Mapping synaptic glutamate transporter dysfunction in vivo to regions surrounding Aß plaques by iGluSnFR two-photon imaging.

Amyloid-ß (Aß) plaques, a hallmark of Alzheimer's disease (AD), are surrounded by regions of neuronal and glial hyperactivity. We use in vivo two-photon and wide-field imaging of the glutamate sensor iGluSnFR to determine whether pathological changes in glutamate dynamics in the immediate vicinity of Aß deposits in APPPS1 transgenic mice could alter neuronal activity in this microenvironment. In regions close to Aß plaques chronic states of high spontaneous glutamate fluctuations are observed and the timing of glutamate responses evoked by sensory stimulation exhibit slower decay rates in two cortical brain areas. GLT-1 expression is reduced around Aß plaques and upregulation of GLT-1 expression and activity by ceftriaxone partially restores glutamate dynamics to values in control regions. We conclude that the toxic microenvironment surrounding Aß plaques results, at least partially, from enhanced glutamate levels and that pharmacologically increasing GLT-1 expression and activity may be a new target for early therapeutic intervention.

Cyclic nucleotide-gated channels contribute to the cholinergic plateau potential in hippocampal CA1 pyramidal neurons.

Plateau potentials are prolonged membrane depolarizations that are observed in hippocampal pyramidal neurons when spiking and Ca(2+) entry occur in combination with muscarinic receptor activation. In this study, we used whole-cell voltage clamping to study the current underlying the plateau potential and to determine the cellular signaling pathways contributing to this current. When combined with muscarinic stimulation, depolarizing command potentials that evoked Ca(2+) influx elicited a prolonged tail current (I(tail)) that had an extrapolated reversal potential of -20 mV. I(tail) was not observed when intracellular Ca(2+) levels were chelated with 10 mm intracellular BAPTA, and I(tail) was reversibly depressed in low external sodium. When I(tail) was evoked at intervals >3 min, current amplitudes were stable for up to 1 hr. However, at shorter intervals, I(tail) was refractory, with a time constant of recovery of 43.5 sec. The inhibitors of soluble guanylate cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and 6-anilino-5,8-quinolinequinone depressed I(tail) and zaprinast, which blocks cGMP-specific phosphodiesterase, enhanced I(tail), suggesting that a component of I(tail) was activated by cGMP. The inhibitors of cyclic nucleotide-gated (CNG) channels l-cis-diltiazem and 2',4'-dichlorobenzamil reversibly depressed I(tail). However, protein kinase G inhibition had no effect. Therefore, these results indicate that a component of I(tail) is attributable to activation of CNG channels. We conclude that Ca(2+) influx when combined with muscarinic receptor activation activates soluble guanylate cyclase and increases cGMP levels. The increased cGMP activates CNG channels and leads to prolonged depolarization. The cation conductance of the CNG channel contributes to the prolonged depolarization of the plateau potential.

P2X7-like receptor activation in astrocytes increases chemokine monocyte chemoattractant protein-1 expression via mitogen-activated protein kinase.

Leukocyte infiltration in the CNS after trauma or inflammation is triggered in part by upregulation of the chemokine, monocyte chemoattractant protein-1 (MCP-1), in astrocytes. However the signals that induce the upregulation of MCP-1 in astrocytes are unknown. We have investigated the roles for ATP P2X7 receptor activation because ATP is an intercellular signaling transmitter that is released in both trauma and inflammation and P2X7 receptors are involved in immune system signaling. Astrocytes in primary cell culture and acutely isolated from the hippocampus were immunopositive for P2X7 receptors. In astrocyte cultures, application of the selective P2X7 agonist, benzoyl-benzoyl ATP (Bz-ATP), activated MAP kinases extracellular signal receptor-activated kinase 1 (ERK1), ERK2, and p38. Purinergic antagonists depressed this activation with a profile suggesting P2X7 receptors. Bz-ATP also increased MCP-1 expression in cultured astrocytes, and again P2X7 antagonists prevented this increase. Blocking either the ERK1/ERK2 or the p38 pathway (with PD98059 or SB203580, respectively) significantly inhibited Bz-ATP-induced MCP-1 expression. Coapplication of both antagonists caused a greater depression. We also tested the roles for ATP receptor activation in inducing MCP-1 upregulation in corticectomy, an in vivo model of trauma. This model of cortical trauma was previously shown to increase MCP-1 expression in vivo principally in astrocytes. Suramin, a wide-spectrum purinergic receptor antagonist, significantly depressed the rapid (3 hr) trauma-induced increase in MCP-1 mRNA. These data indicate that purinergic transmitter receptors in astrocytes are important in regulating chemokine synthesis. The regulation of MCP-1 in astrocytes by ATP may be important in mediating communication with hematopoietic inflammatory cells.

Theta-frequency facilitation of AMPA receptor-mediated synaptic currents in the principal cells of the medial septum.

Recent evidence suggests that Ca(2+)-permeable AMPA receptors display rapid, short-lasting current facilitation. In this study, we investigated the properties of AMPA receptor-mediated synaptic currents in medial septal neurons of the rat in an in vitro slice preparation. Immunocytochemistry with a selective antibody to the GluR2 subunit revealed that both choline acetyltransferase-containing and parvalbumin-containing neurons of the medial septum express no detectable GluR2 subunit immunoreactivity. We used whole cell voltage-clamp recordings to measure synaptically evoked AMPA receptor-mediated currents from medial septal neurons following stimulation of midline afferents. The GYKI 52466 (50 microM)- and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) (20 microM)-sensitive AMPA receptor-mediated component of the synaptic response was isolated by blocking GABA(A)- and N-methyl-D-aspartate receptor-mediated currents with 30 microM bicuculline and 100 microM 2-amino-5-phosphonovaleric acid, respectively. In some cases, patched cells were filled with Lucifer yellow (0.1%) and imaged using 2-photon laser scanning microscopy. AMPA receptor-mediated currents that were observed in large medial septal neurons (20--30 microm) displayed rectification. These currents were sensitive to external application of philanthotoxin-343 (PhTx-343, 50 microM), a potent, high-affinity antagonist of Ca(2+)-permeable, GluR2-lacking AMPA receptors. Rectifying AMPA receptor-mediated currents also displayed a rapid increase in amplitude when evoked five times at low frequency such as 6 Hz. In contrast to currents observed in large medial septal neurons, AMPA-receptor mediated currents evoked in the remaining small (8--11 microm) neurons were nonrectifying and displayed rapid synaptic depression when stimulated five times at 6 Hz. The currents evoked in these cells were unaffected by external application of PhTx-343 and were therefore GluR2-containing AMPA receptors. The results of the present study demonstrate that the principal projection neurons of the medial septum contain PhTx-343-sensitive, GluR2-lacking AMPA receptors that display rapid current facilitation when stimulated at low frequencies.

Serine/threonine protein phosphatases and synaptic inhibition regulate the expression of cholinergic-dependent plateau potentials.

We previously identified cholinergic-dependent plateau potentials (PPs) in CA1 pyramidal neurons that were intrinsically generated by interplay between voltage-gated calcium entry and a Ca(2+)-activated nonselective cation conductance. In the present study, we examined both the second-messenger pathway and the role of synaptic inhibition in the expression of PPs. The stimulation of m1/m3 cholinergic receptor subtypes and G-proteins were critical for activating PPs because selective receptor antagonists (pirenzepine, hexahydro-sila-difenidol hydrochloride, 4-diphenylacetoxy-N-methylpiperidine methiodide) and intracellular guanosine-5'-O-(2-thiodiphosphate) prevented PP generation in carbachol. Intense synaptic stimulation occasionally activated PPs in the presence of oxytremorine M, a cholinergic agonist with preference for m1/m3 receptors. PPs were consistently activated by synaptic stimulation only when oxytremorine M was combined with antagonists at both GABA(A) and GABA(B) receptors. These latter data indicate an important role for synaptic inhibition in preventing PP generation. Both intrinsically generated and synaptically activated PPs could not be elicited following inhibition of serine/threonine protein phosphatases by calyculin A, okadaic acid, or microcystin-L, suggesting that muscarinic-induced dephosphorylation is necessary for PP generation. PP genesis was also inhibited following irreversible thiophosphorylation by intracellular perfusion with ATP-gamma-S. These data indicate that the expression of cholinergic-dependent PPs requires protein phosphatase-induced dephosphorylation via G-protein-linked m1/m3 receptor(s). Moreover, synaptic inhibition via both GABA(A) and GABA(B) receptors normally prevents the synaptic activation of PPs. Understanding the regulation of PPs should provide clues to the role of this regenerative potential in both normal activity and pathophysiological processes such as epilepsy.

Glutamate release through volume-activated channels during spreading depression.

Volume-sensitive organic anion channels (VSOACs) in astrocytes are activated by cell swelling and are permeable to organic anions, such as glutamate and taurine. We have examined the release of glutamate through VSOACs during the propagation of spreading depression (SD). SD was induced by bath application of ouabain in hippocampal brain slices and was monitored by imaging intrinsic optical signals, a technique that provides a measure of cellular swelling. The onset of SD was associated with increased light transmittance, confirming previous studies that cellular swelling occurs during SD. NMDA receptor antagonists, either noncompetitive (MK-801, 10-50 microM) or competitive (CGS-17355, 100 microM), reduced the rate of propagation of SD, indicating that glutamate release contributes to SD onset. SD still occurred in zero Ca(2+)-EGTA (0-Ca(2+)-EGTA) solution, a manipulation that depresses synaptic transmission. HPLC measurements indicated that, even in this solution, there was significant glutamate release. Two lines of experiments indicated that glutamate was released through VSOACs during SD. First, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), a blocker of VSOACs, depressed the rate of propagation of SD in a manner similar to NMDA antagonists. Second, NPPB inhibited the release of glutamate during SD in 0-Ca(2+)-EGTA external solution. These results indicate that cellular swelling during SD causes the activation of VSOACs and the release of glutamate by permeation through this channel. Cellular swelling is a result of neuronal activity and is observed during excitotoxicity. Therefore, glutamate release from VSOAC activation could occur under conditions of cell swelling and contribute to excitotoxic damage.

Imaging spreading depression and associated intracellular calcium waves in brain slices.

Spreading depression (SD) was analyzed in hippocampal and neocortical brain slices by imaging intrinsic optical signals in combination with either simultaneous electrophysiological recordings or imaging of intracellular calcium dynamics. The goal was to determine the roles of intracellular calcium (Ca2+int) waves in the generation and propagation of SD. Imaging of intrinsic optical signals in the hippocampus showed that ouabain consistently induced SD, which characteristically started in the CA1 region, propagated at 15-35 micrometer/sec, and traversed across the hippocampal fissure to the dentate gyrus. In the dendritic regions of both CA1 and the dentate gyrus, SD caused a transient increase in light transmittance, characterized by both a rapid onset and a rapid recovery. In contrast, in the cell body regions the transmittance increase was prolonged. Simultaneous imaging of intracellular calcium and intrinsic optical signals revealed that a slow Ca2+int increase preceded any change in transmittance. Additionally, a wave of increased Ca2+int typically propagated many seconds ahead of the change in transmittance. These calcium increases were also observed in individual astrocytes injected with calcium orange, indicating that Ca2+int waves were normally associated with SD. However, when hippocampal slices were incubated in calcium-free/EGTA external solutions, SD was still observed, although Ca2+int waves were completely abolished. Under these conditions SD had a comparable peak increase in transmittance but a slower onset and a faster recovery. These results demonstrate that although there are calcium dynamics associated with SD, these increases are not necessary for the initiation or propagation of spreading depression.

Biophysical and pharmacological characterization of voltage-dependent Ca2+ channels in neurons isolated from rat nucleus accumbens.

The nucleus accumbens (NA) has an integrative role in behavior and may mediate addictive and psychotherapeutic drug action. Whole cell recording techniques were used to characterize electrophysiologically and pharmacologically high- and low-threshold voltage-dependent Ca2+ currents in isolated NA neurons. High-threshold Ca2+ currents, which were found in all neurons studied and include both sustained and inactivating components, activated at potentials greater than -50 mV and reached maximal activation at approximately 0 mV. In contrast, low-threshold Ca2+ currents activated at voltages greater than -64 mV with maximal activation occurring at -30 mV. These were observed in 42% of acutely isolated neurons. Further pharmacological characterization of high-threshold Ca2+ currents was attempted using nimodipine (Nim), omega-conotoxin-GVIA (omega-CgTx) and omega-agatoxin-IVA (omegaAga), which are thought to identify the L, N, and P/Q subtypes of Ca2+ currents, respectively. Nim (5-10 muM) blocked 18%, omegaCgTx (1-2 muM) blocked 25%, and omegaAga (200 nM) blocked 17% of total Ca2+ current. Nim primarily blocked a sustained high-threshold Ca2+ current in a partially reversible manner. In contrast, omegaCgTx irreversibly blocked both sustained and inactivating components. omegaAga irreversibly blocked only a sustained component. In all three of these Ca2+ channel blockers, plus 5 muM omega-conotoxin-MVIIC to eliminate a small unblocked Q-type Ca2+ current (7%), a toxin-resistant high-threshold Ca2+ current remained that was 32% of total Ca2+ current. This current inactivated much more rapidly than the other high-threshold Ca2+ currents, was depressed in 50 muM Ni2+ and reached maximal activation 5-10 mV negative to the toxin-sensitive high-threshold Ca2+ currents. Thus NA neurons have multiple types of high-threshold Ca2+ currents with a large component being the toxin-resistant "R" component.

Mitogen-activated protein and tyrosine kinases in the activation of astrocyte volume-activated chloride current.

Astrocytes swell during neuronal activity as they accumulate K+ to buffer the increase in external K+ released from neurons. This swelling activates volume-sensitive Cl- channels, which are thought to be important in regulatory volume decrease and in the response of the CNS to trauma and excitotoxicity. Mitogen-activated protein (MAP) kinases also are activated by cell volume changes, but their roles in volume regulation are unknown. We have investigated the role of tyrosine and MAP kinases in the activation of volume-activated Cl- channels in cultured astrocytes, using whole-cell patch-clamp recording and Western immunoblots. As previously described, hypo-osmotic solution induced an outwardly rectifying Cl- current, which was blocked by NPPB and SITS. This Cl- current did not depend on [Ca2+ ]i because it was still observed when 20 mM BAPTA was included in the pipette, but it did exhibit rundown when ATP was omitted. Inhibition of tyrosine kinases with genistein or tyrphostin A23 (but not the inactive agents daidzein and tyrphostin A1) blocked the Cl- current. The MAP kinase kinase (MEK) inhibitor PD 98059 reversibly inhibited activation of the Cl- current by hypo-osmotic solution. Western immunoblots showed that genistein or PD 98059 blocked activation of Erk-1 and Erk-2 by hypo-osmotic solution in astrocytes. Therefore, activation of tyrosine and MAP kinases by swelling is a critical step in the opening of volume-sensitive Cl- channels.

Neurotrophin modulation of NMDA receptors in cultured murine and isolated rat neurons.

Neurotrophin modulation of NMDA receptors in cultured murine and isolated rat neurons. J. Neurophysiol. 78: 2363-2371, 1997. Patch-clamp and calcium imaging techniques were used to assess the acute effects of the neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor (NGF), on the responses of cultured and acutely isolated hippocampal and cultured striatal neurons to the glutamate receptor agonist N-methyl--aspartic acid (NMDA). The effects of BDNF on NMDA-activated currents were examined in greater detail. Currents evoked by NMDA, and the accompanying changes in intracellular calcium, were enhanced by low concentrations of the neurotrophins (1-20 ng/ml). The potentiation by the neurotrophins was rapid in onset and offset (<1 s). The neurotrophins also reduced desensitization of these currents in most cells. The enhancement of NMDA-activated currents by BDNF was observed using both perforated and whole cell patch recording techniques and could be demonstrated in outside-out patches. Furthermore, its effects were not attenuated by pretreatment with the protein kinase inhibitors genistein or 1-(5-isoquinolynesulfony)2-methylpiperazine (H7). Therefore, the actions of BDNF do not appear to be mediated by phosphorylation. Similar enhancements were observed with NT-3 and NT-4 and with NGF despite the fact that hippocampal neurons lack TrkA receptors. All together this evidence suggests that the enhancement of NMDA-evoked currents is unlikely to be mediated through the activation of growth factor receptors. Modulation of NMDA responses by BDNF was dependent on the concentration of extracellular glycine. The most pronounced potentiation by BDNF was observed at low concentrations, whereas no potentiation was observed in saturating concentrations of glycine, suggesting that BDNF may have increased the affinity of the NMDA receptor for glycine. However, the competitive glycine-site antagonist 7-chloro-kynurenic acid blocked the enhancement by BDNF without shifting the dose-inhibition relationship for this antagonist, and Mg2+ consistently depressed the potentiation of NMDA-evoked currents by BDNF, indicating that BDNF does not alter glycine affinity. BDNF also reversibly increased the probability of opening of NMDA channels recorded from outside-out patches taken from cultured hippocampal neurons. Other unrelated peptides including dynorphin and somatostatin also caused a glycine-dependent enhancement of NMDA currents and depressed the currents in saturating concentrations of glycine. In contrast, a shortened analogue dynorphin (6-17), which lacks N-terminus glycine residues, and another peptide met-enkephalin were without effects on NMDA currents recorded in low concentrations of glycine. Our results suggest that neurotrophins and other peptides can serve as glycine-like ligands for the NMDA receptor.

Imaging the induction and spread of seizure activity in the isolated brain of the guinea pig: the roles of GABA and glutamate receptors.

1. The induction and spread of seizure activity was studied using imaging and electrophysiological techniques in the isolated whole brain of the guinea pig. We examined the role of GABA and glutamate receptor subtypes in controlling the spread of seizure activity across the olfactory cortex from a focus in the entorhinal cortex. Seizure spread was monitored by video imaging of intrinsic optical signals (reflectance changes) combined with multiple extracellular recordings. Both the unilateral and bilateral spread of seizure activity was monitored in different experiments. 2. Electrical stimulation of the lateral entorhinal cortex (10-15 V, 5 Hz, 5-10 s) evoked seizure activity that originated in the entorhinal cortex/hippocampus and later spread preferentially toward the posteromedial cortical amygdaloid nucleus ipsilaterally and bilaterally. The pattern of seizure spread in a given brain was highly reproducible. 3. The influence of gamma-aminobutyric acid (GABA) receptors on the spread of seizure activity was monitored at higher resolution on one side of the brain. Perfusion of a low concentration of the GABAA antagonist bicuculline methiodide (20 microM) resulted in spontaneous seizures that spread to the posteromedial cortical amygdaloid nucleus more rapidly than electrically evoked seizures [spread times: 5.5 +/- 3.7 s vs. 15.5 +/- 2.7 s, respectively (means +/- SE)]. Seizure spread was also more extensive in the presence of bicuculline involving the posterior perirhinal cortex and larger areas over the medial amygdala. Higher concentrations of bicuculline (100 microM) resulted in even more widespread propagation of spontaneous seizure activity throughout the olfactory cortex as well as to the perirhinal, insular, and occipital cortices. This concentration of bicuculline also further reduced the time required for seizure activity to spread from the entorhinal cortex to the posteromedial cortical amygdaloid nucleus (spread time = 2.3 +/- 1.7 s). The GABAB antagonist, CGP 35348 (200 microM), in contrast, had no significant effect of seizure induction or propagation. 4. The role of glutamate receptor subtypes in seizure propagation was studied by examining the bilateral spread of seizures. Perfusion of the kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (K/A) receptor antagonist (6-cyano-7-nitroquinoxaline-2,3-dione, CNQX, 20 microM) completely and reversibly suppressed stimulus-evoked seizure activity as detected electrophysiologically and optically. CNQX also reduced the magnitudes of field potentials recorded in the isolated brain in a reversible manner by an average of 70.8 +/- 2.21% of control. The N-methyl-D-aspartate (NMDA) receptor antagonist dibenzocyclohepteneimine (MK-801) did not significantly alter the magnitudes or shapes of field potentials recorded in the isolated brain nor did it significantly alter seizure activity measured optically or electrophysiologically. 5. Perfusion of the metabotropic glutamate receptor agonist [trans-1-amino-(IS,3R)-cyclopentanedicarboxylic acid (trans-ACPD), 150 microM] completely and reversibly suppressed stimulus-evoked seizure activity as detected electrophysiologically and optically. The magnitudes of field potentials recorded in the isolated brain also were reduced by trans-ACPD an average of 75.4 +/- 5.39% of control values. 6. These results demonstrate that GABAA-mediated transmission is functionally present and may play an important role in epileptic tissue in limiting the spread of seizure activity from the entorhinal cortex to the posteromedial cortical amygdaloid nucleus and in creating functional pathways or preferential routes of seizure spread. GABAB-mediated postsynaptic inhibition played no significant role in the induction or spread of seizure activity in this study. K/A receptors but not NMDA receptors are necessary for the induction and subsequent spread of seizure activity originating in the entorhinal cortex/hippocampus.

Cholinergic-dependent plateau potential in hippocampal CA1 pyramidal neurons.

Cholinergic stimulation of the hippocampal formation results in excitation and/or seizure. We report here, using whole-cell patch-clamp techniques in the hippocampal slice (34-35 degrees C), a cholinergic-dependent slow afterdepolarization (sADP) and long-lasting plateau potential (PP). In the presence of 20 microM carbachol, action potential firing evoked by weak intracellular current injection elicited an sADP that lasted several seconds. Increased spike firing evoked by stronger depolarizing stimuli resulted in long-duration PPs maintained close to -20 mV. Removal of either Na+ or Ca2+ from the external media, intracellular Ca2+ ([Ca2+]i) chelation with 10 mM bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid, or the addition of 100 microM Cd2+ to the perfusate abolished both the sADP and PP. The sADP was depressed and the PP was abolished by either 10 microM nimodipine or 1 microM omega-conotoxin, whereas 1.2 microM tetrodotoxin was ineffective. The involvement of a Na+/Ca2+ exchanger was minimal because both the sADP and PP persisted after equimolar substitution of 50 mM Li+ for Na+ in the external media or reduction of the bath temperature to 25 degrees C. Finally in the absence of carbachol the sADP and PP could not be evoked when K+ channels were suppressed, suggesting that depression of K+ conductances alone was not sufficient to unmask the conductance. Based on these data, we propose that a Ca2+-activated nonselective cation conductance was directly enhanced by muscarinic stimulation. The sADP, therefore, represents activation of this conductance by residual [Ca2+]i, whereas the PP represents a novel regenerative event involving the interplay between high-voltage-activated Ca2+ channels and the Ca2+-activated nonselective cation conductance. This latter mechanism may contribute significantly to ictal depolarizations observed during cholinergic-induced seizures.

In vitro ischemia promotes calcium influx and intracellular calcium release in hippocampal astrocytes.

The intracellular calcium concentration ([Ca2+])i of astrocytes within rat hippocampal slices was measured during simultaneous hypoxia and hypoglycemia to examine the early intracellular signaling events induced by this in vitro model of ischemia. Hypoxia-hypoglycemia for 3.3-7.5 min evoked [Ca2+]i increases in astrocytes iontophoretically loaded with calcium orange (11/14 slices; 2.5 min to peak [Ca2+]i, 5 min to > 60 min duration). Calcium elevations also were observed in the absence of extracellular calcium ([Ca2+]o) (4/4 slices), indicative of Ca2+ release from internal stores. Hypoxia-hypoglycemia depolarized astrocytes (51 +/- 16 mV), suggesting additional contribution from voltage-gated Ca2+ influx. Depolarization of a similar magnitude (51 +/- 4 mV) by 50 mM extracellular potassium ([K+]o triggered [Ca2+]i increases (20/24 slices), which were blocked by removal of [Ca2+]o (8/8 slices) indicating that depolarization promoted Ca2+ influx. Voltage-gated Ca2+ influx and internal release were measured in accurately isolated astrocytes during in vitro ischemia to examine these processes in the absence of surrounding neurons. Hypoxia-hypoglycemia (7.5-34.0 min) induced only modest, slow increases in the basal [Ca2+]i of Fura-2-loaded isolated astrocytes (average 12% increase in Fura-2 ratio R340/380 after 10 min) that were blocked by [Ca2+]o removal. Voltage-gated Ca2+ influx was still functional under ischemia, however, as 50 mM [K+]o evoked [Ca2+]i increases (14/14 cells, delta R340/380 of 48%) approximately equal to preischemic responses. Isolated neurons displayed large irreversible increases in basal [Ca2+]i after 1.5-6.5 min in vitro ischemia (10/12 cells; average delta R 340/380 of 152%). The absence of significant basal [Ca2+]i increases on isolated astrocytes indicates that ischemia-induced Ca2+ influx and internal release in astrocytes within slices depend on signals released from neurons (K+, neurotransmitters). Ischemic [Ca2+]i elevations may constitute a signaling mechanism for postischemic reactive responses.

Adrenergic calcium signaling in astrocyte networks within the hippocampal slice.

Norepinephrine (NE) and glutamate (Glu) initiate intracellular calcium ([Ca2+]i) transients, oscillations, and intracellular [Ca2+]i waves in cultured astrocytes. To further elucidate the significance of NE- and Glu-evoked astrocytic [Ca2+]i signaling to neuron-astrocyte communication in the mature CNS, [Ca2+]i of astrocyte networks within hippocampal slices (P21-42) was measured during bath application of NE and Glu receptor agonists. Astrocytes in stratum radiatum were identified by highly negative membrane potentials (75 +/- 3 mV), absence of action potentials, and dye coupling following intracellular injection of the [Ca2+]-sensitive dye calcium orange. NE (2-100 microM) evoked [Ca2+]i increases (7 of 8 slices, 24 of 24 cells in responding slices) characterized by an initial rise, 20-50 sec to peak, followed by a slower return to baseline (over approximately 8 min). The alpha 1-agonist phenylephrine (PE) (10-100 microM) evoked complex [Ca2+]i signals (22 of 26 slices, 90 of 90 cells in responding slices) composed of both a prolonged component (5.1 +/- 1.8 min), synchronized in neighboring cells, and multiple, mainly asynchronous [Ca2+]i spikes (25.0 +/- 11.6 sec). PE responses were completely blocked by the alpha 1-antagonist prazosin (200 nM, n = 4 slices), but not by the alpha 2-antagonist yohimbine (n = 3 slices). The alpha 2-agonist clonidine (10-100 microM) did not increase [Ca2+]i (n = 4 slices). alpha 1-mediated [Ca2+]i transients were observed after removal of extracellular [Ca2+]o (n = 8 of 9 slices), indicating PE-induced Ca2+ release from intracellular stores. Adrenergic responses were mediated by alpha 1-receptors localized to astrocytes because PE and NE increased [Ca2+]i of acutely isolated hippocampal astrocytes. Glu (0.75-2.0 mM) did not increase astrocytic [Ca2+]i in slices (0 of 7), even in the presence of the Glu uptake inhibitor L-trans-pyrrollidine-2,4-dicarboxylic acid (PDC) (0 of 5 slices), or in acutely isolated astrocytes (0 of 7 cells). The metabotropic agonist t-ACPD (30 or 50 microM) did not increase astrocytic [Ca2+]i in hippocampal slices (0 of 5), while kainate (200 microM or 1 mM) induced brief (1-2 min) [Ca2+]i increases only rarely (2 of 8 applications in 6 slices). These results support a primary role of NE release and alpha 1-adrenoceptor stimulation in neuron-astrocyte communication in the mature CNS.

GABAA/benzodiazepine receptors in acutely isolated hippocampal astrocytes.

The properties of GABA receptor-mediated responses were examined in noncultured astrocytes, acutely isolated from the mature rat hippocampus. Whole-cell patch clamping revealed a GABA-activated Cl- conductance that was mimicked by the GABAA receptor agonist muscimol and depressed by the GABAA antagonists bicuculline and picrotoxin. The GABAA-activated currents were potentiated by the barbiturate pentobarbital and the benzodiazepine diazepam. The benzodiazepine inverse agonist DMCM either enhanced or depressed the astrocytic GABAA-mediated responses, suggesting receptor heterogeneity with respect to pharmacologic profiles. In addition, GABA evoked an increase in [Ca2+]n measured by indo-1 fluorometry, which was depressed in the presence of verapamil or picrotoxin. A GABAA-induced depolarization, therefore, causes Ca2+ influx through voltage-gated Ca2+ channels. The expression and subcellular localization of GABAA receptors and its subunits were examined using immunohistochemical and fluorescent benzodiazepine binding techniques. Polyclonal antisera raised against the GABAA/benzodiazepine receptor, which recognizes multiple subunit isoforms, labeled receptors on the astrocytic cell body and most large processes. In contrast, antisera generated against either alpha 1 or beta 1 subunit peptides revealed immunoreactivity predominantly on a subset of processes. To determine the subcellular distribution of membrane-bound receptors, a fluorescent benzodiazepine derivative was superfused over live astrocytes and visualized with laser-scanning confocal microscopy. Specific fluorescence was distributed in discrete clusters on the cell soma and a subset of distal processes. Collectively, these data support the view that astrocytes, like neurons, express GABAA receptors and target subunit isoforms to distinct cellular localizations. Astrocytic GABAA receptors may be involved in both [Cl-]o and [pH]o homeostasis, and a GABA-evoked increase in [Ca2+]i could serve as a signal between GABAergic neurons and astrocytes.

Imaging cell volume changes and neuronal excitation in the hippocampal slice.

Brain cell swelling is a consequence of seizure, ischemia or excitotoxicity. Changes in light reflectance from cortical surface are now used to monitor brain activity but these intrinsic signals are poorly understood. The objectives of this study were first, to show that changes in light transmittance were correlated with cell volume and second, to image increases in light transmittance as they related to neuronal activation. Transverse hippocampal slices from the rat were used for the study. Brief exposure (4-6 min) to hypo-osmotic artificial cerebrospinal fluid (-40 mOsm) elevated light transmittance consistently and reversibly in most regions of the slice and particularly in CA1 dendritic regions. Neither zero-Ca2+ artificial cerebrospinal fluid nor tetrodotoxin altered the transmittance increase and its subsequent reversal, suggesting that it was dependent on osmolality but independent of synaptic transmission and neuronal firing. The amplitude of the CA1 population spike evoked from Schaffer collaterals increased concomitantly with the hypo-osmotic increase in light transmittance, providing evidence that the extracellular tissue resistance increased. Hyper-osmotic artificial cerebrospinal fluid (+40 mOsm) containing impermeant mannitol consistently lowered light transmittance and the amplitude of the population spike. Glycerol (+40 mOsm), which is cell permeant, did not have an affect. Taken together these observations indicate that osmotic challenge alters light transmittance by inducing changes in cell volume. Transmittance increases induced by hypo-osmotic artificial cerebrospinal fluid or 10 microM kainate were small in the CA1 cell body region compared to dendritic regions. Similarly, orthodromic stimulation of axons terminating in stratum oriens or in stratum radiatum evoked transmittance increases only in their respective postsynaptic areas. In contrast, the cell body region and its adjacent proximal-apical dendrites (both sites of action potential initiation) could display dramatic increases in light transmittance upon brief exposure to 20 mM K+. The response, which may represent neuronal damage, was blocked in tetrodotoxin. Antidromic stimulation evoked a weak response in these same proximal areas. We conclude that activity-dependent increases in light transmittance across brain slices primarily reveal glial and neuronal swelling associated with excitatory synaptic input and action potential discharge. The signal can be imaged in real time to reveal neuronal activation, not only among hippocampal areas, but among neuronal regions. Cell swelling is a known consequence of excessive neuronal discharge. Therefore, the imaging of changes in light transmittance across brain slices should prove useful in monitoring epileptiform and excitotoxic states.

Potassium-dependent calcium influx in acutely isolated hippocampal astrocytes.

Potassium depolarization can increase the intracellular ionized calcium concentration ([Ca2+]i) of cultured astrocytes, but it is not known if astrocytes that have matured in the intact CNS also exhibit voltage-dependent [Ca2+]i signalling. To address this issue, fluorometric measurements of [Ca2+]i were obtained from astrocytes acutely isolated from young adult rat hippocampus. In control artificial cerebrospinal fluid containing 5 mM [K+]o, average resting [Ca2+]i was 195 nM. Elevation of [K+]o to 50 mM caused [Ca2+]i to increase 150 nM to 1 microM above resting levels. The threshold [K+]o necessary to evoke an elevation in [Ca2+]i was 20-25 mM, and the magnitude of the [Ca2+]i signal grew progressively with increasing [K+]o (up to 50 mM). These [Ca2+]i increases were blocked completely by removal of external Ca2+, and markedly suppressed by the calcium channel blockers verapamil (30 microM and greater) and Co2+ (1 mM). Neither reversal of Na(+)-Ca2+ exchange, nor Ca(2+)-activated Ca2+ release, nor Ca2+ influx through stretch-activated channels contributed to the [Ca2+]i increase. These results suggest that [K+]o-evoked [Ca2+]i signals are mediated by influx through voltage-gated calcium channels. In contrast to results from cultured astrocytes and acutely isolated neurons, these [Ca2+]i increases were insensitive to dihydropyridine compounds. We conclude that increases in interstitial [K+], observed in situ during several pathological conditions, trigger voltage-dependent [Ca2+]i signals in astroglial cells. This may constitute an important form of neuron-to-glial communication.

Astrocytic GABA receptors.

GABA receptors are distributed widely throughout the central nervous system on a variety of cell types. It has become increasingly clear that astrocytes, both in cell culture and tissue slices, express abundant GABAA receptors. In astrocytes, GABA activates Cl(-)-specific channels that are modulated by barbiturates and benzodiazepines; however, the neuronal inverse agonist methyl-4-ethyl-6, 7-dimethoxy-beta-carboline-3-carboxylate enhances the current in a subpopulation of astrocytes. The properties of astrocytic GABAA receptors, therefore, are remarkably similar to their neuronal counterparts, with only a few pharmacological exceptions. In stellate glial cells of the pituitary pars intermedia, GABA released from neuronal terminals activates postsynaptic potentials directly. The physiological significance of astrocytic GABAA-receptor activation remains unknown, but it may be involved in extracellular ion homeostasis and pH regulation. At present, there is considerably less evidence for the presence of GABAB receptors on astrocytes. The data that have emerged, however, indicate a prominent role for second-messenger regulation by this receptor.