Hilar Interneurons Research Articles

Neuropeptide Y May Be an Endogenous Anticonvulsant in Hippocampus

Marked expression of neuropeptide Y (NPY) and its Y2 receptors in hippocampal mossy fibers has been reported in animal models of epilepsy. Because NPY can suppress glutamate release by activating presynaptic Y2 receptors, these changes have been proposed as an endogenous protective mechanism. Therefore, we investigated whether similar changes in the NPY system may also take place in human epilepsy. We investigated Y1 and Y2 receptor binding and NPY immunoreactivity in hippocampal specimens that were obtained at surgery from patients with temporal lobe epilepsy and in autopsy controls. Significant increases in Y2 receptor binding (by 43–48%) were observed in the dentate hilus, sectors CA1 to CA3, and subiculum of specimens with, but not in those without, hippocampal sclerosis. On the other hand, Y1 receptor binding was significantly reduced (by 62%) in the dentate molecular layer of sclerotic specimens. In the same patients, the total lengths of NPY immunoreactive (NPY-IR) fibers were markedly increased (by 115–958%) in the dentate molecular layer and hilus, in the stratum lucidum of CA3, and throughout sectors CA1 to CA3 and the subiculum, as compared with autopsies. In nonsclerotic specimens, increases in lengths of NPY-IR fibers were more moderate and statistically not significant. NPY mRNA was increased threefold in hilar interneurons of sclerotic and nonsclerotic specimens. It is suggested that abundant sprouting of NPY fibers, concomitant upregulation of Y2 receptors, and downregulation of Y1 receptors in the hippocampus of patients with Ammon's horn sclerosis may be endogenous anticonvulsant mechanisms.

Reduction of chromogranin B-like immunoreactivity in distinct subregions of the hippocampus from individuals with schizophrenia

Synaptic disturbances may play a key role in the pathophysiology of schizophrenia. This study was designed to further investigate possible synaptic alterations in the brains of chronic schizophrenic patients. Chromogranin B was applied as a marker for large dense core vesicles and synapsin I as a protein associated with the synaptic vesicle membrane. The distribution and density of chromogranin B-and synapsin I-like immunoreactivity in subregions of the hippocampus was compared between controls ( n=16) and patients with schizophrenia ( n=17). The overall distribution of hippocampal chromogranin B- and synapsin I-like immunoreactivity was similar in controls and in schizophrenic patients with the highest densities in the terminal field of mossy fibers and in the inner molecular layer of the dentate gyrus. In schizophrenic hippocampi, a significant reduction in the density of chromogranin B-like immunoreactivity was found in the CA4 and CA3 but not in the CA1 area of the dentate gyrus based on computerized image analysis. The loss of immunoreactivity was localized to mossy fibers and terminals surrounding hilar interneurons. Double-labelling immunohistochemistry revealed that synapsin I was co-expressed with chromogranin B in these neuronal structures and was also significantly reduced in schizophrenic hippocampi. The present study demonstrates an area-specific reduction of chromogranin B which is paralleled by a decrease of synapsin I. The loss of presynaptic proteins involved in distinct steps of exocytosis may cause complex synaptic disturbances in specific hippocampal subregions resulting in an imbalanced neurotransmitter availability in schizophrenic patients.

Molecular and functional analysis of hyperpolarization-activated pacemaker channels in the hippocampus after entorhinal cortex lesion.

Differential display of hippocampal tissue after entorhinal cortex lesion (ECL) revealed decreases in mRNA encoding the neuronal hyperpolarization-activated, cyclic nucleotide-gated channel HCN1. In situ hybridization confirmed that hippocampal transcripts of HCN1, but not HCN2/3/4, are down-regulated after ECL. Expression recovered at approximately 21 days after lesion (dal). Immunohistochemistry demonstrated a corresponding regulation of HCN1 protein expression in CA1-CA3 dendrites, hilar mossy cells and interneurons, and granule cells. Patch-clamp recordings in the early phase after lesion from mossy cells and hilar interneurons revealed an increase in the fast time constant of current activation and a profound negative shift in voltage activation of Ih. Whereas current activation recovered at 30 dal, the voltage activation remained hyperpolarized in mossy cells and hilar interneurons. Granule cells, however, were devoid of any detectable somatic Ih currents. Hence, denervation of the hippocampus decreases HCN1 and concomitantly the Ih activity in hilar neurons, and the recovery of h-current activation kinetics occurs parallel to postlesion sprouting.

Commissurally projecting inhibitory interneurons of the rat hippocampal dentate gyrus: a colocalization study of neuronal markers and the retrograde tracer Fluoro-gold.

Improved methods for detecting neuronal markers and the retrograde tracer Fluoro-Gold (FG) were used to identify commissurally projecting neurons of the rat hippocampus. In addition to the dentate hilar mossy cells and CA3 pyramidal cells shown previously to transport retrograde tracers after injection into the dorsal hippocampus, FG-positive interneurons of the dentate granule cell layer and hilus were detected in numbers greater than previously reported. FG labeling of interneurons was variable among animals, but was as high as 96% of hilar somatostatin-positive interneurons, 84% of parvalbumin-positive cells of the granule cell layer and hilus combined, and 33% of hilar calretinin-positive cells. By comparison, interneurons of the dentate molecular layer and all hippocampal subregions were conspicuously FG-negative. Whereas hilar mossy cells and CA3 pyramidal cells were FG-labeled throughout the longitudinal axis, FG-positive interneurons exhibited a relatively homotopic distribution. "Control" injections of FG into the neocortex, septum, and ventral hippocampus demonstrated that the homotopic labeling of dentate interneurons was injection site-specific, and that the CA1-CA3 interneurons unlabeled by contralateral hippocampal FG injection were nonetheless able to transport FG from the septum. These data suggest a hippocampal organizing principle according to which virtually all commissurally projecting hippocampal neurons share the property of being monosynaptic targets of dentate granule cells. Because granule cells innervate their exclusively ipsilateral target cells in a highly lamellar pattern, these results suggest that focal granule cell excitation may result in commissural inhibition of the corresponding "twin" granule cell lamella, thereby lateralizing and amplifying the influence of the initiating discharge.

Stereological assessment of vulnerability of immunocytochemically identified striatal and hippocampal neurons after global cerebral ischemia in rats

Detailed quantitative analysis of the vulnerability of different hippocampal and striatal neurons to global forebrain ischemia has not previously been performed. Here we have studied the survival of immunocytochemically identified neurons using an unbiased stereological method in rats subjected to global forebrain ischemia for 30 min and sacrificed 48 h, 1 week or 4 weeks thereafter. Within the hippocampal formation, there was extensive, progressive loss of CA1 pyramidal neurons and dentate hilar neuropeptide Y (NPY)-positive interneurons. In contrast, no reduction of the number of CA3 and CA4 pyramidal neurons or hilar parvalbumin-positive interneurons was detected. In the dorsolateral striatum, the insult caused a major loss of projection neurons immunoreactive to dopamine- and adenosine 3′:5′-monophosphate-regulated phosphoprotein with a molecular weight of 32 kilodalton (DARPP-32). The number of parvalbumin-positive striatal interneurons was significantly reduced, while NPY-positive interneurons were resistant. All striatal cholinergic interneurons survived the ischemic insult. At 48 h following the ischemia, the cholinergic interneurons within the lesioned striatum transiently expressed the p75 neurotrophin receptor (p75 NTR), as shown by double-label immunocytochemistry. Furthermore, there was a significant increase in the number of choline acetyltransferase (ChAT)- and TrkA-immunoreactive interneurons at 4 weeks after the insult. Injections with the cell mitotic division marker BrdU provided no evidence that the elevated cholinergic cell number was due to neurogenesis. Probably, the higher number of ChAT- and TrkA-positive interneurons reflected increased intracellular levels of the corresponding proteins leading to more cells detectable with immunocytochemistry. This study gives the first quantitative description of the vulnerability of defined hippocampal and striatal neurons after global forebrain ischemia. The ischemia-induced increases of p75 NTR, TrkA and ChAT in cholinergic striatal interneurons at various time points after the insult suggest that neurotrophin signaling might be important for the survival and function of these cells in the post-ischemic phase.

Plasticity of Y1 and Y2 Receptors and Neuropeptide Y Fibers in Patients with Temporal Lobe Epilepsy

Marked expression of neuropeptide Y (NPY) and its Y2 receptors in hippocampal mossy fibers has been reported in animal models of epilepsy. Because NPY can suppress glutamate release by activating presynaptic Y2 receptors, these changes have been proposed as an endogenous protective mechanism. Therefore, we investigated whether similar changes in the NPY system may also take place in human epilepsy. We investigated Y1 and Y2 receptor binding and NPY immunoreactivity in hippocampal specimens that were obtained at surgery from patients with temporal lobe epilepsy and in autopsy controls. Significant increases in Y2 receptor binding (by 43-48%) were observed in the dentate hilus, sectors CA1 to CA3, and subiculum of specimens with, but not in those without, hippocampal sclerosis. On the other hand, Y1 receptor binding was significantly reduced (by 62%) in the dentate molecular layer of sclerotic specimens. In the same patients, the total lengths of NPY immunoreactive (NPY-IR) fibers was markedly increased (by 115-958%) in the dentate molecular layer and hilus, in the stratum lucidum of CA3, and throughout sectors CA1 to CA3 and the subiculum, as compared with autopsies. In nonsclerotic specimens, increases in lengths of NPY-IR fibers were more moderate and statistically not significant. NPY mRNA was increased threefold in hilar interneurons of sclerotic and nonsclerotic specimens. It is suggested that abundant sprouting of NPY fibers, concomitant upregulation of Y2 receptors, and downregulation of Y1 receptors in the hippocampus of patients with Ammon's horn sclerosis may be endogenous anticonvulsant mechanisms.

Imaging of 4-AP-induced, GABA(A)-dependent spontaneous synchronized activity mediated by the hippocampal interneuron network.

Under conditions of increased excitability, such as application of the K(+) channel blocker 4-aminopyridine (4-AP, 100 microM), interneurons in the hippocampal slice show an additional form of synchronized activity that is distinct from the ictal and interictal epileptiform activity induced by these manipulations. In principal neurons, i.e., pyramidal and granule cells, this synchronized interneuron activity (SIA) generates large, multi-component synaptic potentials, which have been termed long-lasting depolarizations (LLDs). These LLDs are dependent on GABA(A) receptor-mediated synaptic transmission but not on excitatory amino acid (EAA) receptors. Intracellular recordings from hilar interneurons have shown that depolarizing GABA(A) receptor-mediated synaptic potentials are also largely responsible for the synchronization of interneurons. The spatiotemporal characteristics of this interneuron activity have not been investigated previously. Using a voltage-sensitive dye and optical techniques that are capable of recording spontaneous synchronized activity, we have characterized the spatiotemporal pattern of SIA (in the presence of 4-AP + EAA receptor antagonists) and compared it with interictal epileptiform activity (in 4-AP only). Like interictal activity, SIA could be observed throughout the hippocampal slice. Unlike interictal activity, which originated in area CA2/CA3 and spread from there, SIA was most prominent in area CA1 and originated either there or in the subiculum. In CA1, interictal activity was largest in and near stratum pyramidale, while SIA was mainly located in s. lacunosum moleculare. Furthermore SIA was equally likely to propagate in either direction, and multiple patterns of propagation could be observed within a single brain slice. These studies suggest that hippocampal area CA1 has the highest propensity for SIA, that multiple locations can serve as the site of origin, and that interneurons located in s. lacunosum moleculare or interneurons that specifically project to this region may be particularly important for synchronized interneuron activity.

The KCl cotransporter, KCC2, is highly expressed in the vicinity of excitatory synapses in the rat hippocampus.

Immunocytochemical visualization of the neuron-specific K+/Cl- cotransporter, KCC2, at the cellular and subcellular level revealed an area- and layer-specific diffuse labelling, and a discrete staining outlining the somata and dendrites of some interneurons in all areas of the rat hippocampus. KCC2 was highly expressed in parvalbumin-containing interneurons, as well as in subsets of calbindin, calretinin and metabotropic glutamate receptor 1a-immunoreactive interneurons. During the first 2 postnatal weeks, an increase of KCC2 staining was observed in the molecular layer of the dentate gyrus, correlating temporally with the arrival of entorhinal cortical inputs. Subcellular localization demonstrated KCC2 in the plasma membranes. Immunoreactivity in principal cells was responsible for the diffuse staining found in the neuropil. In these cells, KCC2 was detected primarily in dendritic spine heads, at the origin of spines and, at a much lower level on the somata and dendritic shafts. KCC2 expression was considerably higher in the somata and dendrites of interneurons, most notably of parvalbumin-containing cells, as well as in the thorny excrescences of CA3 pyramidal cells and in the spines of spiny hilar and stratum lucidum interneurons. The data indicate that KCC2 is highly expressed in the vicinity of excitatory inputs in the hippocampus, perhaps in close association with extrasynaptic GABAA receptors. A high level of excitation is known to lead to a simultaneous net influx of Na+ and Cl-, as evidenced by dendritic swelling. KCC2 located in the same microenvironment may provide a Cl- extrusion mechanism to deal with both ion and water homeostasis in addition to its role in setting the driving force of Cl- currents involved in fast postsynaptic inhibition.

Reduced Excitatory Drive onto Interneurons in the Dentate Gyrus after Status Epilepticus

Impaired GABAergic inhibition may contribute to the development of hyperexcitability in epilepsy. We used the pilocarpine model of epilepsy to demonstrate that regulation of excitatory synaptic drive onto GABAergic interneurons is impaired during epileptogenesis. Synaptic input from granule cells (GCs), perforant path, and CA3 inputs onto hilar border interneurons of the dentate gyrus were examined in rat hippocampal slices during the latent period (1-8 d) after induction of status epilepticus (SE). Short-term depression (STD) of GC inputs to interneurons induced by brief (500-800 msec), repetitive (5-20 Hz) stimulation, as well as paired-pulse depression at both GC and CA3 inputs to interneurons, were significantly (p < 0.05) enhanced in SE-experienced rats. In contrast, we found no significant differences between SE-experienced and age-matched control rats in the properties of minimal EPSCs evoked at low frequency (0.3 Hz). Consistent with reduced GABAergic inhibition onto granule cells, paired-pulse depression of perforant path-evoked granule cell population spikes was lost in SE-experienced rats. Enhanced STD was partially mediated by group II metabotropic glutamate receptors, because the selective antagonist, 2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid, attenuated STD in SE-experienced rats but had no effect on STD of GC inputs in the normal adult rat. The group II mGluR agonist, (2S',1R',2R',3R')-2-(2,3-dicarboxylcyclopropyl) glycine (1 micrometer), produced a greater depression of GC input to hilar border interneurons in SE-experienced rats than in controls. These results indicate that, in the SE-experienced rat, excitatory drive to hilar border inhibitory interneurons is weakened through a use-dependent mechanism involving group II metabotropic glutamate receptors.

Microglia Proliferate in Striatum and Cortex but not Hippocampus Following Brief Periods of Ischemia that do not Kill Neurons

61 Because ischemia induced tolerance induces the birth of new neurons following global ischemia, this study determined whether 2.5 minutes of global ischemia and ischemia-induced tolerance would induce the birth of glia in the brain.Following 0, 2.5, or 5 minutes of global ischemia, adult male gerbils were injected with BrdU (50mg/kg) and dividing cells detected using immunocytochemistry. At four days following 2.5 minutes of global ischemia, when cell proliferation was maximal, BrdU labeled cells were detected in striatum and in neocortex, but not in hippocampus. The majority of the BrdU labeled cells were double-labeled with iso-lectin B4, showing that these dividing cells were mainly microglia. Similarly, BrdU labeled microglia were found in striatum and cortex but not in hippocampus of most animals four days following ischemia-induced tolerance (2.5 minutes of global ischemia followed three days later by 5 minutes of global ischemia). Some of the animals subjected to 2.5 minutes of ischemia showed decreased myelin associated glycoprotein (MAG) immunostaining and increased numbers of APC stained oligodendrocytes in the lateral striatum without apparent neuronal loss. Though the CA1 pyramidal and hilar interneurons of hippocampus are the most vulnerable neurons to ischemia, this data suggests that non-neuronal elements in the striatum may be more sensitive to ischemia than the neurons. In some animals proliferation of microglia in striatum following brief global ischemia may occur in response to injury to white matter and oligodendrocytes.

Microglia Proliferate in Striatum and Cortex but not Hippocampus Following Brief Periods of Ischemia that do not Kill Neurons

61 Because ischemia induced tolerance induces the birth of new neurons following global ischemia, this study determined whether 2.5 minutes of global ischemia and ischemia-induced tolerance would induce the birth of glia in the brain.Following 0, 2.5, or 5 minutes of global ischemia, adult male gerbils were injected with BrdU (50mg/kg) and dividing cells detected using immunocytochemistry. At four days following 2.5 minutes of global ischemia, when cell proliferation was maximal, BrdU labeled cells were detected in striatum and in neocortex, but not in hippocampus. The majority of the BrdU labeled cells were double-labeled with iso-lectin B4, showing that these dividing cells were mainly microglia. Similarly, BrdU labeled microglia were found in striatum and cortex but not in hippocampus of most animals four days following ischemia-induced tolerance (2.5 minutes of global ischemia followed three days later by 5 minutes of global ischemia). Some of the animals subjected to 2.5 minutes of ischemia showed decreased myelin associated glycoprotein (MAG) immunostaining and increased numbers of APC stained oligodendrocytes in the lateral striatum without apparent neuronal loss. Though the CA1 pyramidal and hilar interneurons of hippocampus are the most vulnerable neurons to ischemia, this data suggests that non-neuronal elements in the striatum may be more sensitive to ischemia than the neurons. In some animals proliferation of microglia in striatum following brief global ischemia may occur in response to injury to white matter and oligodendrocytes.

Development and persistence of kindling epilepsy are impaired in mice lacking glial cell line-derived neurotrophic factor family receptor alpha 2.

Seizure activity regulates gene expression for glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN), and their receptor components, the transmembrane c-Ret tyrosine kinase and the glycosylphosphatidylinositol-anchored GDNF family receptor (GFR) alpha 1 and alpha 2 in limbic structures. We demonstrate here that epileptogenesis, as assessed in the hippocampal kindling model, is markedly suppressed in mice lacking GFR alpha 2. Moreover, at 6 to 8 wk after having reached the epileptic state, the hyperexcitability is lower in GFR alpha 2 knock-out mice as compared with wild-type mice. These results provide evidence that signaling through GFR alpha 2 is involved in mechanisms regulating the development and persistence of kindling epilepsy. Our data suggest that GDNF and NRTN may modulate seizure susceptibility by altering the function of hilar neuropeptide Y-containing interneurons and entorhinal cortical afferents at dentate granule cell synapses.

Unusual Target Selectivity of Perisomatic Inhibitory Cells in the Hilar Region of the Rat Hippocampus

Perisomatic inhibitory innervation of all neuron types profoundly affects their firing characteristics and vulnerability. In this study we examined the postsynaptic targets of perisomatic inhibitory cells in the hilar region of the dentate gyrus where the proportion of potential target cells (excitatory mossy cells and inhibitory interneurons) is approximately equal. Both cholecystokinin (CCK)- and parvalbumin-immunoreactive basket cells formed multiple contacts on the somata and proximal dendrites of mossy cells. Unexpectedly, however, perisomatic inhibitory terminals arriving from these cell types largely ignored hilar GABAergic cell populations. Eighty-ninety percent of various GABAergic neurons including other CCK-containing basket cells received no input from CCK-positive terminals. Parvalbumin-containing cells sometimes innervated each other but avoided 75% of other GABAergic cells. Overall, a single mossy cell received 40 times more CCK-immunoreactive terminals and 15 times more parvalbumin-positive terminals onto its soma than the cell body of an average hilar GABAergic cell. In contrast to the pronounced target selectivity in the hilar region, CCK- and parvalbumin-positive neurons innervated each other via collaterals in stratum granulosum and moleculare. Our observations indicate that the inhibitory control in the hilar region is qualitatively different from other cortical areas at both the network level and the level of single neurons. The paucity of perisomatic innervation of hilar interneurons should have profound consequences on their action potential generation and on their ensemble behavior. These findings may help explain the unique physiological patterns observed in the hilus and the selective vulnerability of the hilar cell population in various pathophysiological conditions.

Altered hippocampal expression of neuropeptide Y, somatostatin, and glutamate decarboxylase in Ihara's epileptic rats and spontaneously epileptic rats

By in situ hybridization and immunocytochemistry, expression of neuropeptide Y (NPY), somatostatin and glutamate decarboxylase 65 (GAD65) was studied in the hippocampus of two different epileptic mutant rats, Ihara's epileptic rat (IER) and the spontaneously epileptic rat (SER). GAD65 mRNA expression was enhanced in interneurons of the hippocampus in young IER, that had not yet developed generalized seizures. In older IER and older SER that both showed spontaneous seizures, marked increases of NPY mRNA in hippocampal granule cells and interneurons were found, as well as elevated GAD65 mRNA levels in interneurons. NPY immunoreactivity was enhanced in hilar interneurons and the dentate gyrus of older IER. In addition, some older IER stained heavily for NPY in mossy fibers. These findings suggest that up-regulation of NPY and GAD65 synthesis may be important in epileptogenesis.

Beta-adrenergic receptors primarily are located on the dendrites of granule cells and interneurons but also are found on astrocytes and a few presynaptic profiles in the rat dentate gyrus.

In the rat dentate gyrus, beta-adrenergic receptor (beta-AR) activation is thought to be important in mediating the effects of norepinephrine (NE). beta-AR-immunoreactivity (beta-AR-I) was localized in this study by light and electron microscopy in the rat dentate gyrus by using two previously characterized antibodies to the beta-AR. By light microscopy, dense beta-AR-I was observed in the somata of granule cells and a few hilar interneurons. Diffuse and slightly granular beta-AR-I was found in all laminae, although it was most noticeable in the molecular layer. Ultrastructurally, the cytoplasm of granule cell and interneuronal perikarya (some of which contained parvalbumin immunoreactivity) contained beta-AR-I. beta-AR-I was associated primarily with the endoplasmic reticula; however, a few patches were observed near the plasmalemma. Quantitative analysis revealed that the greatest proportion of beta-AR-labeled profiles was found in the molecular layer. The majority of beta-AR-labeled profiles were either dendritic or astrocytic. In dendritic profiles, beta-AR-I was prominent near postsynaptic densities in large dendrites, many of which originated from granule cell somata. Moreover, some beta-AR-I was found in dendritic spines, sometimes affiliated with the spine apparati. Astrocytic profiles with beta-AR-I were commonly found next to unlabeled terminals which formed asymmetric (excitatory-type) synapses with dendritic spines. Additionally, beta-AR-I was observed in a few unmyelinated axons and axon terminals, many of which formed synapses with dendritic spines. Dual-labeling studies revealed that axons and axon terminals containing tyrosine hydroxylase (TH), the catecholamine synthesizing enzyme, often were near both neuronal and glial profiles containing beta-AR-I. These studies demonstrate that hippocampal beta-AR-I is localized: 1) principally in postsynaptic sites on granule cells and a few interneurons (some of which were basket cells); and 2) in glial processes. These observations add further support to the contention that beta-AR-activation modulates synaptic function through disparate pathways: directly, at either postsynaptic densities or presynaptic processes, or indirectly, through adjacent glial processes.

Localization of the A kinase anchoring protein AKAP79 in the human hippocampus.

The phosphorylation state of the proteins, regulated by phosphatases and kinases, plays an important role in signal transduction and long-term changes in neuronal excitability. In neurons, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and calcineurin (CN) are attached to a scaffold protein, A kinase anchoring protein (AKAP), thought to anchor these three enzymes to specific sites of action. However, the localization of AKAP, and the predicted sites of linked phosphatase and kinase activities, are still unknown at the fine structural level. In the present study, we investigated the distribution of AKAP79 in the hippocampus from postmortem human brains and lobectomy samples from patients with intractable epilepsy, using preembedding immunoperoxidase and immunogold histochemical methods. AKAP79 was found in the CA1, presubicular and subicular regions, mostly in pyramidal cell dendrites, whereas pyramidal cells in the CA3, CA2 regions and dentate granule cells were negative both in postmortem and in surgical samples. In some epileptic cases, the dentate molecular layer and hilar interneurons also became immunoreactive. At the subcellular level, AKAP79 immunoreactivity was present in postsynaptic profiles near, but not attached to, the postsynaptic density of asymmetrical (presumed excitatory) synapses. We conclude that the spatial selectivity for the action of certain kinases and phosphatases regulating various ligand- and voltage-gated channels may be ensured by the selective presence of their anchoring protein, AKAP79, at the majority of glutamatergic synapses in the CA1, but not in the CA2/CA3 regions, suggesting profound differences in signal transduction and long-term synaptic plasticity between these regions of the human hippocampus.

GABAB-Receptor-mediated currents in interneurons of the dentate-hilus border.

GABA(B)-receptor-mediated inhibition was investigated in anatomically identified inhibitory interneurons located at the border between the dentate gyrus granule cell layer and hilus. Biocytin staining was used to visualize the morphology of recorded cells. A molecular layer stimulus evoked a pharmacologically isolated slow inhibitory postsynaptic current (IPSC), recorded with whole cell patch-clamp techniques, in 55 of 63 interneurons. Application of the GABA(B) receptor antagonists, CGP 35348 (400 microM) or CGP 55845 (1 microM) to a subset of 25 interneurons suppressed the slow IPSC by an amount ranging from 10 to 100%. In 56% of these cells, the slow IPSC was entirely GABA(B)-receptor-mediated. However, in the remaining interneurons, a component of the slow IPSC was resistant to GABA(B) antagonists. Subtraction of this antagonist resistant current from the slow IPSC isolated the GABA(B) component (IPSC(B)). This IPSC(B) had a similar onset and peak latency to that recorded from granule cells but a significantly shorter duration. The GABA(B) agonist, baclofen (10 microM), produced a CGP 55845-sensitive outward current in 19 of 27 interneurons. In the eight cells that lacked a baclofen current, strong or repetitive ML stimulation also failed to evoke an IPSC(B), indicating that these cells lacked functional GABA(B) receptor-activated potassium currents. In cells that expressed a baclofen current, the amplitude of this current was approximately 50% smaller in interneurons with axons that projected into the granule cell dendritic layer (22.2 +/- 5.3 pA; mean +/- SE) than in interneurons with axons that projected into or near the granule cell body layer (46.1 +/- 10.0 pA). Similarly, the IPSC(B) amplitude was smaller in interneurons projecting to dendritic (9.4 +/- 2.7 pA) than perisomatic regions (34.3 +/- 5.1 pA). These findings suggest that GABA(B) inhibition more strongly regulates interneurons with axons that project into perisomatic than dendritic regions. To determine the functional role of GABA(B) inhibition, we examined the effect of IPSP(B) on action potential firing and synaptic excitation of these interneurons. IPSP(B) and IPSP(A) both suppressed depolarization-induced neuronal firing. However, unlike IPSP(A), suppression of firing by IPSP(B) could be easily overcome with strong depolarization. IPSP(B) markedly suppressed N-methyl-D-aspartate but not AMPA EPSPs, suggesting that GABA(B) inhibition may play a role in regulating slow synaptic excitation of these interneurons. Heterogeneous expression of GABA(B) currents in hilar border interneurons therefore may provide a mechanism for the differential regulation of excitation of these cells and thereby exert an important role in shaping neuronal activity in the dentate gyrus.

Neuroprotective effects of brain-derived neurotrophic factor in seizures during development

Although the immature brain is highly susceptible to seizures, it is more resistant to seizure-induced neuronal loss than the adult brain. The developing brain contains high levels of neurotrophins which are involved in growth, differentiation and survival of neurons. To test the hypothesis that neurotrophins may protect the developing brain from seizure-induced neuronal loss, brain-derived neurotrophic factor up-regulation was blocked by intracerebroventricular infusion of an 18mer antisense oligodeoxynucleotide sequence to brain-derived neurotrophic factor in 19-day-old rats using micro-osmotic pumps. Control rats were infused with sense or missense oligodeoxynucleotide. Status epilepticus was induced by intraperitoneal administration of kainic acid 24 h after the start of oligodeoxynucleotide infusion. Seizure duration was significantly increased in the antisense oligodeoxynucleotide plus kainic acid group compared to groups that received kainic acid alone or kainic acid plus sense or missense oligodeoxynucleotide. There was no difference between groups in the latency to forelimb clonus. A twofold increase in brain-derived neurotrophic factor levels was observed in the hippocampus 20 h following kainic acid-induced seizures. This kainic acid-induced increase was absent in animals receiving infusion of antisense oligodeoxynucleotide to brain-derived neurotrophic factor at time of seizure induction. Hippocampi of rats in this group (antisense oligodeoxynucleotide plus kainic acid) showed a loss of CA1 and CA3 pyramidal cells and hilar interneurons. This neuronal loss was not dependent upon seizure duration since animals injected with diazepam to control seizure activity in the antisense plus kainic acid group also showed similar neuronal loss. Administration of kainic acid or infusion of antisense alone did not produce any cell loss in these regions. Induction of seizures at postnatal day 20, in the presence or absence of antisense oligonucleotide, did not produce an impairment in learning and memory when tested 15 days later in the Morris water maze. The hippocampi of these animals did not show any synaptic reorganization as assessed by growth-associated protein-43 immunostaining and Timm staining. Our findings confirm prior studies demonstrating that seizures in the immature brain are associated with little, if any, cell loss. However, when seizure-induced increase in brain-derived neurotrophic factor is blocked, seizures do result in neuronal loss in the developing brain. Thus, brain-derived neurotrophic factor appears to provide protection against kainic acid seizure-induced neuronal damage in the developing brain.

Regional and cellular distribution of neural visinin-like protein immunoreactivities (VILIP-1 and VILIP-3) in human brain.

Neural visinin-like proteins (VILIPs) are members of the neuronal subfamily of intracellular EF-hand calcium sensor proteins termed the NCS family, which are thought to play important roles in cellular signal transduction. While numerous studies suggest a wide but uneven distribution of these proteins in rat and chicken brain, their location in, and possible significance for, the human brain, remains to be established. We used specific polyclonal antisera to map the human brain for VILIP-1 and VILIP-3 immunoreactivities. VILIP-1 was detected in cortical pyramidal cells and interneurons, septal, subthalamic and hippocampal neurons (subfields CA1 and CA4 pyramidal cells and especially hilar interneurons) as well as in cerebellar Golgi, basket, granule, stellate and dentate nucleus neurons. Purkinje cells were free of immunoreaction. VILIP-3 was more restricted in its distribution. It was identified in cerebellar Purkinje cells and a subpopulation of granule neurons. Further, neurons belonging to different nuclei of the brain stem and multiple subcortical nerve cells stained for visinin-like protein 3. A weak immunoreaction appeared in cortical and hippocampal neurons. Intracellularly the immunoreactivity appeared in the perikarya, dendrites and some axons. Sometimes, immunostaining was found in the neuropil. Glia did not express visinin-like proteins. Our findings support, from a neuroanatomical viewpoint, the idea that these calcium sensor proteins may be of relevance for neuronal signalling in the human CNS.

Functional interconnections between CA3 and the dentate gyrus revealed by current source density analysis.

The physiological interactions between the dentate gyrus (DG) and CA3 were studied in urethane-anesthetized rats by using field potential recording and current source density (CSD) analysis. Stimulation of CA3b resulted in a short-latency (<2.5-ms onset latency) antidromic population spike in both the DG and CA3c. An excitation (current sink) at the middle molecular layer (MML) was observed at 3-ms latency, possibly mediated by the backfiring of perforant path fibers that projected to both DG and CA3. CA3 stimulation also resulted in a sink at the dendritic layers of CA3c, which was likely mediated by excitatory CA3 recurrent collaterals. It was inferred that the DG was excited at the inner molecular layer (IML) after stimulation near the CA3b/CA3c border. This IML excitation (sink) probably resulted from orthodromic CA3 or hilar projections to the IML and not from mossy fiber backfiring. The IML and the CA3c dendritic sinks were blocked by an intracerebroventricular injection of a non-N-methyl-D-aspartate receptor antagonist, 6-cyano-7-nitroquinoxaline-2, 3-dione, but not by a gamma-aminobutyric acid type A (GABA(A)) receptor antagonist, bicuculline. CA3b stimulation evoked population spike bursts (3-7-ms latency) in both DG and CA3c when GABA(A) inhibition was suppressed by bicuculline, thus confirming that the excitatory afferents project from CA3b to DG and CA3c. A CA3 conditioning stimulus pulse given 30-200 ms before a perforant-path test pulse increased the amplitude of the perforant-path-evoked DG population spike (as compared with the test response without conditioning). After a moderate-intensity stimulation of CA3, a late (<20-ms latency) excitation of the MML of the DG was found. The late DG excitation was blocked by procaine injection at the medial perforant path, suggesting its origin from the medial entorhinal cortex. In conclusion, rich interactions between CA3 and other hippocampal structures were studied quantitatively by CSD analysis in vivo. We infer that CA3 provides an early excitatory feedback path to DG through recurrent collaterals or hilar interneurons and a late feedback through the medial entorhinal cortex.