Charles E. Ribak

Neurons born in the adult dentate gyrus form functional synapses with target cells

Adult neurogenesis occurs in the hippocampus and the olfactory bulb of the mammalian CNS. Recent studies have demonstrated that newborn granule cells of the adult hippocampus are postsynaptic targets of excitatory and inhibitory neurons, but evidence of synapse formation by the axons of these cells is still lacking. By combining retroviral expression of green fluorescent protein in adult-born neurons of the mouse dentate gyrus with immuno-electron microscopy, we found output synapses that were formed by labeled terminals on appropriate target cells in the CA3 area and the hilus. Furthermore, retroviral expression of channelrhodopsin-2 allowed us to light-stimulate newborn granule cells and identify postsynaptic target neurons by whole-cell recordings in acute slices. Our structural and functional evidence indicates that axons of adult-born granule cells establish synapses with hilar interneurons, mossy cells and CA3 pyramidal cells and release glutamate as their main neurotransmitter.

Five types of basket cell in the hippocampal dentate gyrus: a combined Golgi and electron microscopic study.

SummaryFive types of basket cell in the hippocampal denate gyrus of rats were analysed with a combined Golgi and electron microscopic method. Light microscopic observations show that the large somata of these different cell types are located either in the granule cell layer or within 30–50 μm of this layer. The somata of basket cells are pyramidal, horizontal, fusiform or multipolar. Dendrites of basket cells are aspinous or sparsely spinous and are found in all layers of the dentate gyrus. Their axons form an extensive plexus in the granule cell and lower molecular layers.Electron microscopic preparations of Golgi-impregnated, gold-toned basket cells revealed gold-labelled neurons with distinct ultrastructural features. All somata of basket cells displayed an extensive perikaryal cytoplasm with large Nissl bodies and nuclei with infoldings, euchromatin, intranuclear rods and sheets, and large nucleoli. The aspinous dendrites as well as the somata had a mixture of asymmetric and symmetric synapses on their surfaces. Basket cell dendrites located in the hilus were contacted by numerous terminals with characteristics of mossy fibres derived from granule cells. Some of these terminals were identified positively in preparations that also contained impregnated granule cells. The axons of basket cells formed exclusively symmetric synapses. The most common postsynaptic structures to these terminals were the somata and dendrites of granule cells. Dendritic spines were rarely contacted by basket cell axons while the axon hillocks and initial segments of granule cells were never contacted. These findings are consistent with previous immunocytochemical, and physiological data that indicate feedback inhibitory mechanisms in the dentate gyrus are mediated via mossy fibre collaterals which synapse with GABAergic basket cells. In addition, the electron microscopic data for basket cells are similar to those for aspinous stellate cells in the neocortex, another type of cortical, GABAergic local circuit neuron. Thus, the basket cells in the dentate gyrus may have a function similar to other inhibitory, cortical local circuit neurons.

The development ultrastructure and synaptic connections of the mossy cells of the dentate gyrus

SummaryOne of the most distinctive and common cell types in Golgi preparations of the hilus of the rat dentate gyrus is the mossy cell. We have used a variety of techniques including the Golgi method, the combined Golgi and electron microscopic (EM) method and the retrograde transport of horseradish peroxidase (HRP) to study the development, ultrastructure and synaptic connections of this cell type. The mossy cells identified in our light microscopic preparations are characterized by: (1) triangular or multipolar shaped somata; (2) three to four primary dendrites that arise from the soma and bifurcate once or more to produce an extensive dendritic arborization restricted, for the most part, to the hilus; (3) numerous thorny excrescences on their somata and proximal dendrites with typical spines on distal dendrites; and (4) axons that bifurcate and are directed toward the fimbria and the molecular layer of the dentate gyrus.The mossy cells have an immature appearance at birth and on subsequent days their maturation appears to lag somewhat behind that of the hippocampal pyramidal cells. On postnatal day 1, many of the dendrites bear growth cones primarily at their termini and have long, thin filipodia emanating from various points along their lengths. Many of the dendrites enter the molecular layer of the dentate gyrus, though this is rarely seen in the mature brain. Typical pedunculate spines are first commonly seen on the distal dendrites around postnatal day 7 while thorny excrescences are first commonly seen between postnatal days 11 and 14. By postnatal day 21, the dendrites have attained a mature appearance although the density of both typical spines and thorny excrescences is less than that found in adults.Two different retrograde transport methods were used to confirm that mossy cells give rise to the commissural projection to the contralateral dentate gyrus. The first method combined HRP histochemistry with a silver intensification procedure and the second method combined HRP histochemistry with Golgi staining. While the majority of commissurally projecting hilar neurons had the appearance of mossy cells, there were others that were smaller and either ovoid or fusiform.

GABAergic cells in the dentate gyrus appear to be local circuit and projection neurons

SummaryImmunocytochemical results indicate that GAD-positive neurons are found in the molecular and granule cell layers of the dentate gyrus as well as in the hilar region. GAD-positive cells in the molecular and granule cell layers are identified as various types of local circuit neurons. Most of the GAD-positive puncta found throughout the molecular layer and within the granule cell layer are interpreted as axon terminals of these neurons, including five types of basket cells. This interpretation is based on data that indicate the axons of basket cells form synapses with the somata and proximal dendrites of granule cells. The results in the hilus show that 60% of the hilar neurons are GAD-positive. Since previous studies have indicated that 80% of hilar neurons give rise to both associational and commissural pathways, many GABAergic neurons in the hilus are probably projection neurons. This finding is consistent with recent physiological data which suggest that commissural pathway stimulation directly inhibits granule cells. Therefore, GABAergic cells in the dentate gyrus appear to be both projection and local circuit neurons.

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. J. Comp. Neurol. 428:240–253, 2000.

Dentate granule cells form novel basal dendrites in a rat model of temporal lobe epilepsy

Mossy fibre sprouting and re-organization in the inner molecular layer of the dentate gyrus is a characteristic of many models of temporal lobe epilepsy including that induced by perforant-path stimulation. However, neuroplastic changes on the dendrites of granule cells have been less-well studied. Basal dendrites are a transient morphological feature of rodent granule cells during development. The goal of the present study was to examine whether granule cell basal dendrites are generated in rats with epilepsy induced by perforant-path stimulation. Adult Wistar rats were stimulated for 24 h at 2 Hz and with intermittent (1/min) trains (10 s duration) of single stimuli at 20 Hz (20 V, 0.1 ms) delivered 1/min via an electrode placed in the angular bundle. The brains of these experimental rats and age- and litter-matched control animals were processed for the rapid Golgi method. All rats with perforant-path stimulation displayed basal dendrites on many Golgi-impregnated granule cells. These basal dendrites mainly originated from their somata at the hilar side and then extended into the hilus. Quantitative analysis of more than 800 granule cells in the experimental and matched control brains showed that 6-15% (mean=8.7%) of the impregnated granule cells have spiny basal dendrites on the stimulated side, as well as the contralateral side (mean=3.1%, range=2.9-3.9%) of experimental rats, whereas no basal dendrites were observed in the dentate gyrus from control animals. The formation of basal dendrites appears to be an adaptive morphological change for granule cells in addition to the previously described mossy fibre sprouting, as well as dendritic and somatic spine formation observed in the dentate gyrus of animal and human epileptic brains. The presence of these dendrites in the subgranular region of the hilus suggests that they may be postsynaptic targets of the mossy fibre collaterals.

Direct commissural connections to the basket cells of the hippocampal dentate gyrus: anatomical evidence for feed-forward inhibition.

SummaryAfter lesions were placed in the hippocampal commissures, degenerating terminals could be localized above, inside and beneath the granule cell layer of the contralateral dentate gyrus. The terminals formed asymmetric synapses with spines, dendritic shafts and somata of granule cells. Degenerating terminals also formed synapses with dendrites and somata of basket cells identified by the Golgi-electron microscope technique. These basket cells were located either at the hilar border of the granule cell layer or in the molecular layer and each formed an axonal plexus around the somata and proximal dendrites of granule cells. These observations provide an anatomical basis for the recently described feed-forward inhibition in this brain region.

Rapid astrocyte and microglial activation following pilocarpine-induced seizures in rats

Astrocyte and microglial activation occurs following seizures and plays a role in epileptogenesis. However, the precise temporal and spatial response to seizures has not been fully examined. The pilocarpine model of temporal lobe epilepsy was selected to examine glial changes following seizures because morphological changes in the hippocampus closely mimic the human condition. Astrocytic and microglial changes in the hippocampus were examined during the first 5 days after pilocarpine‐induced seizures in rats by analyzing GFAP, Iba1 and S100B‐immunolabeling in CA1, CA3, and the hilus. Also, 3‐dimensional reconstructions of microglial cells from the hilus and granule cell layer were analyzed. Lastly, astrocyte hypertrophy was examined in the hilus using electron microscopy. At 1 day after seizures and continuing throughout the 5 days examined, hypertrophied Iba1‐labeled microglial cells and glial fibrillary acidic protein (GFAP)‐labeled astrocytes were observed. At 1 and 2 days after seizures, significantly greater Iba1 immunolabeling was observed in CA1, CA3, and the hilus. In addition, both the area of Iba1 labeled processes and the number of their endings were increased in the hilus beginning at 1 day after seizures. S100B‐immunolabeling was significantly elevated in CA3 at 1 day, in CA3 and CA1 at 2 days, and in all three hippocampal regions at 3 days after seizures. Electron microscopy confirmed astrocytic hypertrophy and demonstrated astrocytic cell bodies in the location where glial endfeet normally appear on capillaries. The differential response patterns of astrocytes and microglial cells following pilocarpine‐induced seizures may signify their detrimental role in neuroinflammation after seizures.

Calcium-binding proteins are concentrated in the CA2 field of the monkey hippocampus: a possible key to this region's resistance to epileptic damage

SummaryPrevious immunocytochemical studies have shown a heterogeneous distribution of parvalbumin (PA) and calbindin (CB) in the rat hippocampal formation. The results of the present study showed a heterogeneous distribution of PA and CB in primate Ammons horn. The density and intensity of immunoreactivity for both of these calcium-binding proteins was greatest in CA2 as compared to CA1 and CA3. CB-immunoreactivity was localized to the cell bodies, dendrites, and axon initial segments of pyramidal cells whereas PA-immunostaining was found in the axon terminals, dendrites and cell bodies of interneurons that have features similar to GABAergic inhibitory neurons. Based on previous studies that have shown a protective role of calcium-binding proteins in neurons exposed to hyperstimulation, these results suggest that the resistance of CA2 pyramidal cells in temporal lobe epilepsy is due to the high concentration of CB and PA in this region of Ammons horn.

Immunocytochemical localization of GABAergic neurones at the electron microscopical level

SummaryAntibodies prepared to purified brain glutamic acid decarboxylase (GAD), the synthesizing enzyme for the neurotrasmitter, γ-aminobutyric, acid (GABA), have been utilized with an unlabelled antibody method to localize GABAergic neurones in both light and electron microscopic preparations. A modification of Sternbergers peroxidase-antiperoxidase (PAP) complex is used to localize the site of anti-GAD binding, and the PAP complex is visualized with diaminobenzidine and H2O2. The reaction product is visible in both the light and electron microscopes. The ability to localize and identify labelled profiles in the electron microscope provides more functional information than light microscopical preparations. For example, the GAD-positive reaction product occurs mostly in association with synaptic vesicles within axon terminats, and this localization indicates the importance of GAD for the packaging and storage of GABA. The somata and dendrites of neurones giving rise to these terminals are visualized in colchicine-injected material. The GABAergic neurones form axo-somatic, axo-dendritic, axo-axonal and dendro-dendritic synapses in various regions of the rat central nervous system. Pretreatments of animals with anterograde degeneration have shown the significance of some of the GABAergic terminals that form axo-axonal synapses in the spinal cord.An many brain regions, such as the cerebral cortex, hippocampus and olfactory bulb, virtually all of the GABAergic synapses are derived from local circuit neurones. In other regions such as the cerebellum and neostriatum, the GABAergic terminals are derived from both local circuit neurones and the local axon collaterals of projection neurones that have their somata within these regions. A third type of configuration of GABAergic terminals occurs in the globus pallidus and substantia nigra where these terminals are derived from distant brain regions, axon collaterals of projection neurones and from local circuit neurones. Together, these results indicate the complex organization of the GABAergic system of the brain that has been vividly revealed with electron in croscopical immunocytochemistry.