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Cyclic AMP (cAMP)-responsive element-binding protein (CREB) is a transcription factor important in developing nervous system cells and is activated by a variety of signaling molecules. Aroclor 1254 (A1254), a polychlorinated biphenyl mixture, perturbs Ca(2+) homeostasis and increases CREB phosphorylation in rat neonatal cortical cell cultures in a time- and concentration-dependent manner. The present experiments determined that the cell type responding to A1254 with Ca(2+) increases and phosphorylated CREB (phospho-CREB) was predominantly of neuronal morphology and microtubule-associated protein (MAP2)-positive phenotype. Similarly, glutamate (100 microM) increased phospho-CREB immunoreactivity selectively in MAP2-immunopositive cells. Using Western blotting and immunocytochemical techniques, we identified key signal transduction pathways operative in phosphorylating CREB in cortical cell cultures and examined their participation in 3 ppm A1254-induced CREB activation. Cortical cultures treated with glutamate, forskolin or the phorbol ester phorbol 12-myristate 13-acetate exhibited robust increases in phospho-CREB. Tetrodotoxin (1 microM) completely inhibited CREB phosphorylation by A1254, suggesting that synaptic activity is involved in A1254-induced CREB activation. Buffering [Ca(2+)](i) with bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester in the absence of extracellular Ca(2+) partially inhibited A1254-induced CREB phosphorylation. Inhibition of mitogen-activated protein kinase (10 microM U0126) or protein kinase C (PKC; bisindoylmaleimide, 5 microM) activation did not inhibit A1254-induced CREB phosphorylation. By contrast, inhibition of protein kinase A (PKA) with 100 microM PKA inhibitor peptide, PKI, blocked A1254-induced CREB phosphorylation. Thus, we examined whether A1254 activates PKA by increasing cAMP; 10 microM forskolin, but not A1254, elevated intracellular cAMP levels. These results indicate that in neocortical cells in culture, CREB phosphorylation occurs via Ca(2+)-, PKA-, and PKC-dependent pathways. Furthermore, A1254-induced CREB phosphorylation occurs predominantly in neurons, is dependent on synaptic activity and mediated by Ca(2+)- and PKA-dependent pathways.

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The present study assessed intracellular Ca(2+) signaling pathways sensitive to polychlorinated biphenyls (PCBs), xenobiotics that perturb neural development and plasticity. Mobilization of intracellular Ca(2+) stores after acute exposure to a PCB mixture, Aroclor 1254 (A1254), as well as selected PCB congeners, was studied in P0 rat cortical neuronal culture using fluorescence microscopy. Ca(2+) responses to A1254 progressed from a transient intracellular Ca(2+) increase (lasting 3--5 min) at 1 to 2 microM (0.3-0.6 ppm) to a Ca(2+) transient with store-operated Ca(2+) influx and later disturbances of basal Ca(2+) concentration; this latter pattern occurred more often with 10 to 20 microM (3--6 ppm) A1254. Thapsigargin, xestospongin C, and carbachol/Ca(2+)-free buffer blocked significantly the PCB-induced Ca(2+) transient, whereas both ryanodine (to deplete ryanodine-sensitive stores) and the L-type Ca(2+) channel blocker nifedipine were without effect on the A1254 initial Ca(2+) transient. Both thapsigargin and xestospongin also blocked latent elevations (at 0.5 h) in Ca(2+), disturbances that depend upon extracellular Ca(2+) entry via ion channels. Two possible consequences were explored. Phosphorylation of cAMP responsive element binding protein, a Ca(2+)-activated nuclear transcription factor (CREB), occurred in an A1254 concentration-dependent manner and persisted at least 1 h. Cell viability following a 24-h exposure to A1254 (2-20 microM) was decreased at 20 microM, but only in cells cultured >6 days. This cell death did not occur via an apoptotic mechanism. These results indicate that Ca(2+) disturbances following PCB exposure are associated with 1) discrete alterations in IP(3) receptor-mediated signals and 2) activation of downstream events that impact developing cortical cells.

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In this unit, protocols are described for biochemical and optical techniques that have been used by investigators to measure ligand-gated chloride movement in vesicular structures called synaptoneurosomes (also referred to as microsacs), in cultured neurons, and in the acute brain slice. These techniques can be applied to other ions as well. The measurement of uptake and efflux of radioisotopic chloride in synaptoneurosomes is used to study the responses of gamma-aminobutyric acid (GABA) receptors, which are coupled to chloride channels. Similar chloride flux assays for primary neuronal cultures are also presented. Alternatively, the efflux of chloride from synaptoneurosomes and primary neuronal cultures can be studied using fluorescent dyes and photometry. Finally, the measurement of chloride uptake can be studied in individual neurons in brain slices using fluorescent dyes and optical imaging by nonconfocal and confocal microscopy. Several support protocols are provided as well, outlining the preparation of synaptoneurosomes from specific brain regions, and the preparation, loading, and calibration of chloride-sensitive fluorescent dyes.

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Developmental exposure to polychlorinated biphenyls (PCBs), environmental toxicants found throughout the world, results in neurodevelopmental delays and/or deficits. Previous mechanistic studies have demonstrated that PCBs elicit a broad spectrum of biochemical responses that include slow, graded increases in intracellular Ca(2+). Acute exposure of cultures of newborn rodent cortical neurons to the commercial PCB mixture Aroclor 1254 [A1254; 1-20 microM (0.3-6 ppm)], induced recurring oscillations of intracellular Ca(2+) concentration (individual Ca(2+) amplitudes of 200-600 nM). This oscillatory activity was absent in control (0.5 mM Mg(2+)-containing) solution. Ca(2+) oscillations induced by a 1-h exposure to A1254 were concentration dependent, as measured by cell recruitment (proportion of responding cells) as well as by Ca(2+) oscillation frequency and amplitude. Extracellular Ca(2+) entry via L-type voltage-sensitive Ca(2+) channels (VSCCs) was required to elicit the Ca(2+) oscillations because oscillations induced by A1254 were blocked in Ca(2+)-deficient solution or by addition of 1 microM nifedipine. Tetrodotoxin also blocked the Ca(2+) oscillations, suggesting that synaptic activity may activate VSCCs. To examine this further, the role of postsynaptic receptors that indirectly activate L-type VSCCs was examined. At 4 to 5 days in vitro, when GABA exerts a depolarizing action and activates L-type channels, addition of bicuculline blocked Ca(2+) oscillations induced by A1254. After longer maintenance of the cells in vitro (7 days), A1254-induced Ca(2+) oscillations were selectively blocked by a combination of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor antagonists (D-2-amino-5-phosphonopentanoic acid and 2, 3-dihydroxy-6,7-dinitroquinoxaline, respectively). These novel findings show the induction of network activity in an in vitro model by A1254 via activation of excitatory GABAergic and/or glutamatergic synaptic activity, depending on the stage of maturation.

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GABA(A) receptors are targets of highly chlorinated environmental chemicals and have important roles in developing neurons. As such, we examined effects of polychlorinated biphenyls (PCBs) on GABA(A) receptor responses in primary cultures of rat neocortical cells using fluorescence imaging techniques. Between days in vitro (DIV) 5 and 8, the effect of GABA(A) receptor stimulation switched from excitatory (Ca(2+) entry following a Cl(-) efflux; DIV /=7). GABA(A)-receptor-stimulated increases in [Ca(2+)](i) were diminished in a concentration-dependent (1-20 microM) manner following 1 h of exposure to the PCB mixture Aroclor 1254 (A1254), with significant reductions at concentrations as low as 2 microM. A1254 (1-20 microM) also led to concentration-dependent increases in basal [Ca(2+)](i), irrespective of DIV. A1254 (10 and 20 microM) significantly increased basal Ca(2+)(i); the Ca(2+)(i) was elevated to 426 +/- 39 nM by 20 microM A1254 but this concentration was not cytotoxic at 1 h. In addition, the mixture, A1254, as well as ortho- and non-ortho-chlorinated PCB congeners (IUPAC Nos. 4, 15, 126, and 138; 5-10 microM) individually decreased GABA(A)-stimulated Ca(2+)(i) responses and this tended not to depend on increases in basal Ca(2+)(i). In cultures DIV 7 and older, A1254 (20 microM) also impaired inhibitory GABA(A) responses as evidenced by an approximately 50% reduction of GABA(A)-stimulated Cl(-) influx (from approximately 6 to 8 mM net accumulation in controls). The results demonstrate that: (1) GABA(A) receptor increases in Ca(2+)(i) and Cl(-)(i) are inhibited by 2-20 microM A1254, regardless of whether the responses are at excitatory or inhibitory stages of development; (2) Ca(2+)(i) homeostasis in cortical cells is disrupted by 10 microM A1254; yet (3) disruption of excitatory GABA(A) responses by A1254 or PCB congeners does not necessarily depend on impaired Ca(2+) homeostasis. These novel observations suggest that GABA(A) receptor responses are a sensitive target for PCB effects in the rat developing nervous system.

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In brain slice preparations, chloride movements across the cell membrane of living cells are measured traditionally with 36Cl- tracer methods, Cl--selective microelectrodes, or whole-cell recording using patch clamp analysis. We have developed an alternative, noninvasive technique that uses the fluorescent Cl- ion indicator, 6-methoxy-N-ethylquinolinium iodide (MEQ), to study changes in intracellular Cl- by epifluorescence or UV laser scanning confocal microscopy. In brain slices taken from rodents younger than 22 days of age, excellent cellular loading is achieved with the membrane-permeable form of the dye, dihydro-MEQ. Subsequent intracellular oxidation of dihydro-MEQ to the Cl--sensitive MEQ traps the polar form of the dye inside the neurons. Because MEQ is a single-excitation and single-emission dye, changes in intracellular Cl- concentrations can be calibrated from the Stern-Volmer relationship, determined in separate experiments. Using MEQ as the fluorescent indicator for Cl-, Cl- flux through the gamma-aminobutyric acid (GABA)-gated Cl- channel (GABAA receptor) can be studied by dynamic video imaging and either nonconfocal (epifluorescence) or confocal microscopy in the acute brain slice preparation. Increases in intracellular Cl- quench MEQ fluorescence, thereby reflecting GABAA receptor activation. GABAA receptor functional activity can be measured in discrete cells located in neuroanatomically defined populations within areas such as the neocortex and hippocampus. Changes in intracellular Cl- can also be studied under various conditions such as oxygen/glucose deprivation ("in vitro ischemia") and excitotoxicity. In such cases, changes in cell volume may also occur due to the dependence of cell volume regulation on Na+, K+, and Cl- flux. Because changes in cell volume can affect optical fluorescence measurements, we assess cell volume changes in the brain slice using the fluorescent indicator calcein-AM. Determination of changes in MEQ fluorescence versus calcein fluorescence allows one to distinguish between an increase in intracellular Cl- and an increase in cell volume.

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This study describes the pharmacological properties of GABAA receptors expressed in P19-N cells using fluorescence imaging of intracellular chloride with 6-methoxy-N-ethylquinolinium iodide (MEQ). We show that application of the GABA agonist, muscimol (10-200 microM), produces time- and concentration-dependent increases in intracellular [Cl-] that are blocked by bicuculline. Diazepam (10 microM) and pentobarbital (1 mM) potentiate muscimol-stimulation. These receptors exhibit novel pharmacological properties. The neurosteroid, 3alpha-hydroxy-5alpha-pregnane-20-one (1-10 microM) exhibited weak potency in enhancement of muscimol-stimulation. Ethanol (50 and 100 mM) exhibited high efficacy on muscimol responses, a 4- to 5-fold potentiation, respectively, of muscimol (10 microM) alone. GABA and muscimol allosterically modulated specific binding of [3H]flunitrazepam to differentiated P19 cells. Modulation of GABAA receptor mediated increases in intracellular [Cl-] demonstrated stability in response magnitude from 7 to 15 days following removal of retinoic acid. In concert, GABAA receptor subunit mRNA and protein expression patterns in these neuron-like cells were stable over the same period. Using RT-PCR we determined that differentiated P19 cells lack gamma1, gamma2L, alpha6 and delta subunit mRNAs while expressing alpha1, alpha2, alpha3, alpha4, alpha5, beta1, beta2, beta3, gamma2S and gamma3. Furthermore, subunit specific antibody immunocytochemical labeling of cells with a neuronal morphology indicated the presence of alpha1, alpha2, alpha4, and gamma2 subunits (the only subunits tested). Therefore, P19-N cells should prove useful to researchers in need of a model cell culture system in which to study function and regulation of neuronal GABAA receptors.

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The effects of glutamatergic excitotoxins on intracellular Cl- were investigated in the CA1 pyramidal cell layer of the hippocampal slice. Hippocampal slices from rats (14-19 days old) were loaded with 6-methoxy-N-ethylquinolinium chloride (MEQ), a Cl(-)-sensitive fluorescent probe with a fluorescence intensity that correlates inversely with intracellular [Cl-]. Slices were exposed for at least 10 min at 26-28 degrees C to N-methyl-D-aspartate (NMDA; 100 microM) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA; 50 microM). A UV laser scanning confocal microscope was used to measure changes in MEQ fluorescence within area CA1 pyramidal cell soma. Both glutamate receptor agonists produced a rapid decrease in MEQ fluorescence that persisted after washout following a 10-min exposure. The effects of NMDA and AMPA were prevented by the competitive antagonists 2-amino-5-phosphonopentanoic acid and 6,7-dinitroquinoxaline-2,3-dione, respectively. Neither tetrodotoxin nor picrotoxin prevented the effect of NMDA or AMPA, indicating the lack of involvement of presynaptic mechanisms. The effects of NMDA and AMPA on MEQ fluorescence were dependent on the levels of extracellular Cl-, but only NMDA responses were dependent on the levels of extracellular Na+. Removal of Ca2+ from the superfusion medium did not alter the effects of NMDA or AMPA on MEQ fluorescence. In addition, neither the Ca2+ ionophore ionomycin nor the L-type voltage-gated Ca2+ channel agonist (Bay K 8644) decreased MEQ fluorescence. The effects of NMDA and AMPA on cell (somal) volume were also assessed with the fluorescent probe calcein acetoxymethyl ester. Both NMDA and AMPA decreased calcein fluorescence (indicating an increased cell volume), but this was preceded by the decrease in MEQ fluorescence (equivalent to an intracellular accumulation of approximately 20 mM Cl-). Thus, excitotoxins may cause Cl- influx via an anion channel other than the GABA(A) receptor and/or reduce Cl- efflux mechanisms to produce cell swelling. Such anionic shifts may promote neuronal excitability and cell death following an excitotoxic insult to the hippocampal slice.

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The septo-hippocampal pathway contains a major gamma-aminobutyric acid (GABA) projection to dendritic fields within the hippocampus. To determine the importance of the septo-hippocampal pathway in ischemia-induced accumulation of GABA and subsequent cell death in area CA1 of hippocampus, septo-hippocampal deafferentation of adult gerbils was performed. Electrolytic lesions were produced in the medial or medial plus lateral septal regions in gerbils 7 days prior to being subjected to 5 min forebrain ischemia. The extent of deafferentation of the dorsal hippocampus was determined histochemically by acetylcholinesterase staining. Both the medial and medial plus lateral septal lesions produced nearly complete loss of acetylcholinesterase staining in the dorsal hippocampus indicating relatively complete deafferentation. During and following ischemia, in vivo microdialysis was used to measure extracellular GABA accumulation, which reached concentrations up to 1060 +/- 143% of basal. Septo-hippocampal deafferentation in both groups of lesioned animals failed to prevent the accumulation of GABA (and glutamate) induced by ischemia, indicating that ischemia-induced GABA accumulation in area CA1 arises principally from intrinsic GABAergic interneurons. Ischemic animals with medial septal lesions did not demonstrate neuroprotection or increased damage in the stratum pyramidale 7 days after reperfusion. Since the septo-hippocampal pathway provides the source of GABAergic disinhibition within the hippocampus, neither disinhibition nor the septo-hippocampal input appear to play an important role in the development of ischemia-induced neuronal death in the hippocampus.

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The ability of diazepam, a benzodiazepine full agonist, and imidazenil, a benzodiazepine partial agonist, to protect hippocampal area CA1 neurons from death for at least 35 days after cerebral ischemia was investigated. Diazepam (10 mg/kg) administered to gerbils 30 and 90 minutes after forebrain ischemia produced significant protection of hippocampal area CA1 pyramidal neurons 7 days later. In gerbils surviving for 35 days, diazepam produced the same degree of neuroprotection (70% +/- 30%) in the hippocampus compared with 7 days after ischemia. The therapeutic window for diazepam was short; there was no significant neuroprotection when the administration of diazepam was delayed to 4 hours after ischemia. The neuroprotective dose of diazepam also produced hypothermia (approximately 32 degrees C) for several hours after injection. To assess the role of hypothermia in neuroprotection by diazepam, hypothermia depth and duration was simulated using a cold-water spray in separate gerbils. Seven days after ischemia, neuroprotection by hypothermia was similar to that produced by diazepam. However, 35 days after ischemia, there was no significant protection by hypothermia, suggesting that hypothermia does not play a significant role in long-term diazepam neuroprotection. Imidazenil (3 mg/kg), which produced only minimal hypothermia, protected area CA1 of hippocampus to the same degree as that by diazepam 7 days after ischemia. At 35 days after ischemia, significant protection remained, but it was considerably reduced compared with 7 days. Like diazepam, the therapeutic window for imidazenil was short. Imidazenil neuroprotection was lost when the drug was administered as early as 2 hours after ischemia. The ability of ischemia to produce deficits in working memory and of benzodiazepines to prevent the deficits also was investigated. Gerbils trained on an eight-arm radial maze before ischemia demonstrated a significant increase in the number of working errors 1 month after ischemia. The ischemia-induced deficits in working memory were completely prevented by diazepam but not by imidazenil. There was a significant, but weak, negative correlation between the degree of CA1 pyramidal cell survival and the number of working errors in both the diazepam and imidazenil groups. Thus, if given early enough during reperfusion, both benzodiazepine full and partial agonists are neuroprotective for at least 35 days, but the lack of sedating side effects of imidazenil must be weighed against its reduced efficacy.

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We determined if changes in intraneuronal Cl- occur early after ischemia in the hippocampal slice. Slices from juvenile rats (14-19 days old) were loaded with the cell-permeant form of 6-methoxy-N-ethylquinolinium chloride (MEQ), a Cl(-)-sensitive fluorescent dye. Real-time changes in intracellular chloride concentration ([Cl-]i) were measured with UV laser scanning confocal microscopy in multiple neurons within each slice. In vitro ischemia (26-28 degrees C, 10 min) was confirmed by the loss of synaptic transmission (evoked field excitatory postsynaptic potentials) from pyramidal cells in area CA1. After ischemia and reoxygenation (10 min), MEQ fluorescence decreased significantly in CA1 pyramidal cells and interneurons. The decreased fluorescence corresponded to an ischemia-induced increase in [Cl-]i of approximately 10 mM. Pretreatment with the GABA(A)-gated Cl- channel antagonist picrotoxin (100 microM) blocked the ischemia-induced change in [Cl-]i. Analysis of the superfusates indicated that ischemia also caused a transient amino acid (GABA, glutamate, and aspartate) release that was maximal at approximately 10 min, returning to baseline shortly thereafter. Recovery from ischemia was confirmed by the return of synaptic transmission in area CA1, the return toward baseline of the ischemia-induced decrease in MEQ fluorescence, and exclusion of propidium iodide from MEQ fluorescent cells. Furthermore, pyramidal cells did not undergo cell swelling during this early phase of reoxygenation, as indicated by the volume-sensitive dye calcein. Thus, mild ischemia induces the accumulation of [Cl-]i secondary to GABA(A) receptor activation, in the absence of cellular swelling or death. In contrast, depolarization of the slice with K+ (50 mM) decreased MEQ fluorescence significantly but caused cell swelling. Picrotoxin did not prevent the K+-induced increase in [Cl-]i. It is possible that an increased [Cl-]i, following either an ischemic event or an episode of depolarization, would reduce the Cl- driving force and thereby limit synaptic transmission by GABA. To support this hypothesis, ischemia caused a reduction in the ability of the GABA agonist muscimol to increase [Cl-]i after 20-min reoxygenation.

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We have developed a method using UV laser-scanning confocal microscopy and the fluorescent chloride ion indicator, 6-methoxy-N-ethylquinolinium chloride (MEQ), to image GABA-mediated changes in intracellular chloride (Cli-) in individual neurons of the rat acute brain slice. After bath-loading slices with the cell-permeant form (reduced) of MEQ, there was intense fluorescence within neurons of diverse morphologies in the hippocampus, neocortex and cerebellum. MEQ fluorescence localized to the cytosolic compartment of both the somata and proximal dendrites. MEQ fluorescence was calibrated using the ionophores nigericin and tributyltin in the presence of varying extracellular Cl- concentrations. Neuronal MEQ fluorescence was inversely related to intracellular Cl-, with a Stern-Volmer constant of 16 M-1 (50% quench by 61 mM Cl-). Application of GABA in the perfusate produced a concentration-dependent decrease in MEQ fluorescence (EC50 = 40 microM) that was blocked in the presence of the Cl- channel antagonist, picrotoxin. Bath perfusion of hippocampal slices with modulators of the GABAA receptor, pentobarbital and diazepam, potentiated the GABA-mediated response by 85 and 44%, respectively. A regional comparison identified larger GABA responses for both cerebellar Purkinje and granule cells relative to pyramidal neurons of the hippocampus and neocortex and to hippocampal interneurons. Pressure ejection of the GABAA agonist, muscimol (40 microM), from a micropipet onto individual hippocampal neurons allowed the measurement of rapid responses (1-5 s), compared to those obtained with bath application. Thus, optical imaging of [Cl-]i using MEQ and UV-laser-scanning confocal microscopy provides investigators with a new method to study GABAA pharmacology in neighboring neurons and perhaps even in the soma versus dendrites simultaneously, within living brain slices.

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Following transient cerebral ischemia, pyramidal cells within area CA1 of the hippocampus exhibit delayed neuronal death. While interneurons within this sector continue to survive long-term, there is evidence that some interneurons in area CA1 are vulnerable to damage. To determine the nature of vulnerability in a neurochemically heterogeneous population of interneurons throughout area CA1, we examined the labeling of gamma-aminobutyric acid (GABA)ergic interneurons with an antibody to the GABAA receptor alpha 1-subunit 1-35 days following cerebral ischemia in the Mongolian gerbil. Unlike some other GABA interneuron markers, this antibody labels both the dendrites and soma of interneurons, allowing dendritic structure to be examined. Three to four days following ischemia, the pyramidal cells in area CA1 had degenerated, and the alpha 1-subunit-positive interneurons in all layers of area CA1 had developed severely beaded dendrites. At longer survival times (21-35 days), the alpha 1-subunit-immunolabeled dendrites of these interneurons had a fragmented appearance. In contrast, interneurons bordering str. oriens and alveus typically exhibited normal dendritic morphology. Despite the pathologic changes, there was no evidence of interneuron loss in area CA1 up to 35 days post-ischemia. Normal interneuron morphology was also observed in area CA3 and dentate gyrus, regions where neither pyramidal neurons nor granule cells, respectively, die following 5 min of cerebral ischemia. To determine if the ischemia-induced changes in interneuron morphology could be prevented, diazepam was administered 30 and 90 min following ischemia. Diazepam produces long-term neuroprotection of area CA1 pyramidal neurons. In gerbils sacrificed 35 days after ischemia, diazepam markedly attenuated the dendritic beading of the area CA1 interneurons. In addition, the dendrites did not display the fragmented labeling by the alpha 1-subunit antibody. Thus, despite their long-term survival, CA1 hippocampal interneurons in the gerbil can express severe structural abnormalities after transient cerebral ischemia coincident with pyramidal cell degeneration, and the injury to the dendrites can be prevented by the neuroprotectant diazepam.

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The neuroprotective effects of enhancing neuronal inhibition with a gamma-aminobutyric acid (GABA) uptake inhibitor were studied in gerbil hippocampus following transient ischemia. We used in vivo microdialysis to determine a suitable dosing regimen for tiagabine (NNC328) to elevate extracellular levels of GABA within the hippocampus. In anesthetized (normothermic) gerbils, tiagabine (45 mg/kg, i.p.) selectively elevated extracellular GABA levels 450% in area CA1 of the hippocampus. In gerbils subjected to cerebral ischemia via 5-min bilateral carotid occlusion, extracellular GABA levels increased 13-fold in area CA 1 returning to baseline within 30-45 min. When tiagabine was injected 10 min following onset of reperfusion, GABA levels remained elevated (200-470%) for 90 min. In addition, tiagabine significantly reduced the ischemic-induced elevation of glutamate levels in area CA1 during the postischemic period when GABA levels were elevated. There was no effect of postischemic tiagabine on aspartate or six other amino acids. Using the same dosing regimen, we evaluated the degree of neuroprotection in the hippocampus of gerbils 4 and 21 days after ischemia. Tiagabine decreased body temperature a maximum of 2.7 degrees C beginning 30 min into reperfusion and lasting 90 min. In untreated gerbils sacrificed 4 and 21 days after ischemia, there was severe necrosis (99%) of the pyramidal cell layer in area CA1. Whereas tiagabine significantly protected the CA1 pyramidal cell layer in ischemic gerbils at 4 days (overt necrosis confined to about 17% of area CA1), the protection diminished significantly 21 days postischemia. When normothermia was maintained both during and after ischemia in a separate group of tiagabine-treated animals, approximately 77% of the CA1 pyramidal cell layer was necrotic at 4 days. Based on these findings, we suggest that 1) tiagabine slows the development of hippocampal degeneration following ischemia, and 2) that mild, postischemic hypothermia is responsible, in large part, for the neuroprotective actions of this drug. We conclude that the histological outcome after administration of cerebral neuroprotectants should be assessed following long-term survival.

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Pharmacologic blockade of GABA binding sites in the hypothalamus elicits a pattern of physiological and behavioral arousal. The latter outcome implicates a perturbation in the neural functioning of higher brain centers. The effect that hypothalamic GABAA receptor modulation has on the function of cerebral cortical neural substrates linked with responses to stressors was assessed using microinfusion of bicuculline methiodide (BMI) into the medial hypothalamus of freely moving, handling habituated rats. BMI led to rapid increases in frontal cortical dopamine (DA) utilization (calculated from the sum of the levels of the DA metabolites, homovanilic and dihydroxyphenylacetic acids, divided by DA levels) resembling that identified following restraint-induced stress. Also, cortical GABAA receptor function [using chloride (Cl-) enhancement of 3H-flunitrazepam (Flu) binding as an index] was disrupted; i.e. there was a loss of typical Cl- enhancement of 3H-Flu binding in animals after BMI infusions. However, placing animals in restraint after BMI infusion reversed the effects of BMI, with both DA utilization and Cl- facilitated 3H-Flu binding similar to control basal values. Muscimol infusions in separately prepared animals did not alter either frontal cortical DA utilization or GABAA receptor function. The present results implicate GABA in the hypothalamus as "gating" activity of cortical systems involved in sensation of and/or responses to stressors. These findings may have important implications for effects of autonomic arousal on neural substrates involved in mediating stress responses.

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This study examined both the function of the GABAA receptor complex and the expression of its alpha 1, alpha 2 and alpha 3 subunits within the hypothalamus as compared to that of the cerebral cortex. A large number of different GABAA receptor subunit combinations potentially exist in various brain regions which, presumably, would intimate differing receptor structure and function. Here, we present evidence that the average functional characteristics of GABAA receptors within the rat hypothalamus are considerably different from those of the cerebral cortex. We assessed two neurochemical measures of GABAA receptor function: namely, chloride-facilitation of [3H]flunitrazepam binding and GABA-mediated 36chloride uptake. [3H]Flunitrazepam binding in the rat cortex was facilitated by increasing concentrations (12.5-500 mM) of chloride, and this facilitation was responsive to 15 min restraint. Yet, hypothalamic [3H]flunitrazepam binding was not responsive to increasing chloride-concentration in either the basal or restraint conditions. Also, maximal facilitation of GABA-mediated 36chloride uptake was significantly blunted in the hypothalamus relative to cortex (7.4 +/- 0.9 versus 35.8 +/- 1.5 nmoles/mg protein, respectively). While in vitro addition of 10 microM diazepam shifted GABA-mediated 36chloride uptake curves of the cortex to the left, diazepam addition appeared to be without effect in the hypothalamus. However, the blunted maximal facilitation of GABA on hypothalamic 36chloride uptake made accurate determination of the EC50 for the diazepam-potentiation difficult. In addition to these functional disparities between the regions, differences in subunit expression were also apparent. Distributions of alpha 1, alpha 2 and alpha 3 subunit immunoreactivities within cingulate, parietal and temporal cortices and 8 major hypothalamic regions were assessed. Staining of the alpha 1 subunit was prevalent throughout the hypothalamus and cortex, and dense in both regions. However, the alpha 2 and alpha 3 subunits, while of intermediate density in cortex, were of low density or absent (alpha 3) in the hypothalamus. The alpha 2-immunoreactivity was restricted to cell bodies of the arcuate nucleus, dorsomedial nucleus and overlying dorsal area and to neuropil staining of the median eminence. Thus, functional responsiveness of the GABAA receptor differs in the hypothalamus relative to the cortex and this would seem related to the presence of different receptor alpha subunits in homogenate preparations of the two regions.

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The dorsomedial hypothalamus is important for regulation of cardiovascular responses associated with emotional arousal. This region has also been identified as a component of neural circuitry involved in fear/anxiety, yet clear evidence as to the effects of lesioning on stress-related behaviors is missing. In this study, we lesioned the dorsomedial hypothalamic region with the neurotoxin, ibotenic acid (IBO; 2.0 micrograms in 0.2 microliter), and studied the impact on spontaneous and unlearned behavioral responses to stressors. In the open field test, we observed non-generalized increases in motility parameters in the IBO rats with the differences occurring in the latter two-thirds of the test. In the elevated plus-maze, the IBO rats displayed a classic anxiolytic response with a greater proportion of entries into (and greater time spent in) the open arms of the maze. In the environment-specific social interaction (SI) test, the IBO rats showed a normal familiar/unfamiliar environment discrimination with respect to Total SI; however, the composition of the behaviors ('curiosity' vs. physical contact) by the IBO rats was markedly altered, with there being a 2-fold increase in non-violent physical interactions. Additionally, the differences in these traditional indices of anxiety were associated with lesioned animals exhibiting greater acoustic startle responsiveness than controls as a function of prepulse intensity. Overall, the results following IBO lesions indicate an altered responsiveness to sudden stressors, particularly as relates to novelty or exploration-oriented behaviors. The hypothalamic lesion may, therefore, have resulted in a disinhibition of normally suppressed responding to innate fear or challenging stimuli. This study contributes to those that have begun to define neural interactions that are essential for integrated stress responses.

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Catecholamine terminals in the paraventricular nucleus (PVN) of the hypothalamus of 60-day-old rats were destroyed by the stereotaxic injection of 6-hydroxydopamine into the PVN (6-OHDA; 9 micrograms/1.5 microliters bilaterally), and the rats were tested 2 weeks later. Lesions led to a 70% reduction of norepinephrine in the hypothalamus and a loss of dopamine-beta-hydroxylase immunoreactivity in the PVN. Furthermore, 6-OHDA lesions in the hypothalamus disrupted stressor-induced (15 min of restraint) changes in GABAA receptor function in the cerebral cortex (assessed by measuring chloride-facilitated benzodiazepine binding) but did not alter stressor-induced increases in plasma corticosterone levels. Additionally, the lesion did not change the responsiveness of the GABAA receptor to the corticosterone metabolite, allotetrahydrodeoxycorticosterone. These results indicate that stressor-induced changes in cortical GABAA receptor function are not driven by the stressor-induced release of corticosterone. A separate group of animals were tested for behavioral responses to challenge, and while 6-OHDA-induced lesions did not alter total scores in the test of environment-specific social interaction, the lesions did induce a change in composition of the behavior. Lesioned animals demonstrated increased physical (vigorous contact) interactions, similar to behavior previously observed in younger rats. The results of the behavioral study support a role for the GABAA receptor in the cerebral cortex in mediating appropriate behavioral responses to challenge in the adult rat. Thus, a hypothalamic lesion that prevented challenge-induced changes in GABAA receptor function in the cortex (with no change in the corticosterone response to the stressor) also led to altered behavioral responses to challenge.

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Earlier work demonstrated that prenatal exposure to diazepam (DZ) selectively affected the noradrenergic (NE) terminals in the hypothalamus, leading to decreased basal NE levels, turnover rate, and release in adult offspring as well as altered responses to stressors in these NE projections. The exposure also affected plasma hormonal responses to stressors. In the present work, we used immunocytochemistry to study the effects of prenatal DZ exposure on NE terminals and on corticotropin-releasing factor (CRF)-containing neurons in the paraventricular nucleus (PVN) of the hypothalamus. DZ exposure (2.5 or 10 mg/kg over gestational days 14-20) led to a decrease in dopamine-beta-hydroxylase (DBH)-immunoreactivity (-ir) and a decrease in CRF-ir containing cells within the PVN of adult rats. The exposure also decreased DBH-ir in the ventral portion of the bed nucleus of the stria terminalis (BNST) but did not affect CRF-ir in the oval nucleus of BNST. Therefore, this study provides anatomic evidence that targeting benzodiazepine binding sites prenatally affects two neurotransmitter systems involved in responses to stressors.

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