminals were examined with light and electron microscopy. Labeled corticobulbar axons and terminals in the cochlear nucleus are found almost exclusively in the granule cell domain, and the terminals appear as boutons (1–2 μm in diameter) or as small mossy fiber endings (2–5 μm in diameter). These cortical endings contain round synaptic vesicles and form asymmetric synapses on hairy dendritic profiles, from which thin (0.1 μm in diameter), nonsynaptic “hairs” protrude deep into the labeled endings. These postsynaptic dendrites, which are typical of granule cells, surround and receive synapses from large, unlabeled mossy fiber endings containing round synaptic vesicles and are also postsynaptic to unlabeled axon terminals containing pleomorphic synaptic vesicles. No labeled fibers were observed synapsing on profiles that did not fit the characteristics of granule cell dendrites. We describe a circuit in the auditory system by which ascending information in the cochlear nucleus can be modified directly by descending cortical influences. © 1996 Wiley-Liss, Inc.     

Epilepsy induced collateral sprouting of hippocampal mossy fibers: Does it induce the development of ectopic synapses with granule cell dendrites?

In the present study, using Golgi and electron microscopy techniques, experimentally induced epilepsy (kindling and kainate treatment) elicited collateral sprouting of mossy fibers in rat hippocampus. Collateral branches invade the hilus, cross the granule cell layer, and distribute throughout the inner third of the molecular layer. These newly developed collaterals may acquire the typical features of mossy fibers including giant fiber varicosities (mousses), although the mean surface of these mousses was thinner in these collaterals than in terminal branches. Granule cell dendrites may develop giant thorny excrescences, suggesting that the targets of these collaterals are granule cells. Giant synaptic boutons appear in the inner third of molecular layer of epileptic rats. These boutons acquire the morphological features of mossy fiber boutons and made multiple synaptic contacts with dendritic spines. The analysis of the profile types suggests that some of the newly developed collateral mossy fibers made hypotrophic synaptic contacts.     

Hippocampal mossy fiber sprouting and synapse formation after status epilepticus in rats: Visualization after retrograde transport of biocytin

In complex partial epilepsy and in animal models of epilepsy, hippocampal mossy fibers appear to develop recurrent collaterals, that invade the dentate molecular layer. Mossy fiber collaterals have been proposed to subserve recurrent excitation by forming granule cell-granule cell synapses. This hypothesis was tested by visualizing dentate granule cells and their mossy fibers after terminal uptake and retrograde transport of biocytin. Labeling studies were performed with transverse slices of the caudal rat hippocampal formation prepared 2.6–l70.0 weeks after pilocarpine-induced or kainic acid-induced status epilepticus. Light microscopy demonstrated the progressive growth of recurrent mossy fibers into the molecular layer; the densest innervation was observed in slices from pilocarpine-treated rats that had survived 10 weeks or longer after status epilepticus. Thin mossy fiber collaterals originated predominantly from deep within the hilar region, crossed the granule cell body layer, and formed an axonal plexus oriented parallel to the cell body layer within the inner one-third of the molecular layer. When sprouting was most robust, some recurrent mossy fibers at the apex of the dentate gyrus reached the outer two-thirds of the molecular layer. The distribution and density of mossy fiber-like Timm staining correlated with the biocytin labeling. When viewed with the electron microscope, the inner one-third of the dentate molecular layer contained numerous mossy fiber boutons. In some instances, biocytin-labeled mossy fiber boutons were engaged in synaptic contact with biocytin-labeled granule cell dendrites. Granule cell dendrites did not develop large complex spines (“thorny excrescences”) at the site of synapse formation, and they did not appear to have been permanently damaged by seizure activity. These results establish the validity of Timm staining as a marker for mossy fiber sprouting and support the view that status epilepticus provokes the formation of a novel recurrent excitatory circuit in the dentate gyrus. Retrograde labeling with biocytin showed that the recurrent mossy fiber projection often occupies a considerably greater fraction of the dendritic region than previous studies had suggested. © 1995 Wiley-Liss, Inc.     

Ultrastructural features of sprouted mossy fiber synapses in kindled and kainic acid-treated rats

The mossy fiber pathway in the dentate gyrus undergoes sprouting and synaptic reorganization in response to seizures. The types of new synapses, their location and number, and the identity of their postsynaptic targets determine the functional properties of the reorganized circuitry. The goal of this study was to characterize the types and proportions of sprouted mossy fiber synapses in kindled and kainic acid-treated rats. In normal rats, synapses labeled by Timm histochemistry or dynorphin immunohistochemistry were rarely observed in the supragranular region of the inner molecular layer when examined by electron microscopy. In epileptic rats, sprouted mossy fiber synaptic terminals were frequently observed. The ultrastructural analysis of the types of sprouted synapses revealed that 1) in the supragranular region, labeled synaptic profiles were more frequently axospinous than axodendritic, and many axospinous synapses were perforated; 2) sprouted mossy fiber synaptic terminals formed exclusively asymmetric, putatively excitatory synapses with dendritic spines and shafts in the supragranular region and with the soma of granule cells in the granule cell layer; 3) in contrast to the large sprouted mossy fiber synapses in resected human epileptic hippocampus, the synapses formed by sprouted mossy fibers in rats were smaller; and 4) in several cases, the postsynaptic targets of sprouted synapses were identified as granule cells, but, in one case, a sprouted synaptic terminal formed a synapse with an inhibitory interneuron. The results demonstrate that axospinous asymmetric synapses are the most common type of synapse formed by sprouted mossy fiber terminals, supporting the viewpoint that most sprouted mossy fibers contribute to recurrent excitation in epilepsy.     

Different polysialic acid-neural cell adhesion molecule expression patterns in distinct types of mossy fiber boutons in the adult hippocampus.

Dentate granule cells continue to be generated in the adult hippocampus, and the newly generated granule cells express the highly polysialylated neural cell adhesion molecule (PSA-NCAM), which has been shown to be important in neural development and plasticity. In the present study, the PSA-NCAM expression pattern in morphologically distinct types of mossy fiber boutons in adult rats was examined by immunoelectron and confocal laser scanning microscopy. Although many unmyelinated axons within the mossy fiber bundles expressed PSA-NCAM, most of the mature type of mossy fiber boutons were negative for polysialic acid (PSA) but positive for NCAM peptides, suggesting that NCAM is less polysialylated in the mature mossy fiber boutons. On the other hand, PSA was expressed by small round varicosities, irregularly shaped boutons, and the presumptive immature type of mossy fiber boutons. The PSA-positive small boutons were found to make synaptic contacts with CA3 pyramidal cells and nonpyramidal cells. The PSA-expressing presumptive immature boutons contained fewer clear synaptic vesicles and mitochondria, and, in some instances, they were invaginated by the PSA-positive, finger-like dendritic outgrowths arising from the dendritic shafts of the pyramidal cells, which are known to develop into a mossy fiber bouton-thorny excrescence complex. These findings indicate that distinct types of the mossy fiber boutons possess different PSA expression patterns in the adult hippocampus, and they also imply that PSA expression allows the mossy fibers to have the ability to regulate the bouton formation and remodeling that accompany synapse formation at the contact sites with pyramidal cells and nonpyramidal cells.     

Mossy fiber projections from the cuneate nucleus to the cochlear nucleus in the rat

A reciprocal connection is known to exist between the cuneate nucleus, which is a first-order somatosensory nucleus, and the cochlear nucleus, which is a first-order auditory nucleus. We continued this line of study by investigating the fiber endings of this projection in the cochlear nucleus of rats using the neuronal tracer Phaseolus vulgaris leucoagglutinin in combination with ultrastructural and immunocytochemical analyses. In the cochlear nucleus, mossy fiber terminals had been described and named for their morphologic similarity to those in the cerebellum, but their origins had not been discovered. In the present study, we determined that the axonal projections from the cuneate region gave rise to mossy fiber terminals in the granule cell regions of the ipsilateral cochlear nucleus. The cuneate mossy fibers appear to be excitatory in nature, because they are filled with round synaptic vesicles, they make asymmetric synapses with postsynaptic targets, and they are labeled with an antibody to glutamate. The postsynaptic targets of the mossy fibers include dendrites of granule cells. This projection onto the granule cell interneuron circuit of the cochlear nucleus indicates that somatosensory cues are intimately involved with information processing at this early stage of the auditory system. © 1996 Wiley-Liss, Inc.     

Cartwheel neurons of the dorsal cochlear nucleus: a Golgi-electron microscopic study in rat

Cartwheel neurons in rat dorsal cochlear nucleus (DCN) were studied by Golgi impregnation-electron microscopy. Usually situated in layers 1-2, cartwheel neurons (10-14 micrometers in mean cell body diameter) have dendritic trees predominantly in layer 1. The dendrites branch at wide angles. Most primary dendrites are short, nontapering, and bear only a few sessile spines. Secondary and tertiary dendrites are short, curved, and spine-laden. The perikaryon forms symmetric synapses with at least two kinds of boutons containing pleomorphic vesicles. The euchromatic nucleus is indented and has an eccentric nucleolus. The cytoplasm shows several small Nissl bodies, a conspicuous Golgi apparatus, and numerous subsurface and cytoplasmic cisterns of endoplasmic reticulum with a narrow lumen, joined by mitochondria in single or multiple assemblies. In primary dendrites mitochondria are situated peripherally, while in distal branches they become ubiquitous and relatively more numerous. Dendritic shafts usually form symmetric synapses with boutons that contain pleomorphic vesicles. The majority of the dendritic spines are provided with a vesiculo-saccular spine apparatus. All dendritic spines have asymmetric synapses. Most of these are formed with varicosities of thin, unmyelinated fibers (presumably axons of granule cells) running parallel to the long axis of the DCN or radially. These varicosities contain round, clear synaptic vesicles. On the initial axon segment few symmetric synapses are present. The axon acquires a thin myelin sheath after a short trajectory. Cartwheel neurons outnumber all other neurons in layers 1-2 (with the exception of granule cells), and presumably correspond to type C cells with thinly myelinated axons described by Lorente de Nó. The axons of these neurons provide a dense plexus in the superficial layers without leaving the DCN. The possible functional role of cartwheel neurons is discussed.     

Ultrastructural localization of neurotransmitter immunoreactivity in mossy cell axons and their synaptic targets in the rat dentate gyrus

Electrophysiologically identified and intracellularly biocytin-labeled mossy cells in the dentate hilus of the rat were studied using electron microscopy and postembedding immunogold techniques. Ultrathin sections containing a labeled mossy cell or its axon collaterals were reacted with antisera against the excitatory neurotransmitter glutamate and against the inhibitory neurotransmitter γ-aminobutyric acid (GABA). From single- and double-immunolabeled preparations, we found that 1) mossy cell axon terminals made asymmetric contacts onto postsynaptic targets in the hilus and stratum moleculare of the dentate gyrus and showed immunoreactivity primarily for glutamate, but never for GABA; 2) in the hilus, glutamate-positive mossy cell axon terminals targeted GABA-positive dendritic shafts of hilar interneurons and GABA-negative dendritic spines; and 3) in the inner molecular layer, the mossy cell axon formed asymmetric synapses with dendritic spines associated with GABA-negative (presumably granule cell) dendrites. The results of this study support the view that excitatory (glutamatergic) mossy cell terminals contact GABAergic interneurons and non-GABAergic neurons in the hilar region and GABA-negative granule cells in the stratum moleculare. This pattern of connectivity is consistent with the hypothesis that mossy cells provide excitatory feedback to granule cells in a dentate gyrus associational network and also activate local hilar inhibitory elements. Hippocampus 1997;7:559–570. © 1997 Wiley-Liss, Inc.     

Projections of the pontine nuclei to the cochlear nucleus in rats.

In the cochlear nucleus, there is a magnocellular core of neurons whose axons form the ascending auditory pathways. Surrounding this core is a thin shell of microneurons called the granule cell domain (GCD). The GCD receives auditory and nonauditory inputs and projects in turn to the dorsal cochlear nucleus, thus appearing to serve as a central locus for integrating polysensory information and descending feedback. Nevertheless, the source of many of these inputs and the nature of the synaptic connections are relatively unknown. We used the retrograde tracer Fast Blue to demonstrate that a major projection arises from the contralateral pontine nuclei (PN) to the GCD. The projecting cells are more densely located in the ventral and rostral parts of the PN. They also are clustered into a lateral and a medial group. Injections of anterograde tracers into the PN labeled mossy fibers in the contralateral GCD. The terminals are confined to those parts of the GCD immediately surrounding the ventral cochlear nucleus. There is no PN projection to the dorsal cochlear nucleus. These endings have the form of bouton and mossy fiber endings as revealed by light and electron microscopy. The PN represent a key station between the cerebral and cerebellar cortices, so the pontocochlear nucleus projection emerges as a significant source of highly processed information that is introduced into the early stages of the auditory pathway. The cerebropontocerebellar pathway may impart coordination and timing cues to the motor system. In an analogous way, perhaps the cerebropontocochlear nucleus projection endows the auditory system with a timing mechanism for extracting temporal information.     

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.     

Autoradiography of mossy fiber terminals in the fastigial nucleus of the cat

Terminal boutons of mossy fiber collaterals in the fastigial nucleus originating from the nucleus reticularis tegmenti pontis, the bulbar reticular formation, and the medial vestibular nucleus were studied with high-resolution autoradiography in order to examine their ultrastructural features and synaptic relations. Labeled mossy fiber boutons ranged in size from 0.5 to 5 micron in diameter, and they all contained clear and spherical vesicles in an electron-lucent matrix, mitochondria, and some fine tubular elements. These boutons form asymmetric synapses with dendritic profiles of different sizes. No evidence was found for mossy fiber termination on the soma of fastigial neurons. Two types of mossy fiber terminals were distinguished on the basis of the aggregation of synaptic vesicles: one type with clustered vesicles and one type with densely packed vesicles, occurring in equal number from all sources. Furthermore, the applicability of the congruity hypothesis is confirmed for the general identification of terminals.     

Status epilepticus-induced hilar basal dendrites on rodent granule cells contribute to recurrent excitatory circuitry

Mossy fiber sprouting into the inner molecular layer of the dentate gyrus is an important neuroplastic change found in animal models of temporal lobe epilepsy and in humans with this type of epilepsy. Recently, we reported in the perforant path stimulation model another neuroplastic change for dentate granule cells following seizures: hilar basal dendrites (HBDs). The present study determined whether status epilepticus-induced HBDs on dentate granule cells occur in the pilocarpine model of temporal lobe epilepsy and whether these dendrites are targeted by mossy fibers. Retrograde transport of biocytin following its ejection into stratum lucidum of CA3 was used to label granule cells for both light and electron microscopy. Granule cells with a heterogeneous morphology, including recurrent basal dendrites, and locations outside the granule cell layer were observed in control preparations. Preparations from both pilocarpine and kainate models of temporal lobe epilepsy also showed granule cells with HBDs. These dendrites branched and extended into the hilus of the dentate gyrus and were shown to be present on 5% of the granule cells in pilocarpine-treated rats with status epilepticus, whereas control rats had virtually none. Electron microscopy was used to determine whether HBDs were postsynaptic to axon terminals in the hilus, a site where mossy fiber collaterals are prevalent. Labeled granule cell axon terminals were found to form asymmetric synapses with labeled HBDs. Also, unlabeled, large mossy fiber boutons were presynaptic to HBDs of granule cells. These results indicate that HBDs are present in the pilocarpine model of temporal lobe epilepsy, confirm the presence of HBDs in the kainate model, and show that HBDs are postsynaptic to mossy fibers. These new mossy fiber synapses with HBDs may contribute to additional recurrent excitatory circuitry for granule cells.     

Postnatal maturation of rat Purkinje cells cultivated in the absence of two afferent systems: an ultrastructural study.

Organized cultures of newborn rat cerebellum were established in Maximow chambers in order to study the maturation of Purkinje cells in absence of afferent systems. In the first model, standard cultures were devoid of extracerebellar afferents mossy and climbing fibers. Despite this absence, somatic spines appeared upon Purkinje cells during the first week in vitro and maturation proceeded normally except for the almost absence of spiny branchlets. Large dendritic trunks were studded with numerous spines, some of which were naked, a few bearing isolated post-synaptic densities and others occupied by boutons of parallel fibers. Stellate and basket axons made synapses upon the smooth portions of dendrites and soma. In a second model, the cultures were fed the antimitotic drug methylazoxymethanol (MAM) to prevent multiplication of granule cell precursors. Despite the absence of climbing and parallel fibers, the elongation of Purkinje dendrites was not prevented, but again the dendritic arbor consisted of large trunks studded with spines; somatic as well as dendritic spines were contacted by large boutons identified as Purkinje recurrent collaterals (PRC). It is concluded that the Purkinje cell possesses a large autonomy from afferent systems as to the growth of soma and dendrites. Conversely, the geometry of the dendrite and especially the spiny branchlets depend on the presence of both climbing and parallel fibers. One may conclude from the above experiments that specificity of synaptic contacts is maintained as long as postsynaptic sites are not devoid of their normal afferents. Heterologous synapses are formed when postsynaptic sites are present, their normal afferents absent and aberrant ones increasing by collateral sprouting. Such is probably the case in the second model of this study.     

Vessicular glutamate transporters 1 and 2 are differentially associated with auditory nerve and spinal trigeminal inputs to the cochlear nucleus

Projections of glutamatergic somatosensory and auditory fibers to the cochlear nucleus (CN) are mostly nonoverlapping: projections from the spinal trigeminal nucleus (Sp5) terminate primarily in the granule cell domains (GCD) of CN, whereas type I auditory nerve fibers (ANFs) project to the magnocellular areas of the VCN (VCNm) and deep layers of Dorsal CN (DCN). Vesicular glutamate transporters (VGLUTs), which selectively package glutamate into synaptic vesicles, have different isoforms associated with distinct subtypes of excitatory glutamatergic neurons. Here we examined the distributions of VGLUT1 and VGLU2 expression in the CN and their colocalization with Sp5 and ANF terminals following injections of anterograde tracers into Sp5 and the cochlea in the guinea pig. The CN regions that showed the most intense expression of VGLUT1 and VGLUT2 were largely nonoverlapping and were consistent with ANF and Sp5 projections, respectively: VGLUT1 was highly expressed in VCNm and the molecular layer of the DCN, whereas VGLUT2 was expressed predominantly in the GCD. Half (47% +/- 3%) of the Sp5 mossy fiber endings colabeled with VGLUT2, but few (2.5% +/- 1%) colabeled with VGLUT1. In contrast, ANFs colabeled predominantly with VGLUT1. The pathway-specific expression of VGLUT isoforms in the CN may be associated with the intrinsic synaptic properties that are unique to each sensory pathway.     

Roles of afadin in the formation of the cellular architecture of the mouse hippocampus and dentate gyrus

The hippocampal formation with tightly packed neurons, mainly at the dentate gyrus, CA3, CA2, and CA1 regions, constitutes a one-way neural circuit, which is associated with learning and memory. We previously showed that the cell adhesion molecules nectins and its binding protein afadin play roles in the formation of the mossy fiber synapses which are formed between the mossy fibers of the dentate gyrus granule cells and the dendrites of the CA3 pyramidal cells. We showed here that in the afadin-deficient hippocampal formation, the dentate gyrus granules cells and the CA3, CA2, and CA1 pyramidal cells were abnormally located; the mossy fiber trajectory was abnormally elongated; the CA3 pyramidal cells were abnormally differentiated; and the densities of the presynaptic boutons on the mossy fibers and the apical dendrites of the CA3 pyramidal cells were decreased. These results indicate that afadin plays roles not only in the formation of the mossy fiber synapses but also in the formation of the cellular architecture of the hippocampus and the dentate gyrus.     

Kainic acid-induced mossy fiber sprouting and synapse formation in the dentate gyrus of rats

In the kainic acid (KA) model of temporal lobe epilepsy, mossy fibers (MFs) are thought to establish recurrent excitatory synaptic contacts onto granule cells. This hypothesis was tested by intracellular labeling of granule cells with biocytin and identifying their synaptic contacts in the dentate molecular layer with electron microscopic (EM) techniques. Twenty-three granule cells from KA-treated animals and 14 granule cells from control rats were examined 2 to 4 months following KA at the light microscopic (LM) level; four cells showing MF sprouting were further characterized at the EM level. Timm staining revealed a time-dependent growth of aberrant MFs into the dentate inner molecular layer. The degree of sprouting was generally (but not invariably) correlated with the severity and frequency of seizures. LM examination of individual biocytin-labeled MF axon collaterals revealed enhanced collateralization and significantly increased numbers of synaptic MF boutons in the hilus compared to controls, as well as aberrant MF growth into the granule cell and molecular layers. EM examination of serially reconstructed, biocytin-labeled MF collaterals in the molecular layer revealed MF boutons that form asymmetrical synapses with dendritic shafts and spines of granule cells, including likely autaptic contacts on parent dendrites of the biocytin-labeled granule cell. These results constitute ultrastructural evidence for newly formed excitatory recurrent circuits, which might provide a structural basis for enhanced excitation and epileptogenesis in the hippocampus of KA-treated rats.     

Extraordinary synapses of the unipolar brush cell: An electron microscopic study in the rat cerebellum

A neglected type of neuron, termed the unipolar brush cell, was recently characterized in the granular layer of the mammalian cerebellar cortex with several procedures, including light and electron microscopic immunocytochemistry utilizing antibodies to calretinin and neurofilament proteins. Although certain features of the unipolar brush cells were highlighted in these studies, the internal fine structure was partially obfuscated by immunoreaction product. In this study, rat cerebella were prepared for electron microscopy after perfusion fixation and Araldite embedding, and folia of the vestibulo-cerebellum, where unipolar brush cells are known to be enriched, were studied by light microscopy in semithin (0.5–1 μ) sections and by electron microscopy in ultrathin sections. Unipolar brush cells were easily identified in semithin sections immunostained with antibodies to GABA and/or glycine, and cbunterstained with toluidine blue. The unipolar brush cells have a pale cytoplasm and are GABA and glycine negative, while Golgi cells are darker and appear positive for GABA and, for the most part, also for glycine. Sets of identification criteria to differentiate unipolar brush cells from granule and Golgi cells in standard electron micrographs are presented. The unipolar brush cells possess many distinctive features that make them easily distinguishable from other cerebellar neurons and form unusually conspicuous and elaborate synapses with mossy rosettes. The unipolar brush cell has a deeply indented nucleus containing little condensed chromatin. The Golgi apparatus is large and the cytoplasm is rich in neurofilaments, microtubules, mitochondria, and large dense core vesicles, but contains few cisterns of granular endoplasmic reticulum. In addition, unipolar brush cells contain an unusual inclusion, which invariably lacks a limiting membrane and is made up of peculiar ringlet subunits. The cell body usually emits a thin axon and is provided with a single, large dendritic trunk that terminates with a paintbrush-like bunch of branchlets. Numerous nonsynaptic appendages emanate from the cell body, the dendritic stem, and the branchlets. The appendages contain rare organelles and lack neurofilaments. The branchlets contain numerous mitochondria, neurofilaments, large dense core vesicles, and clusters of clear, small, and round synaptic vesicles. They form extensive asymmetric synaptic junctions with of a or two mossy fibers, which indicates minimal convergence of excitatory inputs. Under the postsynaptic densities, the branchlet cytoplasm displays a microfilamentous web. Besides their contact with mossy rosettes, the branchlets form symmetric and asymmetric synaptic junctions with presumed Golgi cell boutons that contain pleomorphic synaptic vesicles, indicating that the unipolar brush cells receive an inhibitory modulation. Some of these junctions are unusually extensive. The branchlets also form asymmetric synapses with granule cell dendrites, in which they represent the presynaptic elements, a feature never described before in the normal cerebellum. A minority of the unipolar brush cells receive mossy fiber contacts directly on the cell body, or on short dendritic branchlets emanating directly from the cell body. Such “enmarron” synapses were previously attributed to Golgi cells. Thus, the unipolar brush cells have complex synaptic features: Besides being specialized to form a powerful link with mossy rosettes, they may also have a paracrine function, and they participate with presynaptic dendrites in the cerebellar microcircuit. © 1994 Wiley-Liss, Inc.     

Ultrastructural localization of dynorphin in the dentate gyrus in human temporal lobe epilepsy: a study of reorganized mossy fiber synapses

Substantial reorganization of mossy fibers from granule cells of the dentate gyrus occurs in a high percentage of humans with medically intractable temporal lobe epilepsy. To identify these fibers and determine their ultrastructural features in human surgical specimens, we used preembedding immunoperoxidase labeling of dynorphin A, an opioid peptide that is abundant in normal mossy fibers. In electron microscopic preparations, dynorphin A immunoreactivity was highly associated with dense core vesicles and was localized predominantly in axon terminals in the inner molecular layer of the dentate gyrus, although some dynorphin-labeled dense core vesicles were also observed in dendritic shafts and spines. The labeled terminal profiles were numerous, and, whereas they varied greatly in size, many were relatively large (2.3 microm in mean major diameter). The terminals contained high concentrations of clear round vesicles and numerous mitochondrial profiles, formed distinct asymmetric synapses, often had irregular shapes, and, thus, exhibited many features of normal mossy fiber terminals. The dynorphin-labeled terminals formed synaptic contacts primarily with dendritic spines, and some of these spines were embedded in large labeled terminals, suggesting that they were complex spines. The labeled terminals frequently formed multiple synaptic contacts with their postsynaptic elements, and perforated postsynaptic densities, with and without spinules, were present at some synapses. These findings suggest that the reorganized mossy fiber terminals in humans with temporal lobe epilepsy form abundant functional synapses in the inner molecular layer of the dentate gyrus, and many of these contacts have ultrastructural features that could be associated with highly efficacious synapses.     

Subdivisions in the Multiple GABAergic Innervation of Granule Cells in the Dentate Gyrus of the Rat Hippocampus

The sources of GABAergic innervation to granule cells were studied to establish how the basic cortical circuit is implemented in the dentate gyrus. Five types of neuron having extensive local axons were recorded electrophysiologically in vitro and filled intracellularly with biocytin (Han et al., 1993). They were processed for electron microscopy in order to reveal their synaptic organization and postsynaptic targets, and to test whether their terminals contained GABA. (1) The hilar cell, with axon terminals in the commissural and association pathway termination field (HICAP cell), formed Gray's type 2 (symmetrical) synapses with large proximal dendritic shafts (n= 18), two-thirds of which could be shown to emit spines, and with small dendritic branches (n= 6). Other boutons of the HICAP neuron were found to make either Gray's type 1 (asymmetrical) synapses (n= 4) or type 2 synapses (n= 6) with dendritic spines. Using a highly sensitive silver-intensified immunogold method for the postembedding visualization of GABA immunoreactivity, both the terminals and the dendrites of the HICAP cell were found to be immunopositive, whereas its postsynaptic targets were GABA-immunonegative. The dendritic shafts of the HICAP cell received synapses from both GABA-negative and GABA-positive boutons; the dendritic spines which densely covered the main apical dendrite in the medial one-third of the molecular layer received synapses from GABA-negative boutons. (2) The hilar cell, with axon terminals distributed in conjunction with the perforant path termination field (HIPP cell), established type 2 synapses with distal dendritic shafts (n= 17), most of which could be shown to emit spines, small-calibre dendritic profiles (n= 2) and dendritic spines (n= 6), all showing characteristics of granule cell dendrites. The sparsely spiny dendrites of the HIPP cell were covered with many synaptic boutons on both their shafts and their spines. (3) The cell with soma in the molecular layer had an axon associated with the perforant path termination field (MOPP cell). This GABA-immunoreactive cell made type 2 synapses exclusively on dendritic shafts (n= 20), 60% of which could be shown to emit spines. The smooth dendrites of the MOPP cell were also restricted to the outer two-thirds of the molecular layer, where they received both GABA-negative and GABA-positive synaptic inputs. (4) The extensive axonal arborization of the dentate basket cell terminated mainly on somata (n= 26) and proximal dendrites (n= 9) in the granule cell layer, and some boutons made synapses on somatic spines (n= 6); all boutons established type 2 synapses. (5) The dentate axo-axonic cell established type 2 synapses (n= 14) exclusively on axon initial segments of granule cells in the granule cell layer, and on initial segments of presumed mossy cells in the hilus. The results demonstrate that granule cells receive inputs from the local circuit axons of at least five distinct types of dentate neuron terminating in mutually exclusive domains of the cell's surface in four out of five cases. Four of the cell types (HICAP cell, MOPP cell, basket cell, axo-axonic cell) contain GABA, and the HIPP cell may also be inhibitory. The specific local inhibitory neurons terminating in conjunction with particular excitatory amino acid inputs to the granule cells (types 1 – 3) are in a position to interact selectively with the specific inputs on the same dendritic segment. This arrangement provides a possibility for the independent regulation of the gain and long-term potentiation of separate excitatory inputs, through different sets of GABAergic local circuit neurons. The pairing of excitatory and inhibitory inputs may also provide a mechanism for the downward reseating of excitatory postsynaptic potentials, thereby extending their dynamic range.