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Metabotropic glutamate receptors (mGluRs) are a group of G-protein-coupled receptors that exert a broad array of modulatory actions at excitatory synapses of the central nervous system. In the hippocampus, the selective activation of the different mGluRs modulates the intrinsic excitability, the strength of synaptic transmission, and induces multiple forms of long-term plasticity. Despite the relevance of mGluRs in the normal function of the hippocampus, we know very little about the changes that mGluRs functionality undergoes during the non-pathological aging. Here, we review data concerning the physiological actions of mGluRs, with particular emphasis on hippocampal area CA3. Later, we examine changes in the expression and functionality of mGluRs during the aging process. We complement this review with original data showing an array of electrophysiological modifications observed in the synaptic transmission and intrinsic excitability of aged CA3 pyramidal cells in response to the pharmacological stimulation of the different mGluRs.

The hippocampal formation (HF) is involved in modulating learning related to drug abuse. While HF-dependent learning is regulated by both endogenous opioids and estrogen, the interaction between these two systems is not well understood. The mossy fiber (MF) pathway formed by dentate gyrus (DG) granule cell axons is involved in some aspects of learning and contains abundant amounts of the endogenous opioid peptide dynorphin (DYN). To examine the influence of ovarian steroids on DYN expression, we used quantitative light microscopic immunocytochemistry to measure DYN levels in normal cycling rats as well as in two established models of hormone-treated ovariectomized (OVX) rats. Rats in estrus had increased levels of DYN-immunoreactivity (ir) in the DG and certain CA3 lamina compared with rats in proestrus or diestrus. OVX rats exposed to estradiol for 24 h showed increased DYN-ir in the DG and CA3, while those with 72 h estradiol exposure showed increases only in the DG. Six hours of estradiol exposure produced no change in DYN-ir. OVX rats chronically implanted with medroxyprogesterone also showed increased DYN-ir in the DG and CA3. Next, dual-labeling electron microscopy (EM) was used to evaluate the subcellular relationships of estrogen receptor (ER) alpha-, ERbeta and progestin receptor (PR) with DYN-labeled MFs. ERbeta-ir was in some DYN-labeled MF terminals and smaller terminals, and had a subcellular association with the plasmalemma and small synaptic vesicles. In contrast, ERalpha-ir was not in DYN-labeled terminals, although some DYN-labeled small terminals synapsed on ERalpha-labeled dendritic spines. PR labeling was mostly in CA3 axons, some of which were continuous with DYN-labeled terminals. These studies indicate that ovarian hormones can modulate DYN in the MF pathway in a time-dependent manner, and suggest that hormonal effects on the DYN-containing MF pathway may be directly mediated by ERbeta and/or PR activation.

Both N- and P/Q-type voltage-dependent calcium channels are involved in fast transmitter release in the hippocampus, but are differentially regulated. Although variable contributions of voltage-dependent calcium channel subtypes to presynaptic Ca 2+ influx have been suggested to give a neural network of great diversity, their presence has only been demonstrated in a culture system and has remained unclear in the brain. Here, the individual large mossy fiber presynaptic terminal was labeled with Ca 2+ /Sr 2+ -sensitive fluorescent dextrans in the hippocampal slice of the mouse. The fractional contribution of voltage-dependent calcium channel subtypes to presynaptic Ca 2+ /Sr 2+ influx was directly measured by the sensitivity of Ca 2+ /Sr 2+ -dependent fluorescent increment to subtype-selective neurotoxins, ω-conotoxin GVIA (an N-type selective blocker), ω-agatoxin IVA (a P/Q-type selective blocker) and SNX-482 (an R-type selective blocker). Synapse-to-synapse comparison of large mossy fiber terminals revealed that the contributions of N- and R-type voltage-dependent calcium channels varied more widely than that of P/Q-type. Even two large mossy fiber presynaptic terminals neighboring on the same axon differed in the fractional contributions of N- and R-type voltage-dependent calcium channels. On the other hand, these terminals were similar in the fractional contributions of P/Q-type voltage-dependent calcium channels. These results provide direct evidence that individual large mossy fiber synapses are differential in the contribution of N- and R-type voltage-dependent calcium channel subtypes to presynaptic Ca 2+ /Sr 2+ influx. We suggest that the synapse-to-synapse variation of presynaptic voltage-dependent calcium channel subtype contributions may be one of the mechanisms amplifying diversity of the hippocampal network.

Like estrogens in female rats, androgens can affect dendritic spine density in the CA1 subfield of the male rat hippocampus [J Neurosci 23:1588 (2003)]. Previous light microscopic studies have shown that androgen receptors (ARs) are present in the nuclei of CA1 pyramidal cells. However, androgens may also exert their effects through rapid non-genomic mechanisms, possibly by binding to membranes. Thus, to investigate whether ARs are at potential extranuclear sites of ARs, antibodies to ARs were localized by light and electron microscopy in the male rat hippocampal formation. By light microscopy, AR immunoreactivity (-ir) was found in CA1 pyramidal cell nuclei and in disperse, punctate processes that were most dense in the pyramidal cell layer. Additionally, diffuse AR-ir was found in the mossy fiber pathway. Ultrastructural analysis revealed AR-ir at several extranuclear sites in all hippocampal subregions. AR-ir was found in dendritic spines, many arising from pyramidal and granule cell dendrites. AR-ir was associated with clusters of small, synaptic vesicles within preterminal axons and axon terminals. Labeled preterminal axons were most prominent in stratum lucidum of the CA3 region. AR-containing terminals formed asymmetric synapses or did not form synaptic junctions in the plane of section analyzed. AR-ir also was detected in astrocytic profiles, many of which apposed terminals synapsing on unlabeled dendritic spines or formed gap junctions with other AR-labeled or unlabeled astrocytes. Collectively, these results suggest that ARs may serve as both a genomic and non-genomic transducer of androgen action in the hippocampal formation.

Ephrins are guidance cues that modulate axonal growth and the subsequent axonal topographic maps in many regions of the CNS. Here we studied the functional roles of ephrin-A/EphA interactions in the layer-specific pattern of axonal projections in the hippocampus by disrupting the ephrin-A signaling by over-expression of a soluble EphA receptor. Tracing experiments in EphA5-Fc over-expressing mice revealed that reduction of ephrin-A/EphA interactions did not affect the proper distribution of the main hippocampal afferents, i.e. entorhinal and commissural projections. However, further ultrastructural analyses showed a reduction in the density of synaptic terminals in the entorhinal and commissural termination layers in these mice. In addition, using anti-calbindin antibodies, we analyzed the dentate mossy fiber projections following disruption of ephrin-A/EphA interactions throughout developing hippocampus. While the main mossy fiber bundle appeared normal, the infrapyramidal bundle formed longer projections that established ectopic contacts in these transgenic mice. Later, the expected specific pruning of the infrapyramidal bundle was not observed at adult stages. Ultrastructural analyses confirmed a higher number of mossy fiber terminals in the infrapyramidal bundle in adult EphA5-Fc transgenic mice and showed that these terminals were larger and established a greater number of contacts than in controls. Our results demonstrate that ephrin-A/EphA interactions regulate the synaptogenesis of hippocampal afferents and the proper development and refinement of granule cell projections.

Zinc is found throughout the CNS in synaptic vesicles of glutamatergic neurons and has been suggested to have a modulatory role in the brain because of its interaction with voltage- and ligand-gated ion channels. We took advantage of zinc transporter 3 knockout mice, which lack vesicular zinc, to study the possible physiological role of this heavy metal in hippocampal mossy fiber neurotransmission. We examined postsynaptic responses evoked by mossy fiber activation, recorded in CA3 pyramidal cells in hippocampal slices prepared from zinc transporter 3 knockout and wild-type mice. Field-potential response threshold and amplitude, input-output curves, and paired-pulse evoked responses were the same in slices from zinc transporter 3 knockout and wild-type mice. Furthermore, neither amplitude nor duration of pharmacologically isolated N -methyl- d -aspartate, non- N -methyl- d -aspartate, GABA A , and GABA B receptor-mediated postsynaptic potentials differed between zinc transporter 3 knockout and wild-type mice. There was no difference in the magnitude of epileptiform discharges evoked by repetitive stimulation or kainic acid application. However, in slices from zinc transporter 3 knockout mice, there was greater attenuation of GABA A -mediated inhibitory postsynaptic potentials during tetanic stimulation compared with slices from wild-type animals. We conclude that lack of vesicular zinc in mossy fibers does not significantly affect the mossy fiber-associated synaptic excitability of CA3 pyramidal cells; however, zinc may modulate GABAergic synaptic transmission under conditions of intensive activation.

Synaptophysin, an integral membrane glycoprotein of synaptic vesicles, has been widely used to investigate synaptogenesis in both animal models and human patients. Kindling is an experimental model of complex partial seizures with secondary generalization, and a useful model for studying activation-induced neural growth in adult systems. Many studies using Timm staining have shown that kindling promotes sprouting in the mossy fiber pathway of the dentate gyrus. In the present study, we used synaptophysin immunohistochemistry to demonstrate activation-induced neural sprouting in non-mossy fiber cortical pathways in the adult rat. We found a significant kindling-induced increase in synaptophysin immunoreactivity in the stratum radiatum of CA1 and stratum lucidum/radiatum of CA3, the hilus, the inner molecular layer of the dentate gyrus, and layer II/III of the piriform cortex, but no significant change in layer II/III of the entorhinal cortex, 4 weeks after the last kindling stimulation. We also found that synaptophysin immunoreactivity was lowest in CA3 near the hilus and increased with increasing distance from the hilus, a reverse pattern to that seen with Timm stains in stratum oriens following kindling. Furthermore, synaptophysin immunoreactivity was lowest in dorsal and greatest in ventral sections of both CA3 and dentate gyrus in both kindled and non-kindled animals. This demonstrates that different populations of sprouting axons are labeled by these two techniques, and suggests that activation-induced sprouting extends well beyond the hippocampal mossy fiber system.

The present experiments investigate the role of postsynaptic neurons in the morphological differentiation of presynaptic terminals that are formed de novo in the adult CNS. Abducens internuclear neurons in the adult cat were chosen as the experimental model. These neurons project onto the contralateral medial rectus motoneurons of the oculomotor nucleus. Abducens internuclear axon terminals were identified by their anterograde labeling with biocytin and analyzed at the electron microscopic level. To promote the formation of new synapses, two different experimental approaches were used. First, after the selective ablation of medial rectus motoneurons with ricin, abducens internuclear neurons reinnervated the neighboring oculomotor internuclear neurons. Second, after axotomy followed by embryonic cerebellar grafting, abducens internuclear axons invaded the implanted tissue and established synaptic connections in both the molecular and granule cell layer. Boutons contacting the oculomotor internuclear neurons developed ultrastructural characteristics that resembled the control synapses on medial rectus motoneurons. In the grafted cerebellar tissue, abducens internuclear axons and terminals did not resemble climbing or mossy fibers but showed similarities with control boutons. However, labeled boutons analyzed in the granule cell layer established a higher number of synaptic contacts than controls. This could reflect a trend towards the mossy fiber phenotype, although labeled boutons significantly differed in every measured parameter with the mossy fiber rosettes found in the graft. We conclude that at least for the abducens internuclear neurons, the ultrastructural differentiation of axon terminals reinnervating novel targets in the adult brain seems to be mainly under intrinsic control, with little influence by postsynaptic cells.

The clinical and basic literature suggest that hilar cells of the dentate gyrus are damaged after seizures, particularly prolonged and repetitive seizures. Of the cell types within the hilus, it appears that the mossy cell is one of the most vulnerable. Nevertheless, hilar neurons which resemble mossy cells appear in some published reports of animal models of epilepsy, and in some cases of human temporal lobe epilepsy. Therefore, mossy cells may not always be killed after severe, repeated seizures. However, mossy cell survival in these studies was not completely clear because the methods did allow discrimination between mossy cells and other hilar cell types. Furthermore, whether surviving mossy cells might have altered physiology after seizures was not examined. Therefore, intracellular recording and intracellular dye injection were used to characterize hilar cells in hippocampal slices from pilocarpine-treated rats that had status epilepticus and recurrent seizures (‘epileptic’ rats). For comparison, mossy cells were also recorded from age-matched, saline-injected controls, and pilocarpine-treated rats that failed to develop status epilepticus. Numerous hilar cells with the morphology, axon projection, and membrane properties of mossy cells were recorded in all three experimental groups. Thus, mossy cells can survive severe seizures, and those that survive retain many of their normal characteristics. However, mossy cells from epileptic tissue were distinct from mossy cells of control rats in that they generated spontaneous and evoked epileptiform burst discharges. Area CA3 pyramidal cells also exhibited spontaneous and evoked bursts. Simultaneous intracellular recordings from mossy cells and pyramidal cells demonstrated that their burst discharges were synchronized, with pyramidal cell discharges typically beginning first. From these data we suggest that hilar mossy cells can survive status epilepticus and chronic seizures. The fact that mossy cells have epileptiform bursts, and that they are synchronized with area CA3, suggest a previously unappreciated substrate for hyperexcitability in this animal model.

Studies were conducted to characterize a chronic epileptic condition that follows recurrent seizures induced by intrahippocampal tetanus toxin injection in infancy. Wistar rat pups received a single injection of tetanus toxin in the right CA3 region on postnatal day 10. Animals were monitored for epileptiform activity by video electroencephalographic or visual observation during the following three to five days. Repeat evaluation six months later demonstrated interictal discharges in 79% (11 of 14) and electrographic seizures in 42% (six of 14) of adult rats with tetanus toxin-induced seizures in infancy. Five of the animals had interictal activity which occurred focally in either the left (n = 2) or right (n = 3) hippocampus. One animal had focal interictal activity independently in these regions and in the left and right cortical regions. The remaining five animals had interictal activity in the hippocampus and synchronously in the ipsilateral cortex or the contralateral hippocampus. Electrographic seizures were focal (nine of 14) or bilateral (five of 14) in onset. The behaviors that accompanied these seizures were quite variable. Clonic face and forelimb movements were observed in some animals. However, a significant portion of rats had electrographic seizures with no associated behavioral change. Timm staining was performed on hippocampal sections from experimental and control animals. There was a significantly greater Timm score (aberrant Timm granules) in the inner molecular layer of the dentate gyrus in tetanus toxin-treated rats than in control rats. Our findings suggest that intrahippocampal tetanus toxin injection in infant rats results in a chronic focal epilepsy that persists for at least six months and is associated with aberrant mossy fiber sprouting in the dentate gyrus. The model described here contributes significantly to the evidence for chronic effects of recurrent seizures in early life, and provides a model for investigation of the molecular and cellular events that contribute to the development of chronic epilepsy.

Sprouting of mossy fibers in the hippocampus of rats that underwent limbic epileptogenesis by amygdala kindling or kainate injection was studied at the light microscopic and ultrastructural levels by cytochemical demonstration of the enzyme 5'-nucleotidase. This adenosine-producing ectoenzyme has previously been shown to characterize malleable terminals during brain development and lesion-induced synaptogenesis, but to be otherwise associated with glial membranes. At the light microscopic level, kainate-treated but not control or kindled rats showed 5'-nucleotidase activity in the CA3 region and in the inner molecular layer of the dentate gyrus. At the ultrastructural level, in control animals, the synapses of the molecular and granular layers were enzyme negative. Only some mossy fiber boutons of the dentate hilus exhibited 5'-nucleotidase activity. In epileptic rats, synaptic labeling within the hilus appeared more intense. Moreover, 5'-nucleotidase-containing terminals within the inner molecular layer, presumably ectopic mossy fiber boutons, were found in both kindled and kainate-treated rats. It is concluded that, in both the normal and epileptic hippocampus, 5'-nucleotidase is associated with axons capable of a plastic sprouting response. The synaptic enzyme may attenuate the glutamatergic transmission of mossy fibers, in particular of the aberrant mossy fibers in epileptic rats, by producing the inhibitory neuromodulator adenosine. Alternatively, 5'-nucleotidase may influence synapse formation by its putative non-enzymatic, adhesive functions.

A role for apolipoprotein E is implicated in regeneration of synaptic circuitry after neural injury. The in vitro mouse organotypic hippocampal slice culture system shows Timm's stained mossy fiber sprouting into the dentate gyrus molecular layer in response to deafferentation of the entorhinal cortex. We show that cultures derived from apolipoprotein E knockout mice are defective in this sprouting response; specifically, they show no sprouting in the dorsal region of the dentate gyrus, yet retain sprouting in the ventral region. Dorsal but not ventral sprouting in cultures from C57B1/6J mice is increased 75% by treatment with 100 pM 17beta-estradiol; this response is blocked by both progesterone and tamoxifen. These results show that neuronal sprouting is increased by estrogen in the same region where sprouting is dependent on apolipoprotein E. Sprouting may be stimulated by estrogen through its up-regulation of apolipoprotein E expression leading to increased recycling of membrane lipids for use by sprouting neurons. Estrogen and apolipoprotein E may therefore interact in their modulation of both Alzheimer's disease risk and recovery from CNS injury.

Neuroplasticity has been investigated considering the neuronal growth-associated phosphoprotein as a marker of neuronal adaptive capabilities. In the present work, studying the hippocampal reorganization observed in the epilepsy model induced by pilocarpine, we carried out quantitative western blotting associated with immunohistochemistry to determine the distribution of growth-associated phosphoprotein in the hippocampus of rats in acute, silent and chronic periods of this epilepsy model. The fibers and punctate elements from the inner molecular layer of the dentate gyrus were strongly immunostained in animals killed 5 h after status epilepticus, compared with the same region in control animals. Rats presenting partial seizures showed no alterations in the immunostaining pattern compared with saline-treated animals. The hippocampal dentate gyrus of animals during the seizure-free period and presenting spontaneous recurrent seizures was also characterized by strong growth-associated phosphoprotein immunostaining of fibers and punctate elements in the inner molecular layer, contrasting with the control group. As determined by western blotting analysis, growth-associated phosphoprotein levels increased following status epilepticus and remained elevated at the later time-points, both during the silent period and during the period of chronic recurring seizures. Pilocarpine-treated animals, which did not develop status epilepticus, showed no change in growth-associated phosphoprotein levels, indicating that status epilepticus is important to induce growth-associated phosphoprotein overexpression. The measurement of this overexpression could represent one of the early signals of hippocampal reorganization due to status epilepticus-induced damage.

Phosphate activated glutaminase is a key enzyme in glutamate synthesis. Here we have employed a quantitative and high-resolution immunogold procedure to analyse the cellular and subcellular expression of this enzyme in the cerebellar cortex. Three main issues were addressed. First, is phosphate activated glutaminase exclusively or predominantly a mitochondrial enzyme, as biochemical data suggest? Second, to what extent is the mitochondrial content of glutaminase dependent on cell type and transmitter identity? Third, can individual neurons maintain a subcellular segregation of mitochondria with different glutaminase content? An attempt was also made to disclose the intramitochondrial localization of glutaminase, and to correlate the content of this enzyme with that of glutamate and glutamine in the same mitochondria (by use of triple labelling). Antisera to the N- and C-termini of glutaminase revealed strong labelling of the putatively glutamatergic mossy fibre terminals. The vast majority of gold particles (approximately 96%) was associated with the mitochondria. Equally high labelling intensities were found in mitochondria of perikarya and dendrites in the pontine nuclei, a major source of mossy fibres. The level of glutaminase immunoreactivity in parallel and climbing fibres (which like the mossy fibres are thought to use glutamate as transmitter) was only about 20% of that in mossy fibres, and similar to that in non-glutamatergic neurons (Purkinje and Golgi cells). Glial cell mitochondria were devoid of specific glutaminase labelling and revealed a much lower glutamate:glutamine ratio than did the mitochondria of mossy fibres. As to the submitochondrial localization of glutaminase, immunogold particles were often found to be aligned with the cristae, suggesting an association of the enzyme with the inner mitochondrial membrane. However, the existence of a glutaminase pool in the mitochondrial matrix could not be excluded. The outer mitochondrial membrane was unlabelled. The present study provides quantitative evidence for a substantial heterogeneity in the mitochondrial content of glutaminase. This heterogeneity applies not only to neurons with different transmitter signatures, but also to different categories of glutamatergic pathways. In terms of the routes involved, the synthesis of transmitter glutamate may be less uniform than previously expected.

The loss of somatostatin-immunoreactive neurons and the sprouting of mossy fibers are typical histopathological abnormalities in the hippocampus in experimental and human temporal lobe epilepsy. To investigate whether the development of seizure-induced alterations is regulated by the subcortical afferent pathways to the hippocampus, we lesioned cholinergic, noradrenergic or serotonergic afferent pathways in rats two days after seizures were induced with kainate. Two months later, somatostatin-immunoreactive neurons were counted in the hilus to assess the severity of neuronal damage. Mossy fiber sprouting was analysed from adjacent Timm-stained sections. Kainate-induced seizures caused a loss of hilar somatostatin-immunoreactive neurons in the septal end of the hippocampus, where 63% of the somatostatin-immunoreactive neurons survived. Even more severe damage was found in the temporal end of the hippocampus (only 21% surviving). Cholinergic deafferentation of the hippocampus (using 192-Ig G–saporin) decreased the overall number of hilar somatostatin-immunoreactive neurons. In control rats that did not receive kainate, 87% (septal end) and 74% (temporal end) of the hilar somatostatin-immunoreactive neurons remained after cholinergic deafferentation. Moreover, seizure-induced damage to hilar somatostatin-immunoreactive neurons was further exacerbated by 192-Ig G–saporin, with only 35% of the neurons remaining in the septal end and 14% in the temporal end of the hippocampus. Noradrenergic [using N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine] or serotonergic (using 5,7-dihydroxytryptamine) lesions did not affect the number of hilar somatostatin-immunoreactive neurons either in control or in kainate-treated rats. The severity and distribution of seizure-induced mossy fiber sprouting were also not affected by any of the lesions. These data suggest that various subcortical afferent pathways may differentially modulate seizure-induced damage to the hippocampus. Damage to cholinergic neurons results in the loss of hilar somatostatin-immunoreactive neurons and exacerbates the seizure-induced loss of somatostatin-immunoreactive neurons.

Somatostatin-, neuropeptide Y-, neurokinin B- and cholecystokinin-containing neurons were investigated in the rat hippocampus in two chronic models of temporal lobe epilepsy, i.e. 30 days after rapid kindling or electrically induced status epilepticus (post-status epilepticus). After rapid kindling, somatostatin immunoreactivity was strongly increased in interneurons and in the outer and middle molecular layer of the dentate gyrus. In four of six post-status epilepticus rats (status epilepticus I rats), somatostatin immunoreactivity was slightly increased in the dorsal but decreased in the ventral dentate gyrus and molecular layer. Somatostatin immunoreactivity decreased in neurons of the dorsal hilus in the two other post-status epilepticus rats investigated, while a complete loss was found in the respective ventral extension (status epilepticus-II rats). These changes were associated with a different extent of neurodegeneration as assessed by Nissl staining. Similarly, neuropeptide Y immunoreactivity was enhanced in neurons of the hilus and in the middle and outer molecular layer of the dentate gyrus in the dorsal hippocampus of rapidly kindled and status epilepticus-I rats. Neuropeptide Y and neurokinin B immunoreactivity was enhanced in the mossy fibers of all post-status epilepticus rats, but not in the rapidly kindled rats. In status epilepticus-II rats, neuropeptide Y-and neurokinin B-positive fibers were also detected in the infrapyramidal region of the stratum oriens of CA3 and in the inner molecular layer of the dentate gyrus in the dorsal and ventral hippocampus respectively, labeling presumably sprouted mossy fibers. Increased staining of neuropeptide Y and neurokinin B was found in the alveus after rapid kindling. Cholecystokinin immunoreactivity was markedly increased in the cerebral cortex, Ammon's horn and the molecular layer of the dentate gyrus in the ventral hippocampus of rapidly kindled and post-status epilepticus rats. The lasting changes in the immunoreactive pattern of various peptides in the hippocampus may reflect functional modifications in the corresponding peptide-containing neurons. These changes may be involved in chronic epileptogenesis, which evolves in response to limbic seizures.

Intracellular current clamp recordings were obtained from mossy cells (n = 6, identified by intracellular injection of biocytin) of the dorsal dentate gyrus from rats under ketamine-xylazine anesthesia. During electroencephalographic theta rhythm (4–6 Hz), recorded with a macroelectrode placed in the contralateral dorsal hippocampus near the fissure, mossy cells displayed intracellular membrane potential oscillations at low frequencies (4–6 Hz) which appeared to be phase locked to the electroencephalographic theta rhythm. The frequency of the intracellular theta rhythm was independent of the membrane potential. However, the phase difference between the intracellular and the electroencephalographic theta rhythms as well as the amplitude of the intracellular theta oscillations were voltage-dependent. These findings are consistent with the hypothesis that rhythmic GABAA receptor-mediated inhibitory postsynaptic potentials contribute to the genesis of the intracellular theta rhythm. Indeed, mossy cells displayed an early, fast inhibitory postsynaptic potential in response to electrical stimulation of the entorhinal cortex, which most likely represents a GABAA receptor-mediated event, indicating that mossy cells possess functional GABAA receptors. At the resting membrane potential, mossy cells did not fire at each cycle of the electroencephalographic theta rhythm but fired only rarely (< 1 Hz). However, when they did fire they did so preferentially in phase with the peak positivity of the electroencephalographic theta rhythm. Reconstruction of two mossy cells with axonal projections to the inner molecular layer showed that the spatial extent of the influence such weakly discharging mossy cells may have on other dentate gyrus neurons during theta oscillations can be several millimeters in the septotemporal direction. In conclusion, these findings show that mossy cells of the rat hilus during ketamine-xylazine anesthesia participate in theta oscillations of the hippocampal formation, during which their low-frequency firing may contribute to the phase-locking of a large number of spatially distributed postsynaptic neurons with postsynaptic sites in the inner molecular layer of the dentate gyrus.

Traditionally studies aimed at elucidating the molecular mechanisms underlying cerebellar motor learning have been focused on plasticity at the parallel fiber to Purkinje cell synapse. In recent years, however, the concept is emerging that formation and storage of memories are both distributed over multiple types of synapses at different sites. Here, we examined the potential role of potentiation at the mossy fiber to granule cell synapse, which occurs upstream to plasticity in Purkinje cells. We show that null-mutants of N-methyl D-aspartate-NR2A receptors (NMDA-NR2A(-/-) mice) have impaired induction of postsynaptic long-term potentiation (LTP) at the mossy fiber terminals and a reduced ability to raise the granule cell synaptic excitation, while the basic excitatory output of the mossy fibers is unaffected. In addition, we demonstrate that these NR2A(-/-) mutants as well as mutants in which the C terminal in the NR2A subunit is selectively truncated (NR2A(Delta C/Delta C) mice) have deficits in phase reversal adaptation of their vestibulo-ocular reflex (VOR), while their basic eye movement performance is similar to that of wild type littermates. These results indicate that NMDA-NR2A mediated potentiation at the mossy fiber to granule cell synapse is not required for basic motor performance, and they raise the possibility that it may contribute to some forms of vestibulo-cerebellar memory formation. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Antisera raised against protein-glutaraldehyde-amino acid conjugates were used to study the light and electron microscopic distribution of GABA, glutamate, glutamine and taurine in organotypic slice cultures of rat hippocampi. In the stratum oriens and radiatum, glutamate-like immunoreactivity was particularly concentrated in nerve endings establishing asymmetric junctions with dendritic spines. Mossy fiber terminals in CA3 and the dentate hilus were also strongly labeled. A quantitative immunogold analysis of the glutamate-immunolabelled profiles showed a pattern that was highly reminiscent of that previously observed in perfusion-fixed hippocampi, including a correspondingly sparse labeling of glial processes and of presynaptic elements in symmetric synapses. GABA-like immunoreactivity was localized predominantly in interneurons and in presynaptic terminals contacting dendritic shafts and neuronal cell bodies, while immunoreactivities for glutamine and taurine were found mainly in astroglial cells and pyramidal cells, respectively. Our data indicate that the major intrinsic fiber systems of the cultured hippocampi have retained their normal transmitter phenotypes.

Excitatory amino acid agonists and antagonists were evaluated for their ability to affect the concomitant release of endogenous l -glutamate and dynorphin A(1–8)-like immunoreactivity from guinea-pig hippocampal mossy fiber synaptosomes. Previous work in this laboratory demonstrated that l (+)2-amino-4-phosphonobutyrate inhibits the potassium-evoked release of these endogenous neurotransmitters from guinea-pig but not rat hippocampal mossy fiber synaptosomes. Therefore, the present study was conducted to evaluate excitatory amino acid agonists as indices to the functional properties of this l (+)2-amino-4-phosphonobutyrate-sensitive glutamatergic autoreceptor on mossy fiber terminals. Low micromolar concentrations of quisqualate, but not kainate, N-methyl- d -aspartate , nor RS-alphaamino-3-hydroxy-5-methyl-4-isoazole-propionic acid, significantly inhibited the potassium-evoked release of both l -glutamate and dynorphin A(1–8)-like immunoreactivity. Quisqualate-induced inhibition of l -glutamate release from mossy fiber terminals was antagonized by the non-N-methyl- d -aspartate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. In contrast, high concentrations of kainate enhanced the potassium-evoked release of l -glutamate and dynorphin A(1–8)-like immunoreactivity, and this potentiation was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. Kainate (1 mM) was the only agonist which significantly enhanced the basal release of l -glutamate, whereas the spontaneous efflux of dynorphin A(1–8)-like immunoreactivity was not affected by any of the agonists tested. The results presented in this paper suggest the existence of inhibitory and excitatory presynaptic glutamatergic autoreceptors that act to modulate the release of endogenous l -glutamate- and prodynor-phin-derived peptides from guinea-pig hippocampal mossy fiber terminals. These inhibitory and excitatory autoreceptors, which are sensitive to quisqualate/ l (+)2-amino-4-phosphonobutyrate or kainate, respectively, may play an important role in regulating synaptic activity at glutamatergic synapses throughout the central nervous system.

The hippocampus provides a suitable area in the brain for the analysis of neuronal plasticity after application of a selective lesioning technique. Using histochemistry and autoradiography, we studied synaptic reorganization in the rat hippocampus with selective CA1 pyramidal cell lesioning caused by transient forebrain ischemia after long-term survival. An autoradiographic study was performed on second messenger systems (3H]inositol 1,4,5-trisphosphate, [3H]forskolin and [3H]phorbol 12,13-dibutyrate binding). One-hundred days after ischemia, depletion of CA1 pyramidal cells and marked shrinkage of the CA1 subfield was noted in spite of unaltered thickness of the CA3 band and of the dentate molecular layers. Although neuronal density in the CA3 region of animals killed seven days after ischemia was not different from the normal group, 78% of animals showed neuronal loss of 30–50% in the stratum pyramidale of the CA3b 100 days after recirculation. Sixty-seven per cent of animals exhibited supragranular mossy fiber sprouting in the dentate gyrus. However, CA3 neuronal loss did not correlate with mossy fiber sprouting. Succinic dehydrogenase was depleted in the CA1 100 days after ischemia, and animals with CA3 damage showed a reduction of succinic dehydrogenase activity in the CA3. In contrast to the unaltered acetylcholinesterase in the animals killed seven days after ischemia, high density bands of acetylcholinesterase activity in the stratum pyramidale of the CA1 were found to be broadened 100 days after ischemia. In the CA1 subfield, subnormal activity of [3H]phorbol 12,13-ditutyrate and [3H]forskolin binding were observed in spite of the depleted [3H]inositol 1,4,5-triphosphate binding. [3H]Forskolin binding in the hilus had increased by 62% 100 days after ischemia, although binding in the stratum lucidum of the CA3 and in the stratum moleculare of the dentate gyrus was unaltered. However, no visible supragranular increase in [3H]forskolin binding was observed. These results indicate that long-term survival after CA1 pyramidal cell depletion caused by transient forebrain ischemia induced the modulation of neuronal activity and synaptic rearrangements in the whole hippocampal formation.

We investigated epileptogenesis after cortical photothrombotic stroke induced with Rose Bengal dye in adult Sprague-Dawley rats. To detect spontaneous seizures, video-electroencephalograms were recorded at 2, 4, 6, 8, and 10 months for 7-14 days (24 h/day). At the end, spatial and emotional learning and memory were assessed using the Morris water-maze and fear-conditioning test, respectively, and the brains were processed for histologic analysis. Seizures were detected in 18% of rats that received photothrombosis. The average seizure frequency was 0.39 seizures per recording day and mean seizure duration was 117 s. Over 60% of seizures occurred during the dark hours. Rats with photothrombotic lesions were impaired in the water-maze (P

The most well-documented synaptic rearrangement associated with temporal lobe epilepsy is mossy fiber sprouting (MFS). MFS is a pronounced expansion of granule cell mossy fiber axons into the inner dentate molecular layer. The recurrent excitatory network formed by MFS is hypothesized to play a critical role in epileptogenesis, which is the transformation of the normal brain into one that is prone to recurrent spontaneous seizures. While many studies have focused on the functional consequences of MFS, relatively few have investigated the molecular mechanisms underlying the increased propensity of mossy fibers to invade the inner molecular layer. We hypothesized that changes in two components of the extracellular matrix, hyaluronan and its primary receptor, CD44, contribute to MFS. Hyaluronan contributes to laminar-specificity in the hippocampus and increases in hyaluronan and CD44 are associated with temporal lobe epilepsy. We tested our hypothesis in an in vitro model of MFS using a combination of histological and biochemical approaches. Application of kainic acid (KA) to organotypic hippocampal slice cultures induced robust MFS into the inner dentate molecular layer compared with vehicle-treated controls. Degradation of hyaluronan with hyaluronidase significantly reduced but did not eliminate KA-induced MFS, suggesting that hyaluronan played a permissive role in MFS, but that loss of hyaluronan signaling alone was not sufficient to block mossy fiber reorganization. Comparison of CD44 expression with MFS revealed that when CD44 expression in the molecular layers was high, MFS was minimal and when CD44 expression/function was reduced following KA treatment or with function blocking antibodies, MFS was increased. The time course of KA-induced reductions in CD44 expression was identical to the temporal progression of KA-induced MFS reported previously in hippocampal slice cultures, suggesting that reduced CD44 expression may help promote MFS. Understanding the molecular mechanisms underlying MFS may lead to therapeutic interventions that limit epileptogenesis.

Entorhinal and commissural/associational projections show a non-overlapping distribution in the hippocampus proper and the dentate gyrus. The expression of Ephrins and their Eph receptors in the developing hippocampus indicates that this family of axonal guidance molecules may modulate the formation of these connections. Here we focused on the role of the ephrin-A5 ligand in the development of the main hippocampal afferents. In situ hybridization showed that ephrin-A5 mRNA was detected mainly in the principal cells of the hippocampus proper and in the dentate gyrus throughout postnatal development. Immunocytochemical analyses revealed prominent expression of the EphA3 receptor, a putative receptor for ephrin-A5, in the main cells and the neuropil of the developing hippocampus. Tracing experiments in ephrin-A5(-/-) mice showed that commissural projections were transiently altered in the hippocampus proper at P5, but they were mistargeted throughout the postnatal development in the dentate gyrus. Immunocytochemistry with anti-calbindin antibodies revealed that the dentate mossy fiber projection was not altered in ephrin-A5(-/-) mice. Electron microscopy studies showed alterations in the density of synapses and spines in commissural/associational layers, but not in entorhinal layers, and in the mossy fibers in these animals. Taken together, these findings indicate that ephrin-A5 signaling is involved in the formation and maturation of synapses in the hippocampus.

As receptivity of the injured hippocampus to cell grafts decreases with time after injury, strategies that improve graft integration are necessary for graft-mediated treatment of chronic neurodegenerative conditions such as temporal lobe epilepsy. We ascertained the efficacy of two distinct graft-augmentation strategies for improving the survival of embryonic day 19 hippocampal CA3 cell grafts placed into the adult hippocampus at 4-months after kainic acid induced injury. The donor cells were labeled with 5'-bromodeoxyuridine, and pre-treated and grafted with either brain-derived neurotrophic factor, neurotrophin-3 and a caspase inhibitor or fibroblast growth factor and caspase inhibitor. The yield of surviving grafted cells and neurons were quantified at 2-months post-grafting. The yield of surviving cells was substantially greater in grafts treated with brain-derived neurotrophic factor, neurotrophin-3 and caspase inhibitor (84%) or fibroblast growth factor and caspase inhibitor (99% of injected cells) than standard cell grafts (26%). Because approximately 85% of surviving grafted cells were neurons, increased yield in augmented groups reflects enhanced survival of grafted neurons. Evaluation of the mossy fiber synaptic re-organization in additional kainic acid-lesioned rats receiving grafts enriched with brain-derived neurotrophic factor, neurotrophin-3 and caspase inhibitor at 3-months post-grafting revealed reduced aberrant dentate mossy fiber sprouting in the dentate supragranular layer than "lesion-only" rats at 4 months post-kainic acid, suggesting that some of the aberrantly sprouted mossy fibers in the dentate supragranular layer withdraw when apt target cells (i.e. grafted neurons) become available in their vicinity. Thus, the yield of surviving neurons from CA3 cell grafts placed into the adult hippocampus at an extended time-point after injury could be enhanced through apt neurotrophic supplementation and caspase inhibition. Apt grafting is also efficacious for reversing some of the abnormal synaptic reorganization prevalent in the hippocampus at later time-points after injury.

We sought to determine the impact of 17beta-estradiol throughout the hippocampal trisynaptic pathway and to investigate the afferent fiber systems within CA1 and CA3 in detail. To achieve this objective, we utilized multielectrode arrays to simultaneously record the field excitatory postsynaptic potentials from the CA1, dentate gyrus, and CA3 of rat hippocampal slices in the presence or absence of 100 pM 17beta-estradiol. We confirmed our earlier findings in CA1, where 17beta-estradiol significantly increased field excitatory postsynaptic potentials amplitude (20%+/-3%) and slope (22%+/-7%). 17beta-Estradiol significantly potentiated the field excitatory postsynaptic potentials in dentate gyrus, amplitude (15%+/-4%) and slope (17%+/-5), and in CA3, amplitude (15%+/-4%) and slope (19%+/-5%). Using a high-density multielectrode array, we sought to determine the source of potentiation in CA1 and CA3 by determining the impact of 17beta-estradiol on the apical afferents and the basal afferents within CA1 and on the mossy fibers and the associational/commissural fibers within CA3. In CA1, 17beta-estradiol induced a modest increase in the amplitude (7%+/-2%) and slope (9%+/-3%) following apical stimulation with similar magnitude of increase following basal stimulation amplitude (10%+/-2%) and slope (12%+/-3%). In CA3, 17beta-estradiol augmented the mossy fiber amplitude (15%+/-3%) and slope (18%+/-6%) and the associational/commissural fiber amplitude (31%+/-13%) and slope (40%+/-15%). These results indicate that 17beta-estradiol potentiated synaptic transmission in each subfield of the hippocampal slice, with the greatest magnitude of potentiation at the associational/commissural fibers in CA3. 17beta-Estradiol regulation of CA3 responses provides a novel site of 17beta-estradiol action that corresponds to the density of estrogen receptors within the hippocampus. The implications of 17beta-estradiol potentiation of the field potential in each of the hippocampal subfields and in particular CA3 associational/commissural fibers for memory function and clinical assessment are discussed.

The neuropeptide cholecystokinin (CCK) is widely distributed in the CNS. We herein investigated the immunocytochemical localization of CCK in the glutamatergic excitatory pathways in the mouse hippocampus, with particular reference to the dorsoventral difference. The intense CCK-like immunoreactivity (CCK-LI) was found in the mossy fiber pathway (stratum lucidum and dentate hilus) and in the inner molecular layer of the dentate gyrus. In the mossy fiber pathway, the CCK-LI was more intense at the ventral level than at the dorsal level. On the other hand, the CCK-LI in the stratum lucidum was more intense in the distal portion than in the proximal portion, both at the dorsal and ventral levels. High-resolution three-dimensional image analysis revealed the coexpression of CCK and synaptoporin (SPO) in the single mossy terminal, where they were spatially segregated but adjacent to each other. Quantitative image analysis indicated the difference in the amount of CCK within the mossy terminals along the dorsoventral and transverse axes of the hippocampus. On the other hand, in the inner molecular layer, CCK- and SPO-positive elements appeared to have little relation to each other. We also examined the postnatal development of the CCK-LI in the mouse hippocampus. The CCK-LI was detected in the inner molecular layer of the ventral dentate gyrus at postnatal day (P) 7. In the mossy fiber pathway, the CCK-LI was first evident at P 14, but it was restricted to the distal portion of the stratum lucidum in the ventral hippocampus. Interestingly, the distributions of the SPO immunoreactivity at P 7 were already similar to those of adult mice. The patterns of expression of CCK-LI at P 28 were almost similar to those of adult mice. The present data demonstrate the heterogeneous expression of CCK-LI in the mouse hippocampus, and provide a baseline to understand the role of CCK in the mouse brain.

Kindling, an animal model of epilepsy, results in an increased volume of the hilus of the dentate gyrus and sprouting of the mossy fiber pathway in the hippocampus. Our previous studies have revealed that chronic infusion of neurotrophins can regulate not only seizure development, but also these kindling-induced structural changes. Kindling, in turn, can alter the expression of neurotrophins and their receptors. We previously showed that intraventricular administration of a synthetic peptide that interferes with nerve growth factor stability and thus its binding to TrkA and p75(NTR) receptors suppressed kindling and sprouting. However, the precise involvement of TrkA, p75(NTR), and downstream signaling effectors of neurotrophins on kindling, sprouting and hilar changes are unknown. One of these downstream effectors is Ras. In the present study, we find that intraventricular infusion of the synthetic peptide Reo3Y, which binds to p65/p95 receptors and causes a rapid inactivation of Ras protein, impairs development of perforant path kindling, reduces the growth in afterdischarge duration, blocks kindling-induced mossy fiber sprouting in area CA3 of hippocampus and in inner molecular layer of the dentate gyrus, and prevents kindling-induced increases in hilar area. These results are consistent with a mediation of neurotrophin effects on kindling, hilar area, and axonal sprouting via Trk receptors, and suggest important roles for Ras in kindling and in kindling-induced structural changes.

Repetitive sound-induced seizures, known as audiogenic kindling (AK), gradually induce the transference of epileptic activity from brainstem to forebrain structures along with behavioral changes. The aim of our work was to correlate the behavioral changes observed during the AK with possible alterations in neuronal proliferation, cell death, hippocampal mossy fiber sprouting and in the EEG pattern of Wistar audiogenic rats, a genetically susceptible strain from our laboratory. Susceptible and non-susceptible animals were submitted to repeated sound stimulations for 14–16 days and hippocampal mitotic activity was studied through the incorporation of bromodeoxyuridine (BrdU). Cell death and mossy fiber sprouting were assessed, respectively, by using Fluoro-Jade and Timm staining, 2 and 32 days after the last kindling stimulation. In addition, we used immunofluorescent double labeling for a glial and a mitotic marker to evaluate newly born cell identity. Some animals had hippocampus and amygdala electrodes for EEG recordings. Our results show that kindled animals with 6–11 generalized limbic seizures (class IV–V) had increased cell proliferation in the dentate gyrus when compared with animals with zero or one to three seizures. BrdU-positive cells labeled on day 2 and on day 32 were both GFAP negative. In the later group, rounded and well-defined BrdU-positive/GFAP-negative nuclei were seen in different portions of the granule cell layer. We did not observe any Fluoro-Jade or differential Timm staining in kindled animals at both killing times. However, EEG recordings showed intense epileptic activity in the hippocampus and amygdala of all animals with limbic seizures. Therefore, our data indicate that AK-induced limbic epileptogenicity is able to increase the hippocampal mitotic rate, even though it does not seem to promote neuronal death or mossy fiber sprouting in the supragranular layer of the dentate gyrus.

Neuronal calcium sensor-1 (NCS-1) is a member of the EF-hand calcium-binding protein superfamily, which is considered to modulate synaptic transmission and plasticity. In this work, we first examined the distribution patterns of NCS-1 in the hippocampus and cerebellum. The intense NCS-1-immunoreactive (IR) elements in the hippocampus were restricted to dendritic layers, while those in the cerebellum occurred in both dendritic and cellular layers. Then, we examined the exact localization of NCS-1 using immunofluorescent double labeling for NCS-1 and synaptophysin, a marker of presynaptic terminals. In the hippocampus, the mossy fiber systems (terminals and bundles) exhibited intense NCS-1 immunoreactivity. On the other hand, the presumed principal cell dendrites were also NCS-1-IR in the stratum lacunosum-moleculare of Ammon’s horn and molecular layer of the dentate gyrus, where NCS-1-IR elements and synaptophysin-IR presynaptic terminals showed characteristic complementary distribution patterns. In the cerebellum, some of the basket cell axon terminals surrounding the somata of Purkinje cells exhibited NCS-1 immunoreactivity, while the pinceau showed consistent labeling for NCS-1. Higher magnification observations revealed that the NCS-1-IR presumed granule cell dendrites and synaptophysin-IR mossy fiber terminals in the glomeruli of the cerebellum showed characteristic complementary distribution patterns. Furthermore, we estimated quantitatively the relative amount of NCS-1 in the presynaptic terminals in individual layers, and confirmed that the mossy fiber terminals in the hippocampus contained comparatively high amounts of NCS-1. These results showed the diverse localization of NCS-1 in pre- and/or postsynaptic elements of the hippocampus and cerebellum, and suggest potential roles in specific synaptic transmission.

Mossy fiber activity can evoke Ca2+ release from internal stores in CA3 neurons, but the physiological conditions under which this occurs and the mechanisms underlying the release are not understood. Using rat hippocampal slices we report here that short trains of mossy fiber stimulation activate group I metabotropic glutamate receptors (mGluRs) on CA3 pyramidal neurons and elicit waves of Ca2+ release from inositol 1,4,5-trisphosphate (IP3) sensitive internal stores that propagate from stratum lucidum to the soma and in some cases distally out the dendrites. Activation of mGluR1,5 receptors by an agonist trans-azetidine-2,4-dicarboxylic acid (tADA) applied to stratum lucidum was also sufficient to induce waves of Ca2+ release. This release was blocked by internal heparin, but not by dantrolene, suggesting the involvement of IP3 rather than ryanodine receptors in not only the initial release but also in the maintenance of the propagating waves. Release could be facilitated by Ca2+ influx through voltage-gated Ca2+ channels, which is consistent with the known Ca2+ sensitivity of IP3 receptors. These results provide insight into the mechanisms and conditions of Ca2+ release in CA3 neurons and demonstrate the powerful influence mossy fiber input can have on these neurons.

Integrins are heterodimeric cell surface receptors composed of different alpha and beta subunits that mediate cell-cell and cell-extracellular matrix interactions. They have been implicated in the regulation of neuronal migration, differentiation, process outgrowth, and plasticity. The alpha8 integrin subunit associates exclusively with the beta1 subunit to form a receptor (alpha8beta1) for fibronectin, vitronectin, tenascin, and osteopontin. In a previous study, we demonstrated that hippocampal dentate hilar neurons are immunoreactive for alpha8. The present study identifies the major types of alpha8-immunoreactive hilar neurons and characterizes the effects of kainic acid-induced seizures on alpha8-immunoreactivity in these cells. Examination of the hilus in normal rats revealed alpha8-immunoreactivity in the somatodendritic compartments of large hilar neurons identified as mossy cells, including a subset of dendritic thorny excrescences that were contacted by large mossy fiber terminals. alpha8-immunoreactivity also was found in approximately 71% of somatostatin-containing hilar cells. Kainic acid-induced seizures dramatically and rapidly altered the levels and distribution of alpha8-immunoreactivity in hilar neurons. After 1.5 h of seizures, alpha8-immunoreactivity in their dendrites was reduced greatly. One day after kainic acid treatment, labeling was diminished throughout the somatodendritic compartments of most hilar cells. This decrease appeared to be transient, since alpha8 labeling returned to normal levels in surviving hilar neurons within 2 weeks of treatment. In addition, many alpha8-immunoreactive hilar neurons, particularly in caudal dentate regions, were lost 3-5 weeks after kainic acid treatment. Our findings suggest that alpha8beta1 may mediate adhesive interactions of the dendritic processes of mossy cells and somatostatin-containing hilar neurons with other cellular elements or with extracellular matrix components. They also suggest that alpha8 may be susceptible to activity-dependent proteolysis that could modulate its function in the somatodendritic compartment of these cells.

The unipolar brush cells are excitatory, cerebellar granular layer interneurons that receive mossy fiber input on their dendritic brushes in the form of a giant glutamatergic synapse. We investigated the postnatal development of the brush of the unipolar brush cell in lobules IX and X by light microscopy and defined the maturation of mossy fiber-unipolar brush cell synapses and mossy fiber-granule cell synapses by electron microscopy using calretinin immunocytochemistry to identify unipolar brush cells. During the first postnatal week, unipolar brush cells possessed one or two short, branched dendrites. The brush differentiated primarily during the successive 21 postnatal (P) days, during which it underwent progressive maturation. This developmental process was subdivided into stages 1–4, which were descriptively termed protodendritic unipolar brush cell (P2–12), filopodial brush (P12–16), intermediate brush (P16–21), and dendriolar brush (P21–28) stages. Electron microscopic measurements of individual mossy fiber-unipolar brush cell and mossy fiber-granule cell synaptic junctions were made at P12, 16, 21, and 28. While the average length of mossy fiber-unipolar brush cell synapses increased during development, that of mossy fiber-granule cell synapses decreased. Comparisons of the lengths of mossy fiber-unipolar brush cell and mossy fiber-granule cell synapses demonstrated that mossy fiber-unipolar brush cell synapses were longer on average than mossy fiber-granule cell synapses for all ages. Frequency distribution histograms also showed that the percentage of mossy fiber-unipolar brush cell synapses longer than 0.5 μm was lower in the pooled P12–P16 groups than in the pooled P21–P28 groups (8 versus 20%). In contrast, mossy fiber-granule cell synapses longer than 0.5 μm were a small minority at P12, 16, and 21, and occurred rarely at P28. The present study indicates that mossy fiber-unipolar brush cell synapses increase in length with the differentiation of the brush dendrioles, while that of mossy fiber-granule cell synapses decrease with differentiation of the granule cell dendritic claws. The finding that mossy fiber-unipolar brush cell synapses were generally longer than mossy fiber-granule cell synapses may indicate that the properties of the postsynaptic targets play a major role in shaping synaptic appositions within cerebellar glomeruli.

Stress, which can precipitate and exacerbate depression, causes atrophy and in severe cases death of hippocampal neurons. Atrophy of the hippocampus has also been observed in patients suffering from recurrent major depression. The present study examines the influence of electroconvulsive seizures, one of the most effective treatments for depression, on the morphology and survival of hippocampal neurons. The results demonstrate that chronic administration of electroconvulsive seizures induces sprouting of the granule cell mossy fiber pathway in the hippocampus. This sprouting is dependent on repeated administration of electroconvulsive seizures, reaches a maximum 12 days after the last treatment and is long lasting (i.e. up to six months). Electroconvulsive seizure-induced sprouting occurs in the absence of neuronal loss, indicating that sprouting is not a compensatory response to cell death. This is different from the sprouting induced by kindling or excitotoxin treatment, which induce cell death along with recurrent seizures. Electroconvulsive seizure-induced sprouting is significantly diminished in brain-derived neurotrophic factor heterozygote knockout mice, indicating that this neurotrophic factor contributes to mossy fiber sprouting. However, infusion of brain-derived neurotrophic factor into the hippocampus does not induce sprouting of the mossy fiber pathway. The results demonstrate that chronic administration of electroconvulsive seizures induces mossy fiber sprouting and suggest that increased expression of brain-derived neurotrophic factor is necessary, but not sufficient for the induction of this sprouting. Although the functional consequences remain unclear, sprouting of the mossy fiber pathway would appear to oppose the actions of stress and could thereby contribute to the therapeutic actions of electroconvulsive seizure therapy.

In our previous study we revealed the presence of clustered large calretinin-immunoreactive multipolar cells in the ventral hilus of the mouse dentate gyrus and indicated that they might be mossy cells, the principal neurons in the dentate hilus. In the present study we confirmed this identification with several methods and analysed further in detail. In Golgi-impregnated samples mossy cells were easily identified by their locations and characteristic thorny excrescences on their proximal dendrites. Golgi-impregnated mossy cells were observed not only in the ventral hilus but also in the dorsal hilus, where no calretinin-immunoreactive large multipolar cells were encountered. Interestingly, mossy cells exhibited dorsoventral differences in the size and complexity of thorny excrescences; mossy cells at the dorsal and middle levels had larger and more complex thorny excrescences, which covered dendritic shafts for a longer distance, while ventral mossy cells had smaller, simpler and shorter thorny excrescences. Confocal laser scanning light microscopic observations at a high magnification showed that the vast majority of calretinin-immunoreactive large neurons in the ventral hilus displayed the thorny excrescences characteristic to mossy cells. Mossy cells identified with the intracellular injection of Lucifer Yellow were calretinin-immunoreactive. Electron microscopic observations clearly revealed that calretinin-immunoreactive elements showed structural features of mossy cells such as thorny excrescences receiving typical synapses from mossy fibre terminals. At the supragranular zone, a well-known target zone of mossy cell axons, a dense calretinin-immunoreactive band was seen, where numerous calretinin-immunoreactive punctae and fibres were packed. Electron microscopic observations revealed that these calretinin-immunoreactive axon terminals in the supragranular zone made asymmetrical synapses on presumed granule cell dendritic spines. Tracer injection studies and lesion experiments indicated that the supragranular calretinin-immunoreactive axon terminals mainly originated from the large calretinin-immunoreactive multipolar cells in the ipsilateral ventral hilus. Fluorescent double immunostaining for calretinin and glutamate receptor 2/3 (GluR2/3) revealed that all large calretinin-immunoreactive hilar cells in the ventral level were GluR2/3-immunoreactive and almost all intensely GluR2/3-immunoreactive hilar cells in the ventral level were calretinin-immunoreactive. In addition intensely GluR2/3-immunoreactive but calretinin-negative large cells were encountered in the dentate hilus at the dorsal level. On the basis of these observations, we concluded that large calretinin-immunoreactive cells in the ventral hilus of the mouse dentate gyrus were really mossy cells and that mossy cells at the dorsal level were calretinin negative. The present study revealed that mouse mossy cells show the dorsoventral difference in the calretinin immunoreactivity and thus they are chemically heterogeneous.

Kindling is an animal model of epileptogenesis, whereby repeated administration of an initially subconvulsive electrical stimulation eventually leads to the development of generalized motor seizures. Once established, the kindling effect is permanent. Although the molecular basis of kindling remains incompletely understood, emerging lines of evidence suggest that the induction of immediate-early genes could represent a link between periodic short-term stimuli and the long-lasting functional and structural alterations in the brain associated with the development of seizures. A recent study showed that null mutation of the immediate-early gene c-fos impairs the structural and functional plasticities in kindling. In the present study, we examined whether two other seizure-inducible immediate-early genes--NGFI-A (also termed EGR-1, zifl268, and Krox-24) and NGFI-B (also termed Nur77)--play requisite roles in kindling. We found that neither the rate of kindling nor seizure-induced granule cell axonal sprouting was affected in mice carrying a null mutation of NGFI-A. Furthermore, double knock-out of NGFI-A and NGFI-B genes does not result in detectable changes in kindling development and associated mossy fibre sprouting. Taken together, our observations indicate that neither constitutive nor seizure-induced expression of NGFI-A or NGFI-B is uniquely required for the establishment of kindling. These findings underscore the specificity of the immediate-early genes whose transcriptional activation contributes to kindling epileptogenesis.

Some glutamatergic synapses in the mammalian central nervous system exhibit high levels of free ionic zinc in their synaptic vesicles. The precise role of this vesicular zinc remains obscure, despite suggestive evidence for zinc as a neuromodulator. As a step towards elucidating the role of free zinc in the brain we have developed a method for imaging zinc release in live brain slices. A newly synthesized zinc-sensitive fluorescent probe, N-(6-methoxy-8-quinolyl)-p-carboxybenzoylsulphonamide (TFLZn), was used to monitor intracellular zinc in live rat hippocampal slices. The dye loaded into the zinc-rich synaptic vesicles of the mossy fibre terminals in the hippocampal formation. Direct electrical stimulation of the mossy fibre pathway diminished the fluorescence in the mossy fibre terminals, consistent with a stimulus-dependent zinc release. The synaptic release of zinc was followed by the rapid replenishment of the zinc levels in vesicles from an as yet unidentified intracellular zinc source. Furthermore, we present evidence that zinc may play a role in a form of long-term potentiation exhibited by the mossy fibre pathway.

In the CA3 region of rat dorsal hippocampus, several σ ligands, such as 1,3-di(2-tolyl)guanidine (DTG), (+)-pentazocine and (+)- N -cyclopropylmethyl- N -methyl-1,4-diphenyl-l-ethyl-but-3-en-1-ylamine hydrochloride (JO-1784), administered intravenously at low doses, potentiate selectively the pyramidal neuron firing activity induced by microiontophoretic applications of N -methyl- d -aspartate, without affecting those induced by quisqualate, kainate or acetylcholine. A similar potentiation of the N -methyl- d -aspartate response has also been found with microiontophoretic applications of neuropeptide Y, an effect exerted via a receptors. The present experiments were carried out to determine the effects of these σ ligands and of neuropeptide Y, in the CAI and CA3 regions following unilateral destruction by a local injection of colchicine of the mossy fiber system, which is a major afference to CA3 pyramidal neurons. In the CAI region, DTG, JO-1784 and neuropeptide Y did not potentiate the activation induced by microiontophoretic applications of N -methyl- d -aspartate. However, ( + )-pentazocine potentiated the N -methyl- d -aspartate response, similarly to its effect in the CA3 region on the intact side. In the CA3 region, on the intact side, (+ )-pentazocine, DTG, JO-1784 and neuropeptide Y induced a selective potentiation of N -methyl- d -aspartate-induced activation, in keeping with previous reports. On the lesioned side, the effect of (+ )-pentazocine on the N -methyl- d -aspartate response was still present, but those of DTG, JO-1784 and neuropeptide Y were abolished. These results suggest that ( + )-pentazocine, on the one hand, and DTG, JO-1784 and neuropeptide Y, on the other, are not acting on the same subtype of a receptors. Since ( + )-pentazocine, JO-1784 and neuropeptide Y have been suggested to act on the a, subtype of receptors, these data suggest the existence of two subtypes ofσ 1 receptors. They also suggest that the receptors on which DTG, JO-1784 and neuropeptide Y are acting are located on the mossy fiber terminals in the CA3 region and are absent in the CA1 region.

The mechanism of delayed death of pyramidal cells in the hippocampal CA1 region and the acute death of various types of hilar neurons after ischemia is still unknown. Excitotoxicity may play a role in ischemic cell death, a prerequisite of which is the development of increased excitability or an enhanced excitatory transmission in the selectively vulnerable subfields of the hippocampus. Such changes may take place upon the loss or malfunction of local inhibitory neurons in the early postischemic period. In the present study we examined the vulnerability of non-pyramidal neurons containing a recently discovered calcium binding protein, calretinin, in the rat hippocampus following 15 min ischemia induced by four-vessel occlusion. Immunostaining for calretinin enabled us to visualize a new type of spiny non-pyramidal cell in the hippocampus specifically associated with the mossy fiber system. This cell type is present exclusively in regions where mossy fiber terminals occur, i.e. in the hilus of the dentate gyrus and in stratum lucidum of the CA3 subfield. A selective loss of immunoreactivity in these neurons was already observed at 12-24 h after ischemia, when the pyramidal cells in the CA1 region showed no signs of damage. At a survival time of two to three days, most if not all spiny calretinin-immunoreactive cells had disappeared from the hippocampus. Other types of calretinin-containing GABAergic neurons were also reduced in number, but only at a time when CA1 pyramidal cells also started to degenerate, i.e. two to three days after ischemia. We speculate that the early loss of spiny calretinin-containing cells, together with other non-pyramidal cells associated with the mossy fiber system (somatostatin-containing neurons and mossy cells of the hilus), may result in pathological network activity in the hippocampus, which may ultimately lead to an increased excitatory transmission and delayed pyramidal cell death in the CA1 region.

Mossy fibre synapses on the CA3 hippocampal neurons in the brain of ground squirrels repeatedly undergo a striking structural transformation during hibernation. In the middle of hibernation bout the giant complex mossy fibre synapses have a reduced number of dendritic spine infoldings that are smaller and have a decreased number of postsynaptic densities in comparison with mossy fibre synapses of active animals. Two hours after arousal all these parameters of mossy fibre synapses increase and significantly exceed their levels not only in torpid but in active euthermic animals between bouts of torpor. The longer postsynaptic densities and the greater proportion of perforated postsynaptic densities were found soon after arousal. These rapid, reversible and repeated changes indicate a cyclic process of partial denervation/reinnervation of hippocampal neurons by mossy fibres in the course of the innate, stereotyped behaviour.

One hundred and eight male mice from nine different inbred strains were tested for two aspects of learning in an eight-arm radial maze. In the first experimental arrangement of the maze, measuring spatial working memory, clear strain differences were found on the fifth day of training. Furthermore, this type of learning showed a high positive correlation with the size of the intra- and infrapyramidal hippocampal mossy fiber terminal field as revealed with Timm's staining. In the second experiment, in which non-spatial reference memory was tested, significant strain differences were found for the learning variables, but there were no significant covariations with the sizes of the intra- and infrapyramidal mossy fiber terminal fields. These results, combined with previous data, suggest that heritable variations of the hippocampal intra- and infrapyramidal mossy fiber projection influence processes determining spatial learning capabilities in mice.

Previous studies have demonstrated that tetanization of hippocampal mossy fibers induces a long-term potentiation of non-tetanized (heterosynaptic) non-mossy fiber afferents (Schaffer collateral/commissural and fimbrial fibers). Tetanization of these non-mossy fiber afferents, in contrast, does not induce mossy fiber long-term potentiation, but induces a long-term depression of mossy fiber responses (Bradler and Barrionuevo, Synapse 4, 132–142, 1989). The synaptic activity necessary to evoke these heterosynaptic alterations of efficacy is not known. Specifically, the dependence of heterosynaptic efficacy on the activation of N-methyl- d -aspartate receptors has not been assessed. In addition, the capability of different afferents to CA3 neurons to support alterations in heterosynaptic efficacy remains largely unknown. In the present study, heterosynaptic alterations of efficacy in the rat did not require the activation of N-methyl- d -aspartate receptors. Mossy fibers supported N-methyl- d -aspartate receptor-indepedent heterosynaptic long-term depression, and N-methyl- d -aspartate receptor-independent long-term potentiation. In contrast, non-mossy fiber afferents expressed N-methyl- d -aspartate receptor-independent heterosynaptic long-term potentiation induced by a mossy fiber tetanus, and an x N-methyl- d -aspartate receptor-independent long-term depression, in addition to N-methyl- d -aspartate receptor-dependent homosynaptic long-term potentiation. The possibility that N--methyl- d -aspartate receptor activity in non-tetanized afferents is necessary for heterosynaptic long-term potentiation induction is discussed. Heterosynaptic long-term depression was induced in the absence of homosynaptic long-term potentiation, suggesting that these concomitant forms of synaptic plasticity rely on different mechanisms.

Axon sprouting typically occurs in a brain region that has been partially denervated. The present study demonstrates, quantitatively, evidence for sprouting outside the region of deafferentation. A modification of the Timm sulfide silver histochemical method was used to monitor an increase in the mossy fiber terminal field in the fascia dentata of adult rats following severe deafferentation of the outer three-fourths of stratum moleculare by unilateral entorhinal lesions. Computer-assisted image processing techniques were used to quantify mossy fiber sprouting. In stratum granulosum, and to a lesser extent in the deep (supragranular) portion of stratum moleculare (areas separated from the zone of deafferentation), there was a three-fold increase in the area of mossy fiber staining on the side of the lesion compared to the non-operated side (and unoperated animals). Much of the increased staining was located near the tip of the infrapyramidal (ventral) blade of the fascia dentata. Since mossy fiber sprouting apparently occurs in the absence of degeneration-produced synaptic dilution in that region, it may represent an example of post-lesion growth initiated by conditions fundamentally different from those normally believed to induce sprouting.

Pyramidal cells of regio inferior in slice cultures of the rat hippocampus were impaled and intracellularly stained with horseradish peroxidase. A correlated light- and electron-microscopic analysis was then performed to study the properties of these neurons under culture conditions with particular emphasis on input synapses onto these cells. Like pyramidal cells in situ, CA3 pyramidal neurons in slice cultures had a triangular cell body with an apical stem dendrite emerging from it. Several basal dendrites and the axon arose from the basal pole of the cell body. The peripheral thin branches of both apical and basal dendrites were covered with small spines, whereas proximal thick dendritic segments and portions of the cell body exhibited large spines or excrescences. The axon gave off numerous fine varicose collaterals which projected to stratum radiatum of CA1 (Schaffer collaterals), to the alveus and to the hilar region. In one case a collateral could be followed to stratum moleculare of the fascia dentata. Electron-microscopic analysis of the injected pyramidal neurons revealed that their cell bodies, dendritic shafts and spines formed synaptic contacts with presynaptic terminals. Mossy fiber endings were identified by their large size and their numerous clear synaptic vesicles with some dense-core vesicles intermingled, and were observed to form synaptic contacts on the large spines or excrescences. Since extrinsic afferents degenerate in slice cultures, the numerous synaptic boutons on the identified pyramidal neurons probably arise from axons of intrinsic neurons that have sprouted in response to deafferentation. This assumption is supported by the finding that collaterals of the injected neurons formed abundant synaptic contacts on dendritic shafts and spines of other cells. These results suggest that, although pyramidal cells under culture conditions retain a remarkable number of their normal characteristics, considerable synaptic reorganization does take place.

Previous reports 655 have indicated that pyramidal cells in the developing rat hippocampal formation are not destroyed by intraventricular or inlraperitoneal administration of kainic acid. We examined the neurotoxic properties of kainic acid and ibotenic aeid following intrahippocampal injection in neonatal rats and found significant pyramidal cell death following injection of 1.0 μg kainic acid in 6, 7 and 9-day-old pups. At doses 2.5 or live times this amount, significant pyramidal cell loss was obtained in 5-day-old rats as well. The susceptibility of pyramidal neurons to kainic acid increased as a function of age. The developing hippocampus was considerably more vulnerable to ibotenic acid compared with kainic acid, in contrast to the order of potency reported in adult rats. The increased sensitivity of CA3 pyramidal cells parallels the development of the mossy fiber innervation to the dendrites of these cells supporting the twofold mechanism suggested by Coyle for kainic acid neurotoxicity; that is, a direct cytotoxic action via postsynaptic receptors as well as increased sensitivity due to the presence of excitatory inputs.

The postnatal development of leucine 5 -enkephalin-like immunoreactivity within the hippocampal formation of the rat has been analyzed using immunocytochemical techniques. From the day of birth to postnatal day three, no intrinsic hippocampal elements exhibit immunoreactivity although labeled axons are found within the fimbria, within the alveus, and in the vicinity of the angular bundle. On postnatal day 4, a few immunoreactive hippocampal neurons can be seen in stratum radiatum of the region CA3 and by postnatal day 8, within the hilus, strata pyramidale and oriens of regio superior, and the subiculum. There is a dramatic increase in the incidence of immunoreactive perikarya between postnatal days 8 and 10 in all fields as well as the appearance of labeled neurons in CA1 stratum pyramidale and stratum granulosum of the dentate gyrus. Two days after the first appearance of immunoreactive perikarya, intensely immunoreactive neurons, labeled much more extensively than is ever seen in the adult, are encountered in each subfield of the hippocampus. The spatio-temporal order in both the emergence of perikaryal immunoreactivity and the transient appearance of intensely immunoreactive neurons follows that of neurogenesis, with immunoreactivity developing 12–14 days after the peak period of last cell division for a given hippocampal region. The incidence of immunoreactive perikarya in the dentate gyrus was quantified in rat pups ranging from postnatal days 8 to 19. The appearance of labeled neurons followed the spatio-temporal gradients that have been described for neurogenesis in this region as well. Immunoreactive perikarya emerged in the suprapyramidal stratum granulosum prior to their emergence in the infrapyramidal zone and in the temporal pole of the dentate earlier than in the mid-dorsoventral dentate. The lateral perforant path and mossy fiber axons, seen to exhibit enkephalin-like immunoreactivity in the adult hippocampal formation, differ in their relative maturity at the age immunoreactivity first appears. Immunoreactivity appears as early as postnatal day 4 in the lateral perforant path, an age at which these axons are just growing into their target field while it is not found within the mossy fibers until after postnatal day 10, an age at which mossy fiber bouton elaboration is well advanced and physiologically competent mossy fiber synapses with the regio inferior pyramidal cells have been established. The latter observation indicates that enkephalin is not necessary for synaptic transmission at the mossy fiber synapse.

The regional distribution of high affinity binding sites for kainic acid has been determined in rat hippocampi by quantitative autoradiography. Selective lesions were made in order to determine the exact localization of these sites in the mossy fiber system, and to evaluate whether the sprouting and synaptic reorganization of the mossy fibers are associated with alterations in the distribution of these binding sites. The results show that kainate binding sites in the stratum lucidum are more vulnerable to destruction of the granules and their mossy fibers by intrahippocampal colchicine injections than to destruction of the CA3/CA4 pyramidal cells by injection of kainate into the amygdala. This suggests that a substantial proportion of the kainate binding sites is associated with the mossy fiber terminals (i.e. the presynaptic elements). Furthermore, in keeping with an earlier study, destruction of the pyramidal neurons of CA3 by intracerebral kainate produced a dark Timm positive band in the supragranular zone which is due to the sprouting of mossy fibers. This was associated with an increase in the density of kainate binding sites, which further stresses the parallelism between the distribution of these sites and mossy fiber terminals.