Low ethanol concentrations enhance GABAergic inhibitory postsynaptic potentials in hippocampal pyramidal neurons only after block of GABAB receptors.

Despite considerable evidence that ethanol can enhance chloride flux through the gamma-aminobutyric acid type A (GABA/A/) receptor-channel complex in several central neuron types, the effect of ethanol on hippocampal GABAergic systems is still controversial. Therefore, we have reevaluated this interaction in hippocampal pyramidal neurons subjected to local monosynaptic activation combined with pharmacological isolation of the various components of excitatory and inhibitory synaptic potentials, using intracellular current- and voltage-clamp recording methods in the hippocampal slice. In accord with our previous findings, we found that ethanol had little effect on compound inhibitory postsynaptic potentials/currents (IPSP/Cs) containing both GABA/A/ and GABA/B/ components. However, after selective pharmacological blockade of the GABA/B/ component of the IPSP (GABA/B/-IPSP/C) by CGP-35348, low concentrations of ethanol (22-66 mM) markedly enhanced the peak amplitude, and especially the area, of the GABA/A/ component (GABA/A/-IPSP/C) in most CA1 pyramidal neurons. Ethanol had no significant effect on the peak amplitude or area of the pharmacologically isolated GABA/B/-inhibitory postsynaptic current (IPSC). These results provide new data showing that activation of GABAB receptors can obscure ethanol enhancement of GABA/A/ receptor function in hippocampus and suggest that similar methods of pharmacological isolation might be applied to other brain regions showing negative or mixed ethanol-GABA interactions.

Operative GABAergic inhibition in hippocampal CA1 pyramidal neurons in experimental epilepsy

Patch–clamp recordings of CA1 interneurons and pyramidal cells were performed in hippocampal slices from kainate- or pilocarpine-treated rat models of temporal lobe epilepsy. We report that γ-aminobutyric acid (GABA)ergic inhibition in pyramidal neurons is still functional in temporal lobe epilepsy because: (i) the frequency of spontaneous GABAergic currents is similar to that of control and (ii) focal electrical stimulation of interneurons evokes a hyperpolarization that prevents the generation of action potentials. In paired recordings of interneurons and pyramidal cells, synchronous interictal activities were recorded. Furthermore, large network-driven GABAergic inhibitory postsynaptic currents were present in pyramidal cells during interictal discharges. The duration of these interictal discharges was increased by the GABA type A antagonist bicuculline. We conclude that GABAergic inhibition is still present and functional in these experimental models and that the principal defect of inhibition does not lie in a complete disconnection of GABAergic interneurons from their glutamatergic inputs.

Electrophysiological evidence for a hyperpolarizing, galanin (1-15)-selective receptor on hippocampal CA3 pyramidal neurons

The effects of the 29-amino acid neuropeptide galanin [GAL (1–29)], GAL(1–15), GAL(1–16), and the GAL subtype 2 receptor agonist d-tryptophan2-GAL(1–29) were studied in the dorsal hippocampus in vitro with intracellular recording techniques. GAL(1–15) induced, in the presence of tetrodotoxin, a dose-dependent hyperpolarization in hippocampal CA3 neurons. Most of the GAL(1–15)-sensitive neurons did not respond to GAL(1–29), GAL(1–16), or d-tryptophan2-GAL(1–29). These results indicate the presence of a distinct, yet-to-be cloned GAL(1–15)-selective receptor on CA3 neurons in the dorsal hippocampus.

Latent N-methyl-D-aspartate receptors in the recurrent excitatory pathway between hippocampal CA1 pyramidal neurons: Ca(2+)-dependent activation by blocking A1 adenosine receptors.

When performed at increased external [Ca2+]/[Mg2+] ratio (2.5 mM/0.5 mM), temporary block of A1 adenosine receptors in hippocampus [by 8-cyclopentyltheophylline (CPT)] leads to a dramatic and irreversible change in the excitatory postsynaptic current (EPSC) evoked by Schaffer collateral/commissural (SCC) stimulation and recorded by in situ patch clamp in CA1 pyramidal neurons. The duration of the EPSC becomes stimulus dependent, increasing with increase in stimulus strength. The later occurring component of the EPSC is carried through N-methyl-D-aspartate (NMDA) receptor-operated channels but disappears under either the NMDA antagonist 2-amino-5-phosphonovaleric acid (APV) or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These findings indicate that the late component of the SCC-evoked EPSC is polysynaptic: predominantly non-NMDA receptor-mediated SCC inputs excite CA1 neurons that recurrently excite each other by predominantly NDMA receptor-mediated synapses. These recurrent connections are normally silent but become active after CPT treatment, leading to enhancement of the late component of the EPSC. The activity of these connections is maintained for at least 2 hr after CPT removal. When all functional NMDA receptors are blocked by dizocilpine maleate (MK-801), subsequent application of CPT leads to a partial reappearance of NMDA receptor-mediated EPSCs evoked by SCC stimulation, indicating that latent NMDA receptors are recruited. Altogether, these findings indicate the existence of a powerful system of NMDA receptor-mediated synaptic contacts in SCC input to hippocampal CA1 pyramidal neurons and probably also in reciprocal connections between these neurons, which in the usual preparation are kept latent by activity of A1 receptors.

Aurintricarboxylic acid prevents GLUR2 mRNA down-regulation and delayed neurodegeneration in hippocampal CA1 neurons of gerbil after global ischemia

Aurintricarboxylic acid (ATA), an inhibitor of endonuclease activity and other protein–nucleic acid interactions, blocks apoptosis in several cell types and prevents delayed death of hippocampal pyramidal CA1 neurons induced by transient global ischemia. Global ischemia in rats and gerbils induces down-regulation of GluR2 mRNA and increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced Ca2+ influx in CA1 before neurodegeneration. This result and neuroprotection by antagonists of AMPA receptors suggests that formation of AMPA receptors lacking GluR2, and therefore Ca2+ permeable, leads to excessive Ca2+ influx in response to endogenous glutamate; the resulting delayed neuronal death in CA1 exhibits many characteristics of apoptosis. In this study, we examined the effects of ATA on expression of mRNAs encoding glutamate receptor subunits in gerbil hippocampus after global ischemia. Administration of ATA by injection into the right cerebral ventricle 1 h before (but not 6 h after) bilateral carotid occlusion prevented the ischemia-induced decrease in GluR2 mRNA expression and the delayed neurodegeneration. These findings suggest that ATA is neuroprotective in ischemia by blocking the transcriptional changes leading to down-regulation of GluR2, rather than by simply blocking endonucleases, which presumably act later after Ca2+ influx initiates apoptosis. Maintaining formation of Ca2+ impermeable, GluR2 containing AMPA receptors could prevent delayed death of CA1 neurons after transient global ischemia, and block of GluR2 down-regulation may provide a further strategy for neuroprotection.

Long-term exposure to high corticosterone levels attenuates serotonin responses in rat hippocampal CA1 neurons

Recent studies indicated that hyperactivity of the hypothalamo-pituitary-adrenal system is a considerable risk factor for the precipitation of affective disorders, most notably of major depression. The mechanism by which this hyperactivity eventually leads to clinical symptoms of depression is unknown. In the present animal study, we tested one possible mechanism, i.e., that long-term exposure to high corticosterone levels alters functional responses to serotonin in the hippocampus, an important area in the etiology of depression. Rats were injected daily for 3 weeks with a high dose of corticosterone; electrophysiological responses to serotonin were recorded intracellularly from CA1 pyramidal neurons in vitro. We observed that daily injections with corticosterone gradually attenuate the membrane hyperpolarization and resistance decrease mediated by serotonin-1A receptors. We next used single-cell antisense RNA amplification from identified CA1 pyramidal neurons to resolve whether the functional deficits in serotonin responsiveness are accompanied by decreased expression levels of the serotonin-1A receptor. It appeared that expression of serotonin-1A receptors in CA1 pyramidal cells is not altered; this result was supported by in situ hybridization. Expression of corticosteroid receptors in the same cells, particularly of the high-affinity mineralocorticoid receptor, was significantly reduced after long-term corticosterone treatment. The present findings indicate that prolonged elevation of the corticosteroid concentration, a possible causal factor for major depression in humans, gradually attenuates responsiveness to serotonin without necessarily decreasing serotonin-1A receptor mRNA levels in pyramidal neurons. These functional changes may occur by a posttranscriptional mechanism or by transcriptional regulation of genes other than the serotonin-1A receptor gene itself.

Differential synaptic localization of two major gamma-aminobutyric acid type A receptor alpha subunits on hippocampal pyramidal cells.

Hippocampal pyramidal cells, receiving domain specific GABAergic inputs, express up to 10 different subunits of the gamma-aminobutyric acid type A (GABAA) receptor, but only 3 different subunits are needed to form a functional pentameric channel. We have tested the hypothesis that some subunits are selectively located at subsets of GABAergic synapses. The alpha 1 subunit has been found in most GABAergic synapses on all postsynaptic domains of pyramidal cells. In contrast, the alpha 2 subunit was located only in a subset of synapses on the somata and dendrites, but in most synapses on axon initial segments innervated by axo-axonic cells. The results demonstrate that molecular specialization in the composition of postsynaptic GABAA receptor subunits parallels GABAergic cell specialization in targeting synapses to a specific domain of postsynaptic cortical neurons.

Very short-term plasticity in hippocampal synapses

Hippocampal pyramidal neurons often fire in bursts of action potentials with short interspike intervals (2–10 msec). These high-frequency bursts may play a critical role in the functional behavior of hippocampal neurons, but synaptic plasticity at such short times has not been carefully studied. To study synaptic modulation at very short time intervals, we applied pairs of stimuli with interpulse intervals ranging from 7 to 50 msec to CA1 synapses isolated by the method of minimal stimulation in hippocampal slices. We have identified three components of short-term paired-pulse modulation, including (i) a form of synaptic depression manifested after a prior exocytotic event, (ii) a form of synaptic depression that does not depend on a prior exocytotic event and that we postulate is based on inactivation of presynaptic N-type Ca2+ channels, and (iii) a dependence of paired-pulse facilitation on the exocytotic history of the synapse.

Surface protein phosphorylation by ecto-protein kinase is required for the maintenance of hippocampal long-term potentiation.

During the induction of long-term potentiation (LTP) in hippocampal slices adenosine triphosphate (ATP) is secreted into the synaptic cleft, and a 48 kDa/50 kDa protein duplex becomes phosphorylated by extracellular ATP. All the criteria required as evidence that these two proteins serve as principal substrates of ecto-protein kinase activity on the surface of hippocampal pyramidal neurons have been fulfilled. This phosphorylation activity was detected on the surface of pyramidal neurons assayed after synaptogenesis, but not in immature neurons nor in glial cells. Addition to the extracellular medium of a monoclonal antibody termed mAb 1.9, directed to the catalytic domain of protein kinase C (PKC), inhibited selectively this surface protein phosphorylation activity and blocked the stabilization of LTP induced by high frequency stimulation (HFS) in hippocampal slices. This antibody did not interfere with routine synaptic transmission nor prevent the initial enhancement of synaptic responses observed during the 1-5 min period immediately after the application of HFS (the induction phase of LTP). However, the initial increase in the slope of excitatory postsynaptic potentials, as well as the elevated amplitude of the population spike induced by HFS, both declined gradually and returned to prestimulus values within 30-40 min after HFS was applied in the presence of mAb 1.9. A control antibody that binds to PKC but does not inhibit its activity had no effect on LTP. The selective inhibitory effects observed with mAb 1.9 provide the first direct evidence of a causal role for ecto-PK in the maintenance of stable LTP, an event implicated in the process of learning and the formation of memory in the brain.

Regulation of excitatory transmission at hippocampal synapses by calbindin D28k.

Distinct subpopulations of neurons in the brain contain one or more of the Ca(2+)-binding proteins calbindin D28k, calretinin, and parvalbumin. Although it has been shown that these high-affinity Ca(2+)-binding proteins can increase neuronal Ca2+ buffering capacity, it is not clear which aspects of neuronal physiology they normally regulate. To investigate this problem, we used a recently developed method for expressing calbindin D28k in the somatic and synaptic regions of cultured hippocampal pyramidal neurons. Ninety-six hours after infection with a replication-defective adenovirus containing the calbindin D28k gene, essentially all cultured hippocampal pyramidal neurons robustly expressed calbindin D28k. Our results demonstrate that while calbindin D28k does not alter evoked neurotransmitter release at excitatory pyramidal cell synapses, this protein has a profound effect on synaptic plasticity. In particular, we show that calbindin D28k expression suppresses posttetanic potentiation.

Developmental regulation of spine motility in the mammalian central nervous system

The function of dendritic spines, postsynaptic sites of excitatory input in the mammalian central nervous system (CNS), is still not well understood. Although changes in spine morphology may mediate synaptic plasticity, the extent of basal spine motility and its regulation and function remains controversial. We investigated spine motility in three principal neurons of the mouse CNS: cerebellar Purkinje cells, and cortical and hippocampal pyramidal neurons. Motility was assayed with time-lapse imaging by using two-photon microscopy of green fluorescent protein-labeled neurons in acute and cultured slices. In all three cell types, dendritic protrusions (filopodia and spines) were highly dynamic, exhibiting a diversity of morphological rearrangements over short (<1-min) time courses. The incidence of spine motility declined during postnatal maturation, but dynamic changes were still apparent in many spines in late-postnatal neurons. Although blockade or induction of neuronal activity did not affect spine motility, disruption of actin polymerization did. We hypothesize that this basal motility of dendritic protrusions is intrinsic to the neuron and underlies the heightened plasticity found in developing CNS.

A decrease of reelin expression as a putative vulnerability factor in schizophrenia

Postmortem prefrontal cortices (PFC) (Brodmann’s areas 10 and 46), temporal cortices (Brodmann’s area 22), hippocampi, caudate nuclei, and cerebella of schizophrenia patients and their matched nonpsychiatric subjects were compared for reelin (RELN) mRNA and reelin (RELN) protein content. In all of the brain areas studied, RELN and its mRNA were significantly reduced (≈50%) in patients with schizophrenia; this decrease was similar in patients affected by undifferentiated or paranoid schizophrenia. To exclude possible artifacts caused by postmortem mRNA degradation, we measured the mRNAs in the same PFC extracts from γ-aminobutyric acid (GABA)A receptors α1 and α5 and nicotinic acetylcholine receptor α7 subunits. Whereas the expression of the α7 nicotinic acetylcholine receptor subunit was normal, that of the α1 and α5 receptor subunits of GABAA was increased when schizophrenia was present. RELN mRNA was preferentially expressed in GABAergic interneurons of PFC, temporal cortex, hippocampus, and glutamatergic granule cells of cerebellum. A protein putatively functioning as an intracellular target for the signal-transduction cascade triggered by RELN protein released into the extracellular matrix is termed mouse disabled-1 (DAB1) and is expressed at comparable levels in the neuroplasm of the PFC and hippocampal pyramidal neurons, cerebellar Purkinje neurons of schizophrenia patients, and nonpsychiatric subjects; these three types of neurons do not express RELN protein. In the same samples of temporal cortex, we found a decrease in RELN protein of ≈50% but no changes in DAB1 protein expression. We also observed a large (up to 70%) decrease of GAD67 but only a small decrease of GAD65 protein content. These findings are interpreted within a neurodevelopmental/vulnerability “two-hit” model for the etiology of schizophrenia.

Modulation of N-methyl-d-aspartate receptor function by glycine transport

The recent discovery of glycine transporters in both the central nervous system and the periphery suggests that glycine transport may be critical to N-methyl-d-aspartate receptor (NMDAR) function by controlling glycine concentration at the NMDAR modulatory glycine site. Data obtained from whole-cell patch–clamp recordings of hippocampal pyramidal neurons, in vitro, demonstrated that exogenous glycine and glycine transporter type 1 (GLYT1) antagonist selectively enhanced the amplitude of the NMDA component of a glutamatergic excitatory postsynaptic current. The effect was blocked by 2-amino-5-phosphonovaleric acid and 7-chloro-kynurenic acid but not by strychnine. Thus, the glycine-binding site was not saturated under the control conditions. Furthermore, GLYT1 antagonist enhanced NMDAR function during perfusion with medium containing 10 μM glycine, a concentration similar to that in the cerebrospinal fluid in vivo, thereby supporting the hypothesis that the GLYT1 maintains subsaturating concentration of glycine at synaptically activated NMDAR. The enhancement of NMDAR function by specific GLYT1 antagonism may be a feasible target for therapeutic agents directed toward diseases related to hypofunction of NMDAR.

Localization and regulation of GLUTx1 glucose transporter in the hippocampus of streptozotocin diabetic rats

We describe the localization of the recently identified glucose transporter GLUTx1 and the regulation of GLUTx1 in the hippocampus of diabetic and control rats. GLUTx1 mRNA and protein exhibit a unique distribution when compared with other glucose transporter isoforms expressed in the rat hippocampus. In particular, GLUTx1 mRNA was detected in hippocampal pyramidal neurons and granule neurons of the dentate gyrus as well as in nonprincipal neurons. With immunohistochemistry, GLUTx1 protein expression is limited to neuronal cell bodies and the most proximal dendrites, unlike GLUT3 expression that is observed throughout the neuropil. Immunoblot analysis of hippocampal membrane fractions revealed that GLUTx1 protein expression is primarily localized to the intracellular compartment and exhibits limited association with the plasma membrane. In streptozotocin diabetic rats compared with vehicle-treated controls, quantitative autoradiography showed increased GLUTx1 mRNA levels in pyramidal neurons and granule neurons; up-regulation of GLUTx1 mRNA also was found in nonprincipal cells, as shown by single-cell emulsion autoradiography. In contrast, diabetic and control rats expressed similar levels of hippocampal GLUTx1 protein. These results indicate that GLUTx1 mRNA and protein have a unique expression pattern in rat hippocampus and suggest that streptozotocin diabetes increases steady-state mRNA levels in the absence of concomitant increases in GLUTx1 protein expression.

Mice transgenically overexpressing sulfonylurea receptor 1 in forebrain resist seizure induction and excitotoxic neuron death

The ability of the sulfonylurea receptor (SUR) 1 to suppress seizures and excitotoxic neuron damage was assessed in mice transgenically overexpressing this receptor. Fertilized eggs from FVB mice were injected with a construct containing SUR cDNA and a calcium-calmodulin kinase IIα promoter. The resulting mice showed normal gross anatomy, brain morphology and histology, and locomotor and cognitive behavior. However, they overexpressed the SUR1 transgene, yielding a 9- to 12-fold increase in the density of [3H]glibenclamide binding to the cortex, hippocampus, and striatum. These mice resisted kainic acid-induced seizures, showing a 36% decrease in average maximum seizure intensity and a 75% survival rate at a dose that killed 53% of the wild-type mice. Kainic acid-treated transgenic mice showed no significant loss of hippocampal pyramidal neurons or expression of heat shock protein 70, whereas wild-type mice lost 68–79% of pyramidal neurons in the CA1–3 subfields and expressed high levels of heat shock protein 70 after kainate administration. These results indicate that the transgenic overexpression of SUR1 alone in forebrain structures significantly protects mice from seizures and neuronal damage without interfering with locomotor or cognitive function.