3D electron microscopic reconstruction of segments of rat cerebellar purkinje cell dendrites receiving ascending and parallel fiber granule cell synaptic inputs

Growing physiological evidence suggests that there are functional differences between synapses made by the ascending and parallel fiber segments of the granule axon on cerebellar Purkinje cells. Supporting this view, our previous electron microscopic studies suggested that these synapses also contacted different regions of the Purkinje cell dendrite, and in particular that ascending segment synapses are made exclusively on the smallest diameter Purkinje cell dendrites. In the current study we used serial electron microscopic techniques to reconstruct Purkinje cell dendritic segments up to almost 10 μm in length. Using a combination of anatomical and immunological labeling techniques we identified the ascending or parallel fiber origins of the excitatory synaptic inputs onto dendritic spines, as well as the location of inhibitory synapses made directly on the dendritic shaft. The results confirmed that there are regions of the Purkinje cell dendrite receiving exclusively ascending or parallel fiber synapses and that ascending segment synapses are only found on small‐diameter dendrites. In addition, we describe for the first time small‐diameter dendritic regions contacted by both types of excitatory synapses. While our data suggest that the majority of inhibitory inputs to the Purkinje cell tree are associated with parallel fiber synaptic inputs, we also found inhibitory inputs on dendritic regions with mixed ascending and parallel fiber inputs, or exclusively parallel fiber inputs. The finding that ascending and parallel fiber inputs can be segregated on the Purkinje cell dendritic tree provides further evidence that these excitatory granule cell synaptic inputs may be functionally distinct. J. Comp. Neurol. 514:583–594, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

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.     

Axon arbors and synaptic connections of a vulnerable population of interneurons in the dentate gyrus in vivo

The predominant gamma-aminobutyric acid (GABA)ergic neuron class in the hilus of the dentate gyrus consists of spiny somatostatinergic interneurons. We examined the axon projections and synaptic connections made by spiny hilar interneurons labeled with biocytin in gerbils in vivo. Axon length was 152-497 mm/neuron. Sixty to 85% of the axon concentrated in the outer two thirds of the molecular layer of the dentate gyrus. The septotemporal span of the axon arbor extended over 48-82% of the total hippocampal length, which far exceeds the septotemporal span of axons of granule cells whose complete axon arbors extended over 15-29%. A three-dimensionally reconstructed 216-microm-long spiny hilar interneuron axon segment in the outer third of the molecular layer formed an average of 1 synapse every 5.1 microm. Of the 42 symmetric (inhibitory) synapses formed by the reconstructed segment, 88% were with spiny dendrites of presumed granule cells, and 67% were with dendritic spines that also receive an asymmetric (excitatory) contact from an unlabeled axon terminal. Postembedding GABA-immunocytochemistry revealed that 55% of the GABAergic synapses in the outer third of the molecular layer were with spines. Therefore, in the outer molecular layer, spiny hilar interneurons form synaptic contacts that appear to be positioned to exert inhibitory control near sites of excitatory synaptic input from the entorhinal cortex to granule cell dendritic spines. These findings demonstrate far-reaching, yet highly specific, connectivity of individual interneurons and suggest that the loss of spiny hilar interneurons, as occurs in temporal lobe epilepsy, may contribute to hyperexcitability in the hippocampus.     

Convergence of excitatory and inhibitory inputs onto CCK-containing basket cells in the CA1 area of the rat hippocampus

The number and distribution of excitatory and inhibitory inputs affect the integrative properties of neurons. These parameters have been studied recently for several hippocampal neuron populations. Besides parvalbumin- (PV) containing cells that include basket and axo-axonic cells, cholecystokinin (CCK)-containing interneurons also form a basket cell population with several properties distinct from PV cells. Here, at the light microscopic level, we reconstructed the entire dendritic tree of CCK-immunoreactive (IR) basket cells to describe their geometry, the total length and laminar distribution of their dendrites. This was followed by an electron microscopic analysis of serial ultrathin sections immunostained against gamma-aminobutyric acid, to estimate the density of excitatory and inhibitory synapses on their somata, axon initial segments and different subclasses of dendrites. The dendritic tree of CCK-IR basket cells has an average length of 6300 microm and penetrates all layers. At the electron microscopic level, CCK basket cells receive dendritic inputs with a density of 80-230 per 100 microm. The ratio of inhibitory inputs is relatively high (35%) and increases towards the soma (83%). The total numbers of excitatory and inhibitory synapses converging onto CCK-IR cells are approximately 8200. Comparison of the two, neurochemically distinct basket cells reveals that CCK-containing basket cells receive much less synaptic input than PV cells; however, the relative weight of inhibition is higher on CCK cells. Additional differences in their anatomical and physiological properties predict that CCK basket cells are under a more diverse, elaborate control than PV basket cells, and thus the function of the two populations must be different.     

Dendrodendritic synapses in the mouse olfactory bulb external plexiform layer

Odor information relayed by olfactory bulb projection neurons, mitral and tufted cells (M/T), is modulated by pairs of reciprocal dendrodendritic synaptic circuits in the external plexiform layer (EPL). Interneurons, which are accounted for largely by granule cells, receive depolarizing input from M/T dendrites and in turn inhibit current spread in M/T dendrites via hyperpolarizing reciprocal dendrodendritic synapses. Because the location of dendrodendritic synapses may significantly affect the cascade of odor information, we assessed synaptic properties and density within sublaminae of the EPL and along the length of M/T secondary dendrites. In electron micrographs the M/T to granule cell synapse appeared to predominate and was equivalent in both the outer and inner EPL. However, the dendrodendritic synapses from granule cell spines onto M/T dendrites were more prevalent in the outer EPL. In contrast, individual gephyrin‐immunoreactive (IR) puncta, a postsynaptic scaffolding protein at inhibitory synapses used here as a proxy for the granule to M/T dendritic synapse was equally distributed throughout the EPL. Of significance to the organization of intrabulbar circuits, gephyrin‐IR synapses are not uniformly distributed along M/T secondary dendrites. Synaptic density, expressed as a function of surface area, increases distal to the cell body. Furthermore, the distributions of gephyrin‐IR puncta are heterogeneous and appear as clusters along the length of the M/T dendrites. Consistent with computational models, our data suggest that temporal coding in M/T cells is achieved by precisely located inhibitory input and that distance from the soma is compensated for by an increase in synaptic density. J. Comp. Neurol. 523:1145–1161, 2015. © 2015 Wiley Periodicals, Inc.     

Ultrastructure and GABA Immunoreactivity in Layers 8 and 9 of the Optic Tectum of Xenopus laevis

This study presents an ultrastructural analysis of layers 8 and 9 in the optic tectum of Xenopus laevis. Retinotectal axons were labelled with horseradish peroxidase and tectal cells were labelled with antibody to GABA. Four distinct axonal and dendritic structures were identified. GABA-negative axon terminals formed asymmetric synapses and were categorized as type a-1 (which included retinotectal axons), characterized by medium size synaptic vesicles and pale mitochondria, and type a-2 (non-retinotectal) with large vesicles and dense mitochondria. GABA-negative dendrites (type d) contained dense mitochondria, microtubules in the dendritic shafts, and dendritic spines devoid of microtubules. GABA-positive structures contained small synaptic vesicles and dense mitochondria. Some dendrites (type D) were not only postsynaptic but were also presynaptic elements, as defined by the presence of vesicles and distinct synaptic clefts with symmetric specializations. GABA-positive presynaptic structures were mostly located in vesicle-filled, bulbous extensions of dendritic shafts and usually terminated onto dendritic spines. Some type D dendrites were the middle element in serial synapses, with input from either GABA-positive or GABA-negative structures and output to GABA-negative structures. Retinotectal terminals were identified as one of the synaptic inputs to GABA-positive processes. Glia were characterized by granular cytoplasm and large mitochondria, often displaying a crystalline matrix structure. These results indicate that GABA-positive neurons are a prominent component of circuitry in the superficial layers of the tectum of Xenopus and that, as in mammals, they participate in serial synaptic arrangements in which retinotectal axons are the first element. These arrangements are consistent with complex processing of visual input to the tectum and a central role for inhibitory processes in the shaping of tectal responses.     

Fine structure of the cell clusters in the cochlear nerve root: Stellate,granule, and mitt cells offer insights into the synaptic organization of local circuit neurons

The small cell shell of the cochlear nucleus contains a complex integrative machinery which can be used to study the roles of interneurons in sensory processing. The cell clusters in the cochlear nerve root of the chinchilla provide the simplest example of this structure. Reported here are the neuronal architecture and synaptic organization of the three principal cell types and the three distinctive neuropil structures that could be characterized with the Nissl and Golgi methods and electron microscopy. Granule cells were characterized by several dendrites with claw-like terminals that received synaptic contacts from multiple excitatory mossy fiber rosettes. Given their relatively large number and their prolific parallel fiber synapses, the granule cells provide a suitable substrate for a tangential spread of excitatory activity, which could build to considerable proportions. The mitt cells had a thickened, single dendrite, its terminal branches arranged in a shape reminiscent of a baseball catcher's mitt. The dendritic mitt enclosed an enormous, convoluted mossy fiber rosette forming many excitatory synapses on just one cell. This could provide for a discrete, comparatively fast input-output relay of signals. Small stellate cells had longer, radiating dendrites that engaged the synaptic nests. These nests were strung in long strands, containing heterogeneous synapses from putative excitatory and inhibitory inputs. Given the prevalence of the synaptic nests, the small stellate cells appear to have the greatest integrative capacity. They provide the main output of the synaptic nests. © 1996 Wiley-Liss, Inc.     

A novel role for MNTB neuron dendrites in regulating action potential amplitude and cell excitability during repetitive firing

Principal cells of the medial nucleus of the trapezoid body (MNTB) are simple round neurons that receive a large excitatory synapse (the calyx of Held) and many small inhibitory synapses on the soma. Strangely, these neurons also possess one or two short tufted dendrites, whose function is unknown. Here we assess the role of these MNTB cell dendrites using patch-clamp recordings, imaging and immunohistochemistry techniques. Using outside-out patches and immunohistochemistry, we demonstrate the presence of dendritic Na⁺ channels. Current-clamp recordings show that tetrodotoxin applied onto dendrites impairs action potential (AP) firing. Using Na⁺ imaging, we show that the dendrite may serve to maintain AP amplitudes during high-frequency firing, as Na⁺ clearance in dendritic compartments is faster than axonal compartments. Prolonged high-frequency firing can diminish Na⁺ gradients in the axon while the dendritic gradient remains closer to resting conditions; therefore, the dendrite can provide additional inward current during prolonged firing. Using electron microscopy, we demonstrate that there are small excitatory synaptic boutons on dendrites. Multi-compartment MNTB cell simulations show that, with an active dendrite, dendritic excitatory postsynaptic currents (EPSCs) elicit delayed APs compared with calyceal EPSCs. Together with high- and low-threshold voltage-gated K⁺ currents, we suggest that the function of the MNTB dendrite is to improve high-fidelity firing, and our modelling results indicate that an active dendrite could contribute to a 'dual' firing mode for MNTB cells (an instantaneous response to calyceal inputs and a delayed response to non-calyceal dendritic excitatory postsynaptic potentials).     

Map of the synapses formed with the dendrites of spiny stellate neurons of cat visual cortex

The synaptic input of six spiny stellate neurons in sublamina 4A of cat area 17 was assessed by electron microscopy. The neurons were physiologically characterized and filled with horseradish peroxidase in vivo. After processing the neurons were reconstructed at the light microscopic level using computer-assisted methods and analyzed quantitatively. The extensive branching of the dendritic tree about 50 μm from the soma meant that the distal branches constituted five times the length of proximal dendrite. Proximal and distal portions of a single dendrite from each neuron were examined in series of ultrathin sections (1,456 sections) in the electron microscope. The majority (79%) of the 263 synapses examined were asymmetric; the remainder (21%) were symmetric. Symmetric synapses formed 35% of synapses sampled on proximal dendrites and were usually located on the shaft. They formed only 4% of synapses sampled on distal dendrites. Spines accounted for less than half of the total asymmetric synapses (45%); the remainder were on shafts. Symmetric synapses formed with four of 92 spines. Nine spines formed no synapses. Spiny stellate neurons in cat visual cortex appear to differ considerably from pyramidal neurons in having a significant asymmetric (excitatory) synaptic input to the dendritic shaft.     

Morphology, Electrophysiology and Pathophysiology of Supragranular Neurons in Rat Primary Somatosensory Cortex

Intracellularly biocytin-labelled neurons in layers 11/111 of adult rat primary somatosensory cortex were analysed for their morphological and electrophysiological properties and studied for their response pattern to transient hypoxia under in vitro conditions. The largest dendritic region is formed by the basal dendrites, which constitute an average area of 0.06 mm² and which can receive synaptic inputs over horizontal distances of more than 300 μm. The dendritic territories formed by the oblique dendrites situated on the apical trunk and by the apical tuft are much smaller. The spine density is highest on the apical trunk, suggesting that large numbers of excitatory synapses are present in this region of the cell. All neurons revealed intrinsic membrane properties of typical regular spiking cells and received an excitatory and a strong biphasic inhibitory input. Whereas a significant correlation could be detected between the cell's input resistance and soma area, no correlation existed between the cell's total dendritic length and input resistance or membrane time constant/input resistance. Neurons responded to transient hypoxia either with an anoxic hyperpolarization with an apparent reversal potential of -82.4 mV, or with a gradual anoxic depolarization which reversed at -56 mV. Oxygen deprivation caused a significant reduction in the extent of axonal collaterals, whereas dendritic proportions and spine density were unaffected. The present study indicates that the dendritic tree is well preserved under in vitro conditions, whereas axonal connections are diminished by oxygen deprivation. Our results further suggest that certain structural properties correlate with the cellular physiology, but that the cell's morphology does not determine its responsiveness to hypoxia.     

Innervation of cat visual areas 17 and 18 by physiologically identified X- and Y- type thalamic afferents. II. Identification of postsynaptic targets by GABA immunocytochemistry and Golgi impregnation

The precise location of physiologically identified specific afferent input on the different types of cell in the visual cortex and the identification of the neurotransmitters of these cells are essential to a better understanding of the first stage of cortical processing. A combination of anatomical, neurochemical, and physiological methods was used to identify the cortical neurones that receive synaptic input from X- and Y-type afferents, which are thought to originate from cells of the lateral geniculate nucleus. One method relied on chance contacts made between single physiologically characterised axons, which had been injected with horseradish peroxidase (HRP), and the processes of cells impregnated by the Golgi method. These experiments revealed that both X and Y axons formed synapses on the dendrites of spiny stellate cells in layer 4. Y axons in both areas 17 and 18 established multiple synaptic contacts on basal dendrites of layer 3 pyramidal cells. One X axon contacted the apical dendrite of a layer 5 pyramidal cell and one Y axon contacted the dendrite of a large cell with smooth dendrites in layer 3. The maximum number of synapses made between one axon and a single postsynaptic cell was eight, although in most cases it was only one. It was concluded that one axon only provides a small fraction of the geniculate afferent input to an individual cell. A second method revealed that the somata in layer 4 in synaptic contact with the HRP-filled axon terminals were GABA-immunoreactive, and therefore might be involved in inhibitory processes. From light microscopic data it was found that somata receiving contacts from X axons in area 17 were significantly smaller (average diameter 15 microns) than those contacted by the Y axons in areas 17 and 18 (average diameter 24 microns). Somatic contacts were extremely rare in layer 6. These data show that the X and Y afferents may activate separate subsets of inhibitory neurones.     

Initial loss but later excess of GABAergic synapses with dentate granule cells in a rat model of temporal lobe epilepsy

Many patients with temporal lobe epilepsy display neuron loss in the dentate gyrus. One potential epileptogenic mechanism is loss of GABAergic interneurons and inhibitory synapses with granule cells. Stereological techniques were used to estimate numbers of gephyrin‐positive punctae in the dentate gyrus, which were reduced short‐term (5 days after pilocarpine‐induced status epilepticus) but later rebounded beyond controls in epileptic rats. Stereological techniques were used to estimate numbers of synapses in electron micrographs of serial sections processed for postembedding GABA‐immunoreactivity. Adjacent sections were used to estimate numbers of granule cells and glutamic acid decarboxylase‐positive neurons per dentate gyrus. GABAergic neurons were reduced to 70% of control levels short‐term, where they remained in epileptic rats. Integrating synapse and cell counts yielded average numbers of GABAergic synapses per granule cell, which decreased short‐term and rebounded in epileptic animals beyond control levels. Axo‐shaft and axo‐spinous GABAergic synapse numbers in the outer molecular layer changed most. These findings suggest interneuron loss initially reduces numbers of GABAergic synapses with granule cells, but later, synaptogenesis by surviving interneurons overshoots control levels. In contrast, the average number of excitatory synapses per granule cell decreased short‐term but recovered only toward control levels, although in epileptic rats excitatory synapses in the inner molecular layer were larger than in controls. These findings reveal a relative excess of GABAergic synapses and suggest that reports of reduced functional inhibitory synaptic input to granule cells in epilepsy might be attributable not to fewer but instead to abundant but dysfunctional GABAergic synapses. J. Comp. Neurol. 518:647–667, 2010. © 2009 Wiley‐Liss, Inc.     

Axon arbors and synaptic connections of hippocampal mossy cells in the rat in vivo

The axon collateralization patterns and synaptic connections of intracellularly labeled and electrophysiologically identified mossy cells were studied in rat hippocampus. Light microscopic analysis of 11 biocytin-filled cells showed that mossy cell axon arbors extended through an average of 57% of the total septotemporal length of the hippocampus (summated two-dimensional length, not adjusted for tissue shrinkage). Axon collaterals were densest in distant lamellae rather than in lamellae near the soma. Most of the axon was concentrated in the inner one-third of the molecular layer, with the hilus containing an average of only 26% of total axon length and the granule cell layer containing an average of only 7%. Ultrastructural analysis was carried out on three additional intracellularly stained mossy cells, in which axon collaterals and synaptic targets were examined in serial sections of chosen axon segments. In the central and subgranular regions of the hilus, mossy cell axons established a low density of synaptic contacts onto dendritic shafts, neuronal somata, and occasional dendritic spines. Most hilar synapses were made relatively close to the mossy cell somata. At greater distances from the labeled mossy cell (1–2 mm along the septotemporal axis), the axon collaterals ramified predominantly within the inner molecular layer and made a high density of asymmetric synaptic contacts almost exclusively onto dendritic spines. Quantitative measurements indicated that more than 90% of mossy cell synaptic contacts in the ipsilateral hippocampus are onto spines of proximal dendrites of presumed granule cells. These results are consistent with a primary mossy cell role in an excitatory associational network with granule cells of the dentate gyrus. © 1996 Wiley-Liss, Inc.     

Localization of a leech inhibitory synapse by photo-ablation of individual dendrites

An inhibitory motor neuron (cell 1) in the leech nervous system has a powerful inhibitory connection onto an excitatory motor neuron (cell 3) that is functionally important in behaviours such as swimming and local bending. The anatomical location of this connection was explored using focal ultraviolet irradiation of cell 3 dendrites filled with Lucifer yellow. Ablation of the main neurite of cell 3 in the middle of the ganglion eliminated 72% of the inhibitory postsynaptic potential (IPSP), showing that most of the synaptic contacts are in the dendritic field contralateral to the cell body. Ablation of a particular dendritic branch (d1), one of several that run anteriorly from the main neurite in the contralateral ganglion, eliminated 70% of the IPSP in some cases but only 4% in others. In these latter cases, subsequent ablation of a more distal dendrite (d2) eliminated from 41% to 83% of the IPSP. These findings suggest that the synapses onto cell 3 from cell 1 are primarily mediated by either one of these dendrites or the other, but not both. This synaptic specificity might be due to a developmental mechanism involving competition between dendrites for occupation of synaptic sites.     

Integration of newly born dentate granule cells into adult brains: hypotheses based on normal and epileptic rodents

The granule cells of the dentate gyrus are a population of neurons continously generated throughout life. In the rat, the morphological development of newly born granule cells generated in the adult share many similarities with granule cells generated during development. These include a specific migration pattern, orientation and progression of neurite outgrowth. It appears as though varied dendritic morphology occurs depending on the position of the granule cells within the granule cell layer. A hypothesis for granule cell migration and differentiation of their dendritic processes is proposed based on normal and epileptic rats. In this hypothesis, the granule cells are generated in the subgranular zone, and then they migrate into the granule cell layer. During this migration, the sequence of neurite outgrowth is described, where the newly born granule cell first sprouts rudimentary processes. One of these processes, the basal dendrite, is transiently present on developing rodent granule cells in rats. However, in seizure-induced rats the basal dendrite often fails to retract, which leads to the formation of hilar basal dendrites, and also perhaps, ectopic granule cells in the hilus. In this review, granule cell development is discussed with relevance to the creation of the recurrent excitatory circuitry in rodent models of temporal lobe epilepsy.     

GABAergic circuitry in the dorsal division of the cat medial geniculate nucleus

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter of the thalamus. We used postembedding immunocytochemistry to examine the synaptic organization of GABA-positive profiles in the dorsal superficial subdivision of the cat medial geniculate nucleus (MGN). Three groups of GABA-positive profiles participate in synapses: axon terminals, dendrites, and presynaptic dendrites. The presynaptic GABA-positive terminals target mainly GABA-negative dendrites. The GABA-positive postsynaptic profiles receive input primarily from GABA-negative axons. The results indicate that the synaptic organization of GABA-positive profiles in the dorsal superficial subdivision of the MGN nucleus is very similar to that in other thalamic nuclei.     

Axo-axonic chandelier cells in the rat fascia dentata: Golgi-electron microscopy and immunocytochemical studies.

Synaptic transmission can be blocked very efficiently by inhibitory synapses on axon initial segments. Inhibitory chandelier cells forming synapses on the axon initial segment of pyramidal neurons have been found in the neocortex and hippocampus proper. Here we describe an axo-axonic local circuit neuron in the rat fascia dentata that establishes synaptic contacts with axon initial segments of numerous dentate granule cells. Examination of a large number of Golgi-impregnated nongranule cells in the fascia dentata of rats revealed a group of neurons with characteristics of chandelier cells. Thus these cells exhibited an extensive axonal plexus within the granular layer that characteristically formed vertical aggregations of axonal varicosities. The cell bodies of these neurons were located in the inner molecular layer or in the outer part of the granular layer. Their dendrites invaded the molecular layer, suggesting an afferent innervation similar to that of the granule cells. Well impregnated putative axo-axonic cells were gold-toned for an electron microscopic analysis. The cell bodies and dendrites of these neurons exhibited characteristic ultrastructural features of nongranule cells, i.e., large amounts of perinuclear cytoplasm, infoldings of the nuclear membrane, and a large number of synaptic contacts on the perikaryon and on the smooth dendritic shafts. The axon originating from the cell body or from a proximal dendrite gave rise to numerous vesicle-filled varicosities that almost exclusively formed symmetric synaptic contacts with axon initial segments. A semiquantitative study of five axonal complexes demonstrated that 92.3% of identified postsynaptic elements were initial segments of granule cell axons. Immunostaining with antibodies against glutamate decarboxylase (GAD) and parvalbumin (PARV) revealed a subpopulation of neurons that very much resembled the Golgi-impregnated axo-axonic cells with regard to cell body location, dendritic arborization, and fine structural characteristics of perikarya and dendrites. GAD and PARV were found to be coexistent in these cells. Moreover, we found GAD- and PARV-immunoreactive terminals in symmetric synaptic contact with axon initial segments of granule cells. The present study has shown a hitherto unknown axo-axonic cell in the rat fascia dentata. On the basis of our immunocytochemical findings, we hypothesize that this cell exerts a strong inhibitory effect on dentate granule cells. This way, signal transmission from the fascia dentata to the hippocampus proper within the "trisynaptic pathway" can efficiently be controlled by a group of highly specialized neurons.     

Afferent and efferent synaptic connections of somatostatin-immunoreactive neurons in the rat fascia dentata

The aim of this study was to determine whether somatostatin (SS)-immunoreactive neurons of the rat fascia dentata are involved in specific excitatory circuitries that may result in their selective damage in models of epilepsy. Synaptic connections of SS-immunoreactive neurons were determined at the electron microscopic level by using normal and colchicine pretreated rats. Vibratome sections prepared from both fascia dentata of control animals and from rats that had received an ipsilateral lesion of the entorhinal cortex 30-36 hours before sacrifice were immunostained for SS by using a monoclonal antibody (SS8). Correlated light and electron microscopic analysis demonstrated that many SS-immunoreactive neurons in the hilus send dendritic processes into the outer molecular layer of the fascia dentata, and dendrites of the same neurons occupy broad areas in the dentate hilar area. The majority of SS-immunoreactive axon terminals form symmetric synapses with the granule cell dendrites in the outer molecular layer and also innervate deep hilar neurons. Via their dendrites in the outer molecular layer, the SS-immunoreactive neurons receive synaptic inputs from perforant pathway axons which were identified by their anterograde degeneration following entorhinal lesions. The axons from the entorhinal cortex are the first segment of the main hippocampal excitatory loop. The hilar dendrites of the same SS-immunoreactive cells establish synapses with the mossy axon collaterals which represent the second member in this excitatory neuronal chain. These observations suggest that SS-immunoreactive neurons in the dentate hilar area may be driven directly by their perforant path synapses and via the granule cells which are known to receive a dense innervation from the entorhinal cortex. These observations demonstrate that SS-immunoreactive neurons in the hilar region are integrated in the main excitatory impulse flow of the hippocampal formation.     

Dendroaxonic synapses in the substantia gelatinosa glomeruli of the spinal trigeminal nucleus of the cat

The glomeruli in the substantia gelatinosa layer of the spinal trigeminal nucleus of the cat contain three kinds of dendritic processes. One of these, the type 2 dendrite, contains large synaptic vesicles in its spine heads and in its shaft. The type 2 dendrite receives axodendritic synapses from primary trigeminal afferent (C) axons and an occasional axodendritic synapse from small axonal (P) endings with small synaptic vesicles. The type 2 dendrites in turn form dendroaxonic synapses on the C endings. The dendroaxonic synapse and the axodendritic synapse of the C ending typically occur in reciprocal pairs. The axodendritic synapse usually lies in the depths of scalloped depressions in the surface of the C ending while the dendroaxonic synapse is found on the rim of the depression. Type 1 spines, i.e., dendritic spines receiving axodendritic synapses from the primary ending and lacking synaptic vesicles, also receive dendrodendritic synapses from type 2 dendrites. The types 2 dendrite with its large, rounded synaptic vesicles is considered to be excitatory at its dendroaxonic and dendrodendritic synapses. The type 2 dendrites course from glomerulus to glomerulus receiving their excitatory input through the axodendritic synapses of C axons. A type 2 dendrite, in response to C axon excitation would activate type 1 spines directly through their dendrodendritic synapses (C→2→1) and indirectly by increasing transmitter release at the axodendritic synapses of the C axonal endings through their dendroaxonic synapses (2→C→1). The type 2 dendrites could serve two functions. First, they may prolong transmitter release from the axodendritic synapses of C axonal endings beyond the time of arrival of incoming action potentials because of the reciprocal pairing of dendroaxonic and axodendritic synapses (C?2). Second, they may extend the spatial range of the excitatory output of active primary afferent axons to type 1 spines of glomeruli whose primary afferent axons may be inactive (C→2→1).