Temporal profiles of the in vitro phosphorylation rate and immunocontent of glial fibrillary acidic protein (GFAP) after kainic acid-induced lesions in area CA1 of the rat hippocampus: demonstration of a novel phosphoprotein associated with gliosis

Kainate-induced seizure activity causes persistent changes in the hippocampus that include synaptic reorganization and functional changes in the mossy fibers. Using in situ hybridization histochemistry, the expression of PKC alpha, PKC beta, PKC gamma, PKC delta and PKC epsilon mRNAs was investigated in the hippocampus of adult rats following seizures induced by a s.c. injection of kainic acid. In CA1 and CA3, we found a significant decrease in PKC gamma mRNA 1 day after kainic acid which persisted for a 2nd day in CA1. None of the other PKC isoform mRNAs were altered in CA1 or CA3. In granule cells, a significant up-regulation specific to PKC epsilon mRNA was observed. One week after kainic acid administration, a marked increase in PKC epsilon immunoreactivity was found that persisted 2 months after kainic acid administration. PKC epsilon immunoreactivity was found associated with mossy fibers projecting to the hilus of the dentate gyrus and to the stratum lucidum of the CA3 field and presumably with the newly sprouted mossy fibers projecting to the supragranular layer. These data provide the first evidence for a long-lasting increase of the PKC epsilon in the axons of granule cells caused by kainate-induced seizures and suggest that PKC epsilon may be involved in the functional and/or structural modifications of granule cells that occur after limbic seizures.     

GABAergic cells are the major postsynaptic targets of mossy fibers in the rat hippocampus

Dentate granule cells communicate with their postsynaptic targets by three distinct terminal types. These include the large mossy terminals, filopodial extensions of the mossy terminals, and smaller en passant synaptic varicosities. We examined the postsynaptic targets of mossy fibers by combining in vivo intracellular labeling of granule cells, immunocytochemistry, and electron microscopy. Single granule cells formed large, complex "mossy" synapses on 11-15 CA3 pyramidal cells and 7-12 hilar mossy cells. In contrast, GABAergic interneurons, identified with immunostaining for substance P-receptor, parvalbumin, and mGluR1a-receptor, were selectively innervated by very thin (filopodial) extensions of the mossy terminals and by small en passant boutons in both the hilar and CA3 regions. These terminals formed single, often perforated, asymmetric synapses on the cell bodies, dendrites, and spines of GABAergic interneurons. The number of filopodial extensions and small terminals was 10 times larger than the number of mossy terminals. These findings show that in contrast to cortical pyramidal neurons, (1) granule cells developed distinct types of terminals to affect interneurons and pyramidal cells and (2) they innervated more inhibitory than excitatory cells. These findings may explain the physiological observations that increased activity of granule cells suppresses the overall excitability of the CA3 recurrent system and may form the structural basis of the target-dependent regulation of glutamate release in the mossy fiber system.     

Altered hippocampal kainate-receptor mRNA levels in temporal lobe epilepsy patients

This study determined whether hippocampal kainate (KA) receptor mRNA levels were increased or decreased in temporal lobe epilepsy patients compared with nonseizure autopsies. Hippocampal sclerosis (HS; n = 17), nonsclerosis (non-HS; n = 11), and autopsy hippocampi (n = 9) were studied for KA1-2 and GluR5-7 mRNA levels using semiquantitative in situ hybridization techniques, along with neuron densities. Compared with autopsy hippocampi, HS and non-HS cases showed decreased GluR5 and GluR6 hybridization densities per CA2 and/or CA3 pyramid. Furthermore, HS patients demonstrated increased KA2 and GluR5 hybridization densities per granule cell compared with autopsy hippocampi. These findings indicate that chronic temporal lobe seizures were associated with differential changes in hippocampal KA1-2 and GluR5-7 hybridization densities that vary by subfield and pathology group. In temporal lobe epilepsy patients, these results support the hypothesis that pyramidal cell GluR5 and GluR6 mRNA levels are decreased as a consequence of seizures, and in HS patients granule cell KA2 and GluR5 mRNA levels are increased in association with aberrant fascia dentata mossy fiber sprouting and/or hippocampal neuronal loss.     

Synaptic reorganization in explanted cultures of rat hippocampus

Due to loss of afferent innervation, synaptic reorganization occurs in organotypic hippocampal slice cultures. With extra- and intracellular recordings, we confirm that the excitatory loop from the dentate gyrus (DG) to CA3 and further to CA1 is preserved. However, hilar stimulation evoked antidromic population spikes in the DG which were followed by a population postsynaptic potential (PPSP); intracellularly, an antidromic spike with a broad shoulder or EPSP/IPSP sequences were induced. Synaptic responses were blocked by glutamate receptor antagonists. Stimulation of CA1 induced a PPSP in DG. Dextranamine stained pyramidal cells of CA1 were shown to project to DG. After removal of area CA3, DG's and mossy fibers' (MF) stimulation still elicited PPSPs and EPSP/IPSP sequences in area CA1 which disappeared when a cut was made through the hippocampal fissure. During bicuculline perfusion, hilar stimulation caused EPSPs in granule cells and spontaneous and evoked repetitive firing appeared even after its isolation from areas CA3 and CA1. Collateral excitatory synaptic coupling between granule cells was confirmed by paired recordings. Besides the preservation of the trisynaptic pathway in this preparation, new functional synaptic contacts appear, presumably due to MF collateral sprouting and formation of pathways between areas CA1 and DG.     

Consequences of neonatal seizures in the rat: morphological and behavioral effects

Whereas neonatal seizures are a predictor of adverse neurological outcome, there is controversy regarding whether seizures simply reflect an underlying brain injury or can cause damage. We subjected neonatal rats to a series of 25 brief flurothyl-induced seizures. Once mature the rats were compared with control littermates for spatial learning and activity level. Short-term effects of recurrent seizures on hippocampal excitation were assessed by using the intact hippocampus formation preparation and long-term effects by assessing seizure threshold. Brains were analyzed for neuronal loss, sprouting of granule cell axons (mossy fibers), and neurogenesis. Compared with controls, rats subjected to neonatal seizures had impaired learning and decreased activity levels. There were no differences in paired-pulse excitation or inhibition or duration of afterdischarges in the intact hippocampal preparation. However, when studied as adults, rats with recurrent flurothyl seizures had a significantly lower seizure threshold to pentylenetetrazol than controls. Rats with recurrent seizures had greater numbers of dentate granule cells and more newly formed granule cells than the controls. Rats with recurrent seizures also had sprouting of mossy fibers in CA3 and the supragranular region. Recurrent brief seizures during the neonatal period have long-term detrimental effects on behavior, seizure susceptibility, and brain development.     

Differential regulation by MK801 of immediate-early genes, brain-derived neurotrophic factor and trk receptor mRNA induced by a kindling after-discharge

Transient changes in immediate-early genes and neurotrophin expression produced by kindling stimulation may mediate secondary downstream events involved in kindling development. Recent experiments have demonstrated conclusively that both kindling progression and mossy fibre sprouting are significantly impaired by administration of the N-methyl-D-aspartate (NMDA) receptor antagonist MK801. To further examine the link between kindling, changes in gene expression and the NMDA receptor, we examined the effects of MK801 on neuronal induction of immediate-early genes, brain-derived neurotrophic factor (BDNF) and trk receptor mRNA expression produced by a single electrically induced hippocampal after-discharge in rats. The after-discharge produced a rapid (after 1 h) increase in Fos, Jun-B, c-Jun, Krox-24 mRNA and protein and Krox-20 protein in dentate granule neurons and a delayed, selective expression of Fos, Jun-D and Krox-24 in hilar interneurons. MK801 pretreatment produced a very strong inhibition of Fos, Jun-D and Krox-20 increases in dentate neurons but had a much smaller effect on Jun-B and c-Jun expression. MK801 did not inhibit Krox-24 expression in granule neurons or the delayed expression of Fos, Jun-D and Krox-24 in hilar interneurons. BDNF protein and trk B and trk C mRNA expression were also strongly induced in dentate granule cells 4 h following an after-discharge. MK801 abolished the increase in BDNF protein and trk B, but not trk C mRNA in granule cells at 4 h. These results demonstrate that MK801 differentially regulates the AD-increased expression of a group of genes previously identified as being likely candidates for an involvement in kindling. Because MK801 significantly retards the development of kindling and mossy fibre sprouting, it can be argued that those genes whose induction is not significantly attenuated by MK801 are unlikely to play an important role in the MK801-sensitive component of kindling and the changes in neural connectivity (mossy fibre sprouting) associated with kindling. Conversely, the role in kindling of those genes whose expression was significantly attenuated by MK801 (Fos, Jun-D, Krox-20, trkB and BDNF) requires further examination.     

Evidence from simultaneous intracellular recordings in rat hippocampal slices that area CA3 pyramidal cells innervate dentate hilar mossy cells

1. Simultaneous intracellular recordings of area CA3 pyramidal cells and dentate hilar "mossy" cells were made in rat hippocampal slices to test the hypothesis that area CA3 pyramidal cells excite mossy cells monosynaptically. Mossy cells and pyramidal cells were differentiated by location and electrophysiological characteristics. When cells were impaled near the border of area CA3 and the hilus, their identity was confirmed morphologically after injection of the marker Neurobiotin. 2. Evidence for monosynaptic excitation of a mossy cell by a pyramidal cell was obtained in 7 of 481 (1.4%) paired recordings. In these cases, a pyramidal cell action potential was followed immediately by a 0.40 to 6.75 (mean, 2.26) mV depolarization in the simultaneously recorded mossy cell (mossy cell membrane potentials, -60 to -70 mV). Given that pyramidal cells used an excitatory amino acid as a neurotransmitter (Cotman and Nadler 1987; Ottersen and Storm-Mathisen 1987) and recordings were made in the presence of the GABAA receptor antagonist bicuculline (25 microM), it is likely that the depolarizations were unitary excitatory postsynaptic potentials (EPSPs). 3. Unitary EPSPs of mossy cells were prone to apparent "failure." The probability of failure was extremely high (up to 0.72; mean = 0.48) if the effects of all presynaptic action potentials were examined, including action potentials triggered inadvertently during other spontaneous EPSPs of the mossy cell. Probability of failure was relatively low (as low as 0; mean = 0.24) if action potentials that occurred during spontaneous activity of the mossy cell were excluded. These data suggest that unitary EPSPs produced by pyramidal cells are strongly affected by concurrent synaptic inputs to the mossy cell. 4. Unitary EPSPs were not clearly affected by manipulation of the mossy cell's membrane potential. This is consistent with the recent report that area CA3 pyramidal cells innervate distal dendrites of mossy cells (Kunkel et al. 1993). Such a distal location also may contribute to the high incidence of apparent failures. 5. Characteristics of unitary EPSPs generated by pyramidal cells were compared with the properties of the unitary EPSPs produced by granule cells. In two slices, pyramidal cell and granule cell inputs to the same mossy cell were compared. In other slices, inputs to different mossy cells were compared. In all experiments, unitary EPSPs produced by granule cells were larger in amplitude but similar in time course to unitary EPSPs produced by pyramidal cells. Probability of failure was lower and paired-pulse facilitation more common among EPSPs triggered by granule cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Marked decrease of neuropeptide Y Y2 receptor binding sites in the hippocampus in murine prion disease

Using autoradiographic binding methodology with monoiodinated peptide YY together with the agonists neuropeptide Y (NPY) and NPY (13-36), as well as in situ hybridization with oligonucleotide probes complementary to the NPY Y2 receptor (Y2-R) mRNA, we have studied whether or not intracerebral prion inoculation affects Y2-Rs in male CD-1 mice. Monoiodinated peptide YY binding, mainly representing Y2-Rs, was down-regulated by 85% in the CA1 strata oriens and radiatum and by 50-65% in the CA3 stratum oriens 110-140 days postinoculation. In the CA3 stratum radiatum, where the mossy fibers from the dentate granule cells project, there was a significant decrease in PYY binding at 110-120 days. Y2-R mRNA, moderately expressed both in the CA1 and CA3 pyramidal cell layers and the granule cell layer in the dentate gyrus, showed a slight, but not significant, decrease in CA3 neurons 130 days postinoculation. The results indicate that the accumulation of the scrapie prion protein in the CA1-3 region strongly inhibits NPY binding at the Y2-Rs, which, however, is only marginally due to reduced Y2-R mRNA expression. The loss of the ability of NPY to bind to inhibitory Y2-Rs may cause dysfunction of hippocampal circuits and may contribute to the clinical symptoms in mouse scrapie.     

Hippocampal AMPA and NMDA mRNA levels correlate with aberrant fascia dentata mossy fiber sprouting in the pilocarpine model of spontaneous limbic epilepsy

There is considerable controversy whether aberrant fascia dentata (FD) mossy fiber sprouting is an epiphenomena related to neuronal loss or a pathologic abnormality responsible for spontaneous limbic seizures. If mossy fiber sprouting contributes to seizures, then reorganized axon circuits should alter postsynaptic glutamate receptor properties. In the pilocarpine-status rat model, this study determined if changes in alpha amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and n-methyl-D-aspartic acid (NMDA) receptor subunit mRNA levels correlated with mossy fiber sprouting. Sprague-Dawley rats were injected with pilocarpine (320 mg/kg; i.p.) and maintained in status epilepticus for 6 to 8 hours (pilocarpine-status). Rats were killed during the: (1) latent phase after neuronal loss but before spontaneous limbic seizures (day 11 poststatus; n = 7); (2) early seizure phase after their first seizures (day 25; n = 7); and (3) chronic seizure phase after many seizures (day 85; n = 9). Hippocampi were studied for neuron counts, inner molecular layer (IML) neo-Timm's staining, and GluR1-3 and NMDAR1-2b mRNA levels. Compared with controls, pilocarpine-status rats in the: (1) latent phase showed increased FD GluR3, NMDAR1, and NMDAR2b; greater CA4 and CA1 NMDAR1; and decreased subiculum GluR1 hybridization densities; (2) early seizure phase showed increased FD GluR3, increased CA1 NMDAR1, and decreased subiculum NMDAR2b densities; and (3) chronic seizure phase showed increased FD GluR2; increased FD and CA4 GluR3; decreased CA1 GluR2; and decreased subiculum GluR1, GluR2, NMDAR1, and NMDAR2b levels. In multivariate analyses, greater IML neo-Timm's staining: (1) positively correlated with FD GluR3 and NMDAR1 and (2) negatively correlated with CA1 and subiculum GluR1 and GluR2 mRNA levels. These results indicate that: (1) hippocampal AMPA and NMDA receptor subunit mRNA levels changed as rats progressed from the latent to chronic seizure phase and (2) certain subunit alterations correlated with mossy fiber sprouting. Our findings support the hypothesis that aberrant axon circuitry alters postsynaptic hippocampal glutamate receptor subunit stoichiometry; this may contribute to limbic epileptogenesis.     

Granule cell mRNA levels for BDNF, NGF, and NT-3 correlate with neuron losses or supragranular mossy fiber sprouting in the chronically damaged and epileptic human hippocampus

This study determined in temporal lobe epilepsy patients if there were correlations among hippocampal granule cell expression of neurotrophin mRNAs, aberrant supragranular mossy fiber sprouting, and neuron losses. Consecutive surgically resected hippocampi (n = 9) and comparison tissue from autopsies (n = 3) were studied for: 1. Granule cell mRNA levels using in situ hybridization for brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3); 2. neo-Timm supragranular mossy fiber sprouting; and 3. Ammon's horn neuron densities. Clinically, patients were classified into those with hippocampal sclerosis (HS; n = 7) and non-HS cases (i.e., mass lesions and autopsies; n = 5). Results showed that compared to non-HS cases, HS patients showed increased granule cell mRNA levels for BDNF, NGF, and NT-3 (p = 0.035, p = 0.04, p = 0.045 respectively; one-tail directional test). Moreover, granule cell BDNF mRNA levels correlated inversely with Ammon's horn neuron densities (p = 0.02) and correlated positively with greater supragranular mossy fiber sprouting (p = 0.02). NGF mRNA levels correlated inversely with Ammon's horn neuron densities (p = 0.02), and NT-3 mRNA levels correlated inversely with age at surgery (p = 0.04) and correlated positively with greater mossy fiber sprouting (p = 0.026). These results indicate in the chronically damaged human hippocampus that granule cells express neurotrophin mRNAs, and mRNA levels correlate with either hippocampal neuron losses or aberrant supragranular mossy fiber sprouting. These data support the hypothesis that in the epileptic human hippocampus, there may be pathophysiologic associations among mossy fiber synaptic plasticity, hippocampal neuron damage, and granule cell mRNA neurotrophin levels.     

Feed-forward and feed-back activation of the dentate gyrus in vivo during dentate spikes and sharp wave bursts

Intermittently occurring field events, dentate spikes (DS), and sharp waves (SPW) in the hippocampus reflect population synchrony of principal cells and interneurons along the entorhinal cortex-hippocampus axis. We have investigated the cellular-synaptic generation of DSs and SPWs by intracellular recording from granule cells, pyramidal cells, and interneurons in anesthetized rats. The recorded neurons were anatomically identified by intracellular injection of biocytin. Extracellular recording electrodes were placed in the hilus to record field DSs and multiple units and in the CA1 pyramidal cell layer to monitor SPW-associated fast field oscillations (ripples) and unit activity. DSs were associated with large depolarizing potentials in granule cells, but they rarely discharged action potentials. When they were depolarized slightly with intracellular current injection, bursts of action potentials occurred concurrently with extracellularly recorded DSs. Two interneurons in the hilar region were also found to discharge preferentially with DSs. In contrast, CA1 pyramidal cells, recorded extracellularly and intracellularly, were suppressed during DSs. In association with field SPWs, extracellular recordings from the CA1 pyramidal layer and the hilar region revealed synchronous bursting of these cell populations. Intracellular recordings from CA3 and CA1 pyramidal cells, granule cells, and from a single CA3 region interneuron revealed SPW-concurrent depolarizing potentials and action potentials. These findings suggest that granule cells may be discharged anterogradely by entorhinal input or retrogradely by the CA3-mossy cell feedback pathway during DSs and SPWs, respectively. Although both of these intermittent population patterns can activate granule cells, the impact of DSs and SPWs is diametrically opposite on the rest of the hippocampal circuitry. Entorhinal cortex activation of the granule cells during DSs induces a transient decrease in the hippocampal output, whereas during SPW bursts every principal cell population of the hippocampal formation may be recruited into the population event.     

Status epilepticus-induced alterations in metabotropic glutamate receptor expression in young and adult rats

In adult rats, kainic acid induces status epilepticus and delayed, selective cell loss of pyramidal neurons in the hippocampal CA3. In pup rats, kainate induces status epilepticus but not the accompanying neuronal cell death. The precise mechanisms underlying this age-dependent vulnerability to seizure-induced cell death are not understood. Metabotropic glutamate receptors (mGluRs) are developmentally and spatially regulated throughout the hippocampus and are implicated in seizure-induced damage. In the present study we used in situ hybridization to examine possible changes in mGluR expression at the level of the hippocampus after status epilepticus in postnatal day 10 (P10) pup and adult (P40) rats. Status epilepticus did not alter expression of mGluR1, mGluR3, or mGluR5 mRNAs. In pup and adult rats, status epilepticus induced a reduction in expression of mGluR2 mRNA in granule cells of the dentate gyrus. This change could lead to augmented glutamate release at mossy fiber synapses on CA3 pyramidal cells and thereby promote hyperexcitation. In pup but not adult rats, mGluR4 mRNA expression was enhanced in CA3 pyramidal neurons. Upregulation of presynaptic mGluR4 in pup CA3 neurons could lead to reduced transmitter release from CA3 axons, including recurrent collaterals, thereby reducing vulnerability of neonatal CA3 neurons to seizure-induced damage. These findings indicate that status epilepticus affects mGluR expression in a gene- and cell-specific manner, and that these changes vary with the developmental stage.     

Physiological unmasking of new glutamatergic pathways in the dentate gyrus of hippocampal slices from kainate-induced epileptic rats

In humans with temporal lobe epilepsy and kainate-treated rats, the mossy fibers of the dentate granule cells send collateral axons into the inner molecular layer. Prior investigations on kainate-treated rats demonstrated that abnormal hilar-evoked events can occasionally be observed in slices with mossy fiber sprouting when gamma-aminobutyric acid-A (GABAA)-mediated inhibition is blocked with bicuculline. However, these abnormalities were observed infrequently, and it was unknown whether these rats were epileptic. Wuarin and Dudek reported that in slices from kainate-induced epileptic rats (3-13 mo after treatment), hilar stimulation evoked abnormal events in most slices with mossy fiber sprouting exposed simultaneously to bicuculline and elevated extracellular potassium concentration [K+]o. Using the same rats, extracellular recordings were obtained from granule cells in hippocampal slices to determine whether 1) hilar stimulation could evoke abnormal events in slices with sprouting in normal artificial cerebrospinal fluid (ACSF), 2) adding only bicuculline could unmask hilar-evoked abnormalities and glutamate-receptor antagonists could block these events, and 3) increasing only [K+]o could unmask these abnormalities. In normal ACSF, hilar stimulation evoked abnormal field potentials in 27% of slices with sprouting versus controls without sprouting (i.e., saline-treated or only 2-4 days after kainate treatment). In bicuculline (10 microM) alone, hilar stimulation triggered prolonged field potentials in 84% of slices with sprouting, but not in slices from the two control groups. Addition of the N-methyl-D-aspartate (NMDA) receptor antagonist, DL-2-amino-5-phosphonopentanoic acid (AP5), either blocked the bursts or reduced their probability of occurrence. The alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), always eliminated the epileptiform bursts. In kainate-treated rats with sprouting, but not in saline-treated controls, abnormal hilar-evoked responses were also revealed in 6-9 mM [K+]o. Additionally, 63% of slices with sprouting generated spontaneous bursts lasting 1-40 s in ACSF containing 9 mm [K+]o; similar bursts were not observed in controls. These results indicate that 1) mossy fiber sprouting is associated with new glutamatergic pathways, and although NMDA receptors are important for propagation through these circuits, AMPA receptor activation is crucial, 2) modest elevations of [K+]o, in a range that would have relatively little effect on granule cells, can unmask these new excitatory circuits and generate epileptiform bursts, and 3) this new circuitry underlies an increased electrographic seizure susceptibility when inhibition is depressed or membrane excitability is increased.     

Biosynthesis and metabolism of native and oxidized neuropeptide Y in the hippocampal mossy fiber system

Neuropeptide Y (NPY) gene expression is known to be modulated in the mossy fiber projection of hippocampal granule cells following seizure. We investigated NPY biosynthesis and metabolism in an attempt to characterize NPY biochemically as a neurotransmitter in the granule cell mossy fiber projection. NPY biosynthesis was compared in normal control animals and in animals that had experienced a single pentylenetetrazole-induced seizure. In situ hybridization analysis established the postseizure time course of preproNPY mRNA expression in the hippocampal formation, localizing the majority of increased preproNPY mRNA content to the hilus of the dentate gyrus. Radioimmunoassay analysis of the CA3/mossy fiber terminal subfield confirmed a subsequent increase in NPY peptide content. Biosynthesis of NPY peptide by granule cells and transport to the CA3/mossy fiber subfield was demonstrated by in vivo radiolabel infusion to the dentate gyrus/hilus followed by sequential HPLC purification of identified radiolabeled peptide from the CA3/mossy fiber terminal subfield. Additional in vivo radiolabeling studies revealed a postseizure increase in an unidentified NPY-like immunoreactive (NPY-LI) species. HPLC/radioimmunoassay analyses of CA3 subfield tissue extracts comparing normal control animals and pentylenetetrazole-treated animals confirmed the increased total NPY-LI, and demonstrated that the increased NPY-LI was comprised of a minor increase in native NPY and a major increase in the unknown NPY-LI. Data from subsequent and separate analyses incorporating immunoprecipitation with anti-C-terminal flanking peptide of NPY, further HPLC purification, and matrix-assisted laser desorption/ionization mass spectrometry support the conclusion that the unknown NPY-LI is methionine sulfoxide NPY. NPY and NPY-sulfoxide displayed differential calcium sensitivity for release from mossy fiber synaptosomes. Similar to NPY, NPY sulfoxide displayed high-affinity binding to each of the cloned Y1, Y2, Y4, and Y5 receptor subtypes. Postrelease inactivation of NPY was demonstrated in a mossy fiber synaptosomal preparation. Thus, the present study in combination with previously reported electrophysiological activity of NPY in the CA3 subfield demonstrates that NPY fulfills the classical criteria for a neurotransmitter in the hippocampal granule cell mossy fiber projection, and reveals the presence of two molecular forms of NPY that display differential mechanisms of release while maintaining similar receptor potencies.     

Dynorphin and the kappa 1 ligand [3H]U69,593 binding in the human epileptogenic hippocampus

The distribution of dynorphin (DYN), one of its binding sites (kappa 1 receptor) and their relationship to neuronal loss and granule cell hyperexcitability was examined in hippocampi from patients with temporal lobe epilepsy (TLE). In hippocampi that were not the seizure focus (mass associated temporal lobe epilepsy, MaTLE; and paradoxical temporal lobe epilepsy, PTLE) DYN-like immunoreactivity was localized in the dentate granule cells and their mossy fiber terminals within the hilus and area CA3. In hippocampi that were the seizure focus (MTLE), 89% showed an additional band of immunoreactivity confined to the inner molecular layer (IML) of the dentate gyrus, representing recurrent mossy fiber collaterals. In 11% of MTLE patients no staining was found in the IML (MTLE/DYN-). The MTLE/DYN- hippocampi were also characterized by a significantly lower degree of cell loss than in MTLE hippocampi in the dentate granule cell layer, the hilus and CA3. Both MTLE and MTLE/DYN- hippocampi showed evoked epileptiform bursting in granule cells while MTLE showed greater polysynaptic EPSPs and spontaneous excitatory activity. Thus granule cell recurrent collateral sprouting may account for only some aspects of hyperexcitability. In 30% of the MTLE group, hilar neurons of a variety of morphological types expressed DYN immunoreactivity in their somata and dendrites. The density of [3H]U69,593 binding sites in MaTLE and PTLE patients was highest in areas CA1 and the subiculum-regions having little or no DYN-staining. In the dentate molecular layer, hilus and CA3--regions with the most DYN immunoreactivity--there was a low density of ligand binding. The significance of this transmitter/receptor mismatch is yet unknown.     

Distribution of alpha 1A, alpha 1B and alpha 1E voltage-dependent calcium channel subunits in the human hippocampus and parahippocampal gyrus

The distribution of voltage-dependent calcium channel subunits in the central nervous system may provide information about the function of these channels. The present study examined the distribution of three alpha-1 subunits, alpha 1A, alpha 1B and alpha 1E, in the normal human hippocampal formation and parahippocampal gyrus using the techniques of in situ hybridization and immunocytochemistry. All three subunit mRNAs appeared to be similarly localized, with high levels of expression in the dentate granule and CA pyramidal layer. At the protein level, alpha 1A, alpha 1B and alpha 1E subunits were differentially localized. In general, alpha 1A-immunoreactivity was most intense in cell bodies and dendritic processes, including dentate granule cells, CA3 pyramidal cells and entorhinal cortex pre-alpha and pri-alpha cells. The alpha 1B antibody exhibited relatively weak staining of cell bodies but stronger staining of neuropil, especially in certain regions of high synaptic density such as the polymorphic layer of the dentate gyrus and the stratum lucidum and radiatum of the CA regions. The alpha 1E staining pattern shared features in common with both alpha 1A and alpha 1B, with strong immunoreactivity in dentate granule, CA3 pyramidal and entorhinal cortex pri-alpha cells, as well as staining of the CA3 stratum lucidum. These findings suggest regions in which particular subunits may be involved in synaptic communication. For example, comparison of alpha 1B and alpha 1E staining in the CA3 stratum lucidum with calbindin-immuno-reactivity suggested that these two calcium channels subunits may be localized presynaptically in mossy fibre terminals and therefore may be involved in neurotransmitter release from these terminals.     

Ultrastructural localization of zinc transporter-3 (ZnT-3) to synaptic vesicle membranes within mossy fiber boutons in the hippocampus of mouse and monkey

Zinc transporter-3 (ZnT-3), a member of a growing family of mammalian zinc transporters, is expressed in regions of the brain that are rich in histochemically reactive zinc (as revealed by the Timm's stain), including entorhinal cortex, amygdala, and hippocampus. ZnT-3 protein is most abundant in the zinc-enriched mossy fibers that project from the dentate granule cells to hilar and CA3 pyramidal neurons. We show here by electron microscopy that ZnT-3 decorates the membranes of all clear, small, round synaptic vesicles (SVs) in the mossy fiber boutons of both mouse and monkey. Furthermore, up to 60-80% of these SVs contain Timm's-stainable zinc. The coincidence of ZnT-3 on the membranes of SVs that accumulate zinc, and its homology with known zinc transporters, suggest that ZnT-3 is responsible for the transport of zinc into SVs, and hence for the ability of these neurons to release zinc upon excitation.     

Human cytochrome P4502B6: interindividual hepatic expression, substrate specificity, and role in procarcinogen activation

In situ hybridization was combined with serotonin (5-hydroxytryptamine, 5-HT) or tyrosine hydroxylase immunocytochemistry and with Fluoro-Gold retrograde labeling of bulbo-spinal pathways in order to investigate the expression of GAP-43 mRNA in monoamine cell groups of the adult rat brain stem. Consistent with previous reports, GAP-43 mRNA was observed in serotonin and dopamine cell groups in the pons. In addition, GAP-43 expressing cells were observed in all the major monoamine cell groups in the medulla. Thus the B1, B2 and B3 serotonin cell groups all showed high GAP-43 expression in all contained many GAP-43 expressing serotonin cells with spinal cord projections. The A1, A2, A5 and A6 noradrenaline cell groups also showed high GAP-43 expression, although cells with spinal cord projections were largely restricted to the A5 group and A6 subcoeruleus region. In all areas, GAP-43 expressing cells with spinal cord projections were also observed which were not serotonergic or noradrenergic.     

Specialized electrophysiological properties of anatomically identified neurons in the hilar region of the rat fascia dentata

Because of their strategic position between the granule cell and pyramidal cell layers, neurons of the hilar region of the hippocampal formation are likely to play an important role in the information processing between the entorhinal cortex and the hippocampus proper. Here we present an electrophysiological characterization of anatomically identified neurons in the fascia dentata as studied using patch-pipette recordings and subsequent biocytin-staining of neurons in slices. The resting potential, input resistance (RN), membrane time constant (taum), "sag" in hyperpolarizing responses, maximum firing rate during a 1-s current pulse, spike width, and fast and slow afterhyperpolarizations (AHPs) were determined for several different types of hilar neurons. Basket cells had a dense axonal plexus almost exclusively within the granule cell layer and were distinguishable by their low RN, short taum, lack of sag, and rapid firing rates. Dentate granule cells also lacked sag and were identifiable by their higher RN, longer taum, and lower firing rates than basket cells. Mossy cells had extensive axon collaterals within the hilus and a few long-range collaterals to the inner molecular layer and CA3c and were characterized physiologically by small fast and slow AHPs. Spiny and aspiny hilar interneurons projected primarily either to the inner or outer segment of the molecular layer and had a dense intrahilar axonal plexus, terminating onto somata within the hilus and CA3c. Physiologically, spiny hilar interneurons generally had higher RN values than mossy cells and a smaller slow AHP than aspiny interneurons. The specialized physiological properties of different classes of hilar neurons are likely to be important determinants of their functional operation within the hippocampal circuitry.