Rochelle D. Schwartz-Bloom

γ-Aminobutyric acidA neurotransmission and cerebral ischemia

In this review, we present evidence for the role of γ‐aminobutyric acid (GABA) neurotransmission in cerebral ischemia‐induced neuronal death. While glutamate neurotransmission has received widespread attention in this area of study, relatively few investigators have focused on the ischemia‐induced alterations in inhibitory neurotransmission. We present a review of the effects of cerebral ischemia on pre and postsynaptic targets within the GABAergic synapse. Both in vitro and in vivo models of ischemia have been used to measure changes in GABA synthesis, release, reuptake, GABAA receptor expression and activity. Cellular events generated by ischemia that have been shown to alter GABA neurotransmission include changes in the Cl− gradient, reduction in ATP, increase in intracellular Ca2+, generation of reactive oxygen species, and accumulation of arachidonic acid and eicosanoids. Neuroprotective strategies to increase GABA neurotransmission target both sides of the synapse as well, by preventing GABA reuptake and metabolism and increasing GABAA receptor activity with agonists and allosteric modulators. Some of these strategies are quite efficacious in animal models of cerebral ischemia, with sedation as the only unwanted side‐effect. Based on promising animal data, clinical trials with GABAergic drugs are in progress for specific types of stroke. This review attempts to provide an understanding of the mechanisms by which GABA neurotransmission is sensitive to cerebral ischemia. Furthermore, we discuss how dysfunction of GABA neurotransmission may contribute to neuronal death and how neuronal death can be prevented by GABAergic drugs.

The Chloride Transporter Na+-K+-Cl− Cotransporter Isoform-1 Contributes to Intracellular Chloride Increases after In Vitro Ischemia

Ischemic episodes in the CNS cause significant disturbances in neuronal ionic homeostasis. To directly measure changes in intracellular Cl− concentration ([Cl−]i) during and after ischemia, we used Clomeleon, a novel ratiometric optical indicator for Cl−. Hippocampal slices from adult transgenic mice expressing Clomeleon in hippocampal neurons were subjected to 8 min of oxygen-glucose deprivation (OGD) (an in vitro model for ischemia) and reoxygenated in the presence of glucose. This produced mild neuronal damage 3 h later that was prevented when the extracellular [Cl−] was maintained at 10 mm during reoxygenation. OGD induced a transient decrease in fluorescence resonance energy transfer within Clomeleon, indicating an increase in [Cl−]i. During reoxygenation, there was a partial recovery in [Cl−]i, but [Cl−]i rose again 45 min later. To investigate sources of Cl− accumulation, we examined the effects of Cl− transport inhibitors on the rises in [Cl−]i during and after OGD. Bumetanide and furosemide, which inhibit Cl− influx through the Na+-K+-Cl− cotransporter isoform-1 (NKCC-1) and efflux through the K+-Cl− cotransporter isoform-2, were unable to inhibit the first rise in [Cl−]i, yet entirely prevented the secondary rise in [Cl−]i during reoxygenation. In contrast, picrotoxin, which blocks the GABA-gated Cl− channel, did not inhibit the secondary rise in [Cl−]i after OGD. [Cl−]i increases during reoxygenation were accompanied by an increase in phosphorylation of NKCC-1, an indication of increased NKCC-1 activity after OGD. We conclude that NKCC-1 plays an important role in OGD-induced Cl− accumulation and subsequent neuronal damage.

Distribution of GABAA, GABAB, and glycine receptors in the central auditory system of the big brown bat, Eptesicus fuscus

Quantitative autoradiographic techniques were used to compare the distribution of GABAA, GABAB, and glycine receptors in the subcortical auditory pathway of the big brown bat, Eptesicus fuscus. For GABAA receptors, the ligand used was 35S‐t‐butylbicyclophosphorothionate (TBPS); for GABAB receptors, 3H‐GABA was used as a ligand in the presence of isoguvacine to block binding to GABAA sites; for glycine, the ligand used was 3H‐strychnine. In the subcortical auditory nuclei there appears to be at least a partial complementarity in the distribution of GABAA receptors labeled with 35S‐TBPS and glycine receptors labeled with 3H‐strychnine. GABAA receptors were concentrated mainly in the inferior colliculus (IC) and medial geniculate nucleus, whereas glycine receptors were concentrated mainly in nuclei below the level of the IC. Within the IC, there was a graded spatial distribution of 35S‐TBPS binding; the most dense labeling was in the dorsomedial region, but very sparse labeling was observed in the ventrolateral region. There was also a graded spatial distribution of 3H‐strychnine binding. The most dense labeling was in the ventral and lateral regions and the weakest labeling was in the dorsomedial region. Thus, in the IC, the distribution of 35S‐TBPS was complementary to that of 3H‐strychnine. GABAB receptors were distributed at a low level throughout the subcortical auditory nuclei, but were most prominent in the dorsomedial part of the IC.

Changes in Intracellular Chloride after Oxygen–Glucose Deprivation of the Adult Hippocampal Slice: Effect of Diazepam

Ischemic injury to the CNS results in loss of ionic homeostasis and the development of neuronal death. An increase in intracellular Ca2+ is well established, but there are few studies of changes in intracellular Cl– ([Cl–]i) after ischemia. We used an in vitro model of cerebral ischemia (oxygen–glucose deprivation) to examine changes in [Cl–]i and GABAA receptor-mediated responses in hippocampal slices from adult rats. Changes in [Cl–]i were measured in area CA1 pyramidal neurons using optical imaging of 6-methoxy-N-ethylquinolinium chloride, a Cl–-sensitive fluorescent indicator. Oxygen–glucose deprivation induced an immediate rise in [Cl–]i, which recovered within 20 min. A second and more prolonged rise in [Cl–]i occurred within the next hour, during which postsynaptic field potentials failed to recover. The sustained increase in [Cl–]i was not blocked by GABAA receptor antagonists. However, oxygen–glucose deprivation caused a progressive downregulation of the K+–Cl– cotransporter (KCC2), which may have contributed to the Cl– accumulation. The rise in [Cl–]i was accompanied by an inability of the GABAA agonist muscimol to cause Cl– influx. In vivo, diazepam is neuroprotective when given early after ischemia, although the mechanism by which this occurs is not well understood. Here, we added diazepam early after oxygen–glucose deprivation and prevented the downregulation of KCC2 and the accumulation of [Cl–]i. Consequently, both GABAA responses and synaptic transmission within the hippocampus were restored. Thus, after oxygen–glucose deprivation, diazepam may decrease neuronal excitability, thereby reducing the energy demands of the neuron. This may prevent the activation of downstream cell death mechanisms and restore Cl– homeostasis and neuronal function.

Diazepam promotes ATP recovery and prevents cytochrome c release in hippocampal slices after in vitro ischemia

Abstract: Benzodiazepines protect hippocampal neurons when administered within the first few hours after transient cerebral ischemia. Here, we examined the ability of diazepam to prevent early signals of cell injury (before cell death) after in vitro ischemia. Ischemia in vitro or in vivo causes a rapid depletion of ATP and the generation of cell death signals, such as the release of cytochrome c from mitochondria. Hippocampal slices from adult rats were subjected to 7 min of oxygen‐glucose deprivation (OGD) and assessed histologically 3 h after reoxygenation. At this time, area CA1 neurons appeared viable, although slight abnormalities in structure were evident. Immediately following OGD, ATP levels in hippocampus were decreased by 70%, and they recovered partially over the next 3 h of reoxygenation. When diazepam was included in the reoxygenation buffer, ATP levels recovered completely by 3 h after OGD. The effects of diazepam were blocked by picrotoxin, indicating that the protection was mediated by an influx of Cl‐ through the GABAA receptor. It is interesting that the benzodiazepine antagonist flumazenil did not prevent the action of diazepam, as has been shown in other studies using the hippocampus. Two hours after OGD, the partial recovery of ATP levels occurred simultaneously with an increase of cytochrome c (∼400%) in the cytosol. When diazepam was included in the reoxygenation buffer, it completely prevented the increase in cytosolic cytochrome c. Thus, complete recovery of ATP and prevention of cytochrome c release from mitochondria can be achieved when diazepam is given after the loss of ATP induced by OGD.

Optical imaging of hippocampal neurons with a chloride-sensitive dye : Early effects of in vitro ischemia

Abstract: 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°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 ∼10 mM. Pretreatment with the GABAA‐gated Cl− channel antagonist picrotoxin (100 µM) 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 ∼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 GABAA 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.

Rapid down-regulation of GABAA receptors in the gerbil hippocampus following transient cerebral ischemia.

Abstract: During transient cerebral ischemia, there is a temporary and robust accumulation of extracellular GABA in the hippocampus. We examined whether the acute exposure of GABAA/benzodiazepine receptors to high concentrations of GABA early after ischemia results in receptor down‐regulation as observed in vitro. Gerbils were killed 30 and 60 min following a 5‐min bilateral carotid occlusion, and their brains were prepared for receptor autoradiography. The hydrophilic GABAA receptor antagonist [3H]SR‐95531 and the hydrophobic benzodiazepine agonist [3H]flunitrazepam were used to distinguish between cell surface and internalized receptors. Ischemia significantly decreased [3H]SR‐95531 binding in hippocampal areas CA1 and CA3 and in the dentate gyrus 30 min after ischemia. Scatchard analysis in area CA1 revealed that ischemia decreased the Bmax as low as 44%. The affinity of the remaining sites was increased substantially (72% decrease in KD). As expected, there were no changes in the binding of [3H]flunitrazepam to hippocampus in the early postischemic period because the benzodiazepine could bind to both internalized receptors and those on the cell surface. We hypothesize that prolonged exposure (∼30–45 min) of GABAA receptors to high concentrations of synaptic GABA in vivo causes receptor down‐regulation, perhaps via receptor internalization.

Activation of excitatory amino acid receptors in the rat hippocampal slice increases intracellular Cl- and cell volume.

Abstract: 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°C to N‐methyl‐d‐aspartate (NMDA; 100 µM) or α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA; 50 µM). 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 ∼20 mM Cl−). Thus, excitotoxins may cause Cl− influx via an anion channel other than the GABAA 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.

Long-Term Neuroprotection by Benzodiazepine: Full Versus Partial Agonists after Transient Cerebral Ischemia in the Gerbil

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 (~32°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.

Chloride transport inhibitors influence recovery from oxygen-glucose deprivation-induced cellular injury in adult hippocampus.

Cerebral ischemia in vivo or oxygen-glucose deprivation (OGD) in vitro are characterized by major disturbances in neuronal ionic homeostasis, including significant rises in intracellular Na(+), Ca(2+), and Cl(-) and extracellular K(+). Recently, considerable attention has been focused on the cation-chloride cotransporters Na-K-Cl cotransporter isoform I (NKCC-1) and K-Cl cotransporter isoform II (KCC2), as they may play an important role in the disruption of ion gradients and subsequent ischemic damage. In this study, we examined the ability of cation-chloride transport inhibitors to influence the biochemical (i.e. ATP) and histological recovery of neurons in adult hippocampal slices exposed to OGD. In the hippocampus, 7 min of OGD caused a loss of ATP that recovered partially (approximately 50%) during 3 h of reoxygenation. Furosemide, which inhibits the NKCC-1 and KCC2 cotransporters, and bumetanide, a more specific NKCC-1 inhibitor, enhanced ATP recovery when measured 3 h after OGD. Furosemide and bumetanide also attenuated area CA1 neuronal injury after OGD. However, higher concentrations of these compounds appear to have additional non-specific toxic effects, limiting ATP recovery following OGD and promoting neuronal injury. The KCC2 cotransporter inhibitor DIOA and the Cl(-) ATPase inhibitor ethacrynic acid caused neuronal death even in the absence of OGD and promoted cytochrome c release from isolated mitochondria, indicating non-specific toxicities of these compounds.