Deconstructing the neural and ionic involvement of seizure-like events in the striatal network.

Seizures occur in the basal ganglia (BG) of epileptic patients and in animal models of epilepsy, but there is relatively little known about how these events are gated and/or propagated through this structure. Here, we present and characterize a model of in vitro seizure-like events (SLEs) in the striatum by applying chemostimulants to brain slices from young rat pups. We found that bath perfusion of artificial cerebral spinal fluid (aCSF) containing 0.25 mM MgCl(2), 5mM KCl and 100 µM 4-aminopyridine (LM/HK/4AP) elicited recurrent hyper-excitability in striatal medium spiny neurons (MSNs) in the form of paroxysmal depolarization shifts (PDSs) with an amplitude of 27.8 ± 2.1 mV and a duration of 29.4 ± 3.7s. PDSs coincided with SLEs in the striatal network with an amplitude of 106.5 ± 11.3 µV, duration of 23.6 ± 3.2 s, and a spiking frequency of 7.9 ± 1.3 Hz. Notably, chemostimulant-induced MSN PDSs were predominantly observed at earlier ages (P7-11), whereas occurrence of MSN PDSs declined to 50% by P12 and were no longer noted after P14; antagonism of the cannabinoid receptor (CB1) with 10 µM LY 320135 along with perfusion of LM/HK/4AP in older animals (P14-15) was unable to elicit MSN PDSs and SLEs. PDSs and SLEs were blocked with 60 µM 2-amino-5-phosphonopentanoate (APV), an N-methyl-d-aspartate receptor (NMDAR) blocker, or with traditional anticonvulsants such as 100 µM phenytoin or 50 µM carbamazepine. Conversely, blockade of 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid receptors (AMPARs) with 10 µM CNQX or T- and L-type Ca(2+) channels with 50 µM NiCl(2) or 50 µM nimodipine, respectively, did not significantly change MSN PDS and SLE amplitudes, durations and frequencies seen with LM/HK/4AP treatment alone. Striatal SLEs were driven by MSN hyper-excitability and synchrony since neither the presence of 1µM scopolamine, a muscarinic acetylcholine (ACh) receptor inhibitor, nor selective inhibition of fast-spiking interneurons (FSIs) with 50µM IEM1460 had any significant effect on MSN PDSs and SLEs. Next, we physically isolated the striatum from cortical and thalamic input and found that the striatum was intrinsically capable of manifesting NMDAR-dependent SLEs. Altogether, the present study is the first to deconstruct how SLEs can form in the striatum by examining how MSN activity coincides with SLEs. It also highlights a previously unrecognized potential for the striatum to manifest SLEs in vitro, without involving the cortex and thalamus. From these findings, further hypotheses can be developed for studying the BG's role in seizure generation and propagation, which may lead to novel pharmacological targets for the treatment of epilepsy.

Elevated potassium elicits recurrent surges of large GABAA-receptor-mediated post-synaptic currents in hippocampal CA3 pyramidal neurons.

Previously, we found that rat hippocampal CA3 interneurons become hyperactive with increasing concentrations of extracellular K(+) up to 10 mM. However, it is unclear how this enhanced interneuronal activity affects pyramidal neurons. Here we voltage-clamped rat hippocampal CA3 pyramidal neurons in vitro at 0 mV to isolate γ-aminobutyric acid (GABA)-activated inhibitory post-synaptic currents (IPSCs) and measured these in artificial cerebrospinal fluid (aCSF) and with 10 mM K(+) bath perfusion. In aCSF, small IPSCs were present with amplitudes of 0.053 ± 0.007 nA and a frequency of 0.27 ± 0.14 Hz. With 10 mM K(+) perfusion, IPSCs increased greatly in frequency and amplitude, culminating in surge events with peak amplitudes of 0.56 ± 0.08 nA, that appeared and disappeared cyclically with durations lasting 2.02 ± 0.37 min repeatedly, up to 10 times over a 30-min bath perfusion of elevated K(+). These large IPSCs were GABA(A)-receptor mediated and did not involve significant desensitization of this receptor. Perfusion of a GABA transporter inhibitor (NO-711), glutamate receptor inhibitors CNQX and APV, or a gap junctional blocker (carbenoxolone) prevented the resurgence of large IPSCs. Pressure ejected sucrose resulted in the abolishment of subsequent surges. No elevated K(+)-mediated surges were observed in CA3 interneurons from the stratum oriens layer. In conclusion, these cyclic large IPSC events observable in CA3 pyramidal neurons in 10 mM KCl may be due to transient GABA depletion from continuously active interneuronal afferents.

Characterizing the persistent CA3 interneuronal spiking activity in elevated extracellular potassium in the young rat hippocampus.

Seizures coincide with an increase in extracellular potassium concentrations [K(+)](e) yet little information is available regarding this phenomenon on the firing pattern, frequency and neuronal properties of inhibitory neurons responsible for modulating network excitability. Therefore, we investigated the effects of elevating [K(+)](e) from 2.5 to 12.5mM on CA3 rat hippocampal interneurons in vitro using whole-cell patch-clamp recordings. We found that the majority of interneurons (21/25) in artificial cerebral spinal fluid (aCSF) exhibited spontaneous tonic spiking activity. As the [K(+)](e) increased to 12.5mM, interneurons exhibited a tonic, irregular, burst firing activity, or a combination of these. The input resistance decreased significantly to 59+/-18% at 7.5mM K(+) and did not further change at higher [K(+)](e) while the amount of K(+)-induced depolarization significantly increased from 5 to 12.5mM K(+) perfusion; a depolarization block occurred in 4 of the 12 interneurons at 12.5mM. Also, as [K(+)](e) increased, a transition from lower (1.3+/-0.6Hz) to higher dominant peak frequency (15.0+/-5.0Hz) was observed. We found that non-fast spiking (NFS) interneurons represented the majority of cells recorded and exhibited mostly tonic firing activity in raised K(+). Fast spiking (FS) interneurons predominately had a tonic firing pattern with very few exhibiting bursting activity in elevated K(+). In conclusion, we report that raised [K(+)](e) in amounts observed during seizures increases hippocampal CA3 interneuronal activity and suggests that a loss or impairment of inhibitory function may be present during these events.

Adenosine A1 receptor activation mediates NMDA receptor activity in a pertussis toxin-sensitive manner during normoxia but not anoxia in turtle cortical neurons.

Adenosine is a defensive metabolite that is critical to anoxic neuronal survival in the freshwater turtle. Channel arrest of the N-methyl-d-aspartate receptor (NMDAR) is a hallmark of the turtle's remarkable anoxia tolerance and adenosine A1 receptor (A1R)-mediated depression of normoxic NMDAR activity is well documented. However, experiments examining the role of A1Rs in regulating NMDAR activity during anoxia have yielded inconsistent results. The aim of this study was to examine the role of A1Rs in the normoxic and anoxic regulation of turtle brain NMDAR activity. Whole-cell NMDAR currents were recorded for up to 2 h from turtle cortical pyramidal neurons exposed to pharmacological A1R or Gi protein modulation during normoxia (95% O(2)/5% CO2) and anoxia (95% N2/5% CO2). NMDAR currents were unchanged during normoxia and decreased 51+/-4% following anoxic exposure. Normoxic agonism of A1Rs with adenosine or N6-cyclopentyladenosine (CPA) decreased NMDAR currents 57+/-11% and 59+/-6%, respectively. The A1R antagonist 8-cyclopentyl-1,3-dimethylxanthine (DPCPX) had no effect on normoxic NMDAR currents and prevented the adenosine and CPA-mediated decreases in NMDAR activity. DPCPX partially reduced the anoxic decrease at 20 but not 40 min of treatment. The Gi protein inhibitor pertussis toxin (PTX) prevented both the CPA and anoxia-mediated decreases in NMDAR currents and calcium chelation or blockade of mitochondrial ATP-sensitive K+ channels also prevented the CPA-mediated decreases. Our results suggest that the long-term anoxic decrease in NMDAR activity is activated by a PTX-sensitive mechanism that is independent of A1R activity.

Mitochondrial ATP-sensitive K+ channels regulate NMDAR activity in the cortex of the anoxic western painted turtle.

Hypoxic mammalian neurons undergo excitotoxic cell death, whereas painted turtle neurons survive prolonged anoxia without apparent injury. Anoxic survival is possibly mediated by a decrease in N-methyl-d-aspartate receptor (NMDAR) activity and maintenance of cellular calcium concentrations ([Ca(2+)](c)) within a narrow range during anoxia. In mammalian ischaemic models, activation of mitochondrial ATP-sensitive K(+) (mK(ATP)) channels partially uncouples mitochondria resulting in a moderate increase in [Ca(2+)](c) and neuroprotection. The aim of this study was to determine the role of mK(ATP) channels in anoxic turtle NMDAR regulation and if mitochondrial uncoupling and [Ca(2+)](c) changes underlie this regulation. In isolated mitochondria, the K(ATP) channel activators diazoxide and levcromakalim increased mitochondrial respiration and decreased ATP production rates, indicating mitochondria were 'mildly' uncoupled by 10-20%. These changes were blocked by the mK(ATP) antagonist 5-hydroxydecanoic acid (5HD). During anoxia, [Ca(2+)](c) increased 9.3 +/- 0.3% and NMDAR currents decreased 48.9 +/- 4.1%. These changes were abolished by K(ATP) channel blockade with 5HD or glibenclamide, Ca(2+)(c) chelation with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or by activation of the mitochondrial Ca(2+) uniporter with spermine. Similar to anoxia, diazoxide or levcromakalim increased [Ca(2+)](c) 8.9 +/- 0.7% and 3.8 +/- 0.3%, while decreasing normoxic whole-cell NMDAR currents by 41.1 +/- 6.7% and 55.4 +/- 10.2%, respectively. These changes were also blocked by 5HD or glibenclamide, BAPTA, or spermine. Blockade of mitochondrial Ca(2+)-uptake decreased normoxic NMDAR currents 47.0 +/- 3.1% and this change was blocked by BAPTA but not by 5HD. Taken together, these data suggest mK(ATP) channel activation in the anoxic turtle cortex uncouples mitochondria and reduces mitochondrial Ca(2+) uptake via the uniporter, subsequently increasing [Ca(2+)](c) and decreasing NMDAR activity.

AMPA receptors undergo channel arrest in the anoxic turtle cortex.

Without oxygen, all mammals suffer neuronal injury and excitotoxic cell death mediated by overactivation of the glutamatergic N-methyl-D-aspartate receptor (NMDAR). The western painted turtle can survive anoxia for months, and downregulation of NMDAR activity is thought to be neuroprotective during anoxia. NMDAR activity is related to the activity of another glutamate receptor, the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR). AMPAR blockade is neuroprotective against anoxic insult in mammals, but the role of AMPARs in the turtle's anoxia tolerance has not been investigated. To determine whether AMPAR activity changes during hypoxia or anoxia in the turtle cortex, whole cell AMPAR currents, AMPAR-mediated excitatory postsynaptic potentials (EPSPs), and excitatory postsynaptic currents (EPSCs) were measured. The effect of AMPAR blockade on normoxic and anoxic NMDAR currents was also examined. During 60 min of normoxia, evoked peak AMPAR currents and the frequencies and amplitudes of EPSPs and EPSCs did not change. During anoxic perfusion, evoked AMPAR peak currents decreased 59.2 +/- 5.5 and 60.2 +/- 3.5% at 20 and 40 min, respectively. EPSP frequency (EPSP(f)) and amplitude decreased 28.7 +/- 6.4% and 13.2 +/- 1.7%, respectively, and EPSC(f) and amplitude decreased 50.7 +/- 5.1% and 51.3 +/- 4.7%, respectively. In contrast, hypoxic (Po(2) = 5%) AMPAR peak currents were potentiated 56.6 +/- 20.5 and 54.6 +/- 15.8% at 20 and 40 min, respectively. All changes were reversed by reoxygenation. AMPAR currents and EPSPs were abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In neurons pretreated with CNQX, anoxic NMDAR currents were reversibly depressed by 49.8 +/- 7.9%. These data suggest that AMPARs may undergo channel arrest in the anoxic turtle cortex.

Calcium and protein phosphatase 1/2A attenuate N-methyl-D-aspartate receptor activity in the anoxic turtle cortex.

Excitotoxic cell death (ECD) is characteristic of mammalian brain following min of anoxia, but is not observed in the western painted turtle following days to months without oxygen. A key event in ECD is a massive increase in intracellular Ca(2+) by over-stimulation of N-methyl-d-aspartate receptors (NMDARs). The turtle's anoxia tolerance may involve the prevention of ECD by attenuating NMDAR-induced Ca(2+) influx. The goal of this study was to determine if protein phosphatases (PPs) and intracellular calcium mediate reductions in turtle cortical neuron whole-cell NMDAR currents during anoxia, thereby preventing ECD. Whole-cell NMDAR currents did not change during 80 min of normoxia, but decreased 56% during 40 min of anoxia. Okadaic acid and calyculin A, inhibitors of serine/threonine PP1 and PP2A, potentiated NMDAR currents during normoxia and prevented anoxia-mediated attenuation of NMDAR currents. Decreases in NMDAR activity during anoxia were also abolished by inclusion of the Ca(2+) chelator -- BAPTA and the calmodulin inhibitor -- calmidazolium. However, cypermethrin, an inhibitor of the Ca(2+)/calmodulin-dependent PP2B (calcineurin), abolished the anoxic decrease in NMDAR activity at 20, but not 40 min suggesting that this phosphatase might play an early role in attenuating NMDAR activity during anoxia. Our results show that PPs, Ca(2+) and calmodulin play an important role in decreasing NMDAR activity during anoxia in the turtle cortex. We offer a novel mechanism describing this attenuation in which PP1 and 2A dephosphorylate the NMDAR (NR1 subunit) followed by calmodulin binding, a subsequent dissociation of alpha-actinin-2 from the NR1 subunit, and a decrease in NMDAR activity.

Effect of anoxia and pharmacological anoxia on whole-cell NMDA receptor currents in cortical neurons from the western painted turtle.

The mammalian brain undergoes rapid cell death during anoxia that is characterized by uncontrolled Ca(2+) entry via N-methyl-D-aspartate receptors (NMDARs). In contrast, the western painted turtle is extremely anoxia tolerant and maintains close-to-normal [Ca(2+)](i) during periods of anoxia lasting from days to months. A plausible mechanism of anoxic survival in turtle neurons is the regulation of NMDARs to prevent excitotoxic Ca(2+) injury. However, studies using metabolic inhibitors such as cyanide (NaCN) as a convenient method to induce anoxia may not represent a true anoxic stress. This study was undertaken to determine whether turtle cortical neuron whole-cell NMDAR currents respond similarly to true anoxia with N(2) and to NaCN-induced anoxia. Whole-cell NMDAR currents were measured during a control N(2)-induced anoxic transition and a control NaCN-induced transition. During anoxia with N(2) normalized, NMDAR currents decreased to 35.3%+/-10.8% of control values. Two different NMDAR current responses were observed during NaCN-induced anoxia: one resulted in a 172%+/-51% increase in NMDAR currents, and the other was a decrease to 48%+/-14% of control. When responses were correlated to the two major neuronal subtypes under study, we found that stellate neurons responded to NaCN treatment with a decrease in NMDAR current, while pyramidal neurons exhibited both increases and decreases. Our results show that whole-cell NMDAR currents respond differently to NaCN-induced anoxia than to the more physiologically relevant anoxia with N(2).

Gap junctions do not underlie changes in whole-cell conductance in anoxic turtle brain.

An acute reduction in cell membrane permeability could provide an effective strategy to prolong anoxic survival. A previous study has shown that in the western painted turtle whole-cell neuronal conductance (G(w)) decreases during anoxia, which may be mediated by the activation of adenosine A(1) receptors and calcium. Reduction in G(w) is thought to be the result of ion channel closure, but closure of gap junctions could also be responsible for this phenomenon. In our study, antibody staining of connexin 32 and 43 (Cx32 and Cx43) suggested the presence of gap junctional components in the turtle cortex. To examine if gap junctions were involved in the previously measured anoxic decrease in G(w), neuronal connectivity was assessed through the measurement of whole-cell capacitance (C(w)). Turtle cortical sheets were perfused with normoxic (95%O(2)/5%CO(2)), anoxic (95%N(2)/5%CO(2)), high calcium (4 mM) and adenosine (200 microm) artificial cerebral spinal fluid (aCSF). No significant change in C(w) was observed under any of the above conditions. However, during hypo-osmotic aCSF perfusion C(w) decreased significantly, with the lowest value of 50+/-10.4 pF (P<0.05) occurring at 30 min. To visualize changes in gap junction permeability lucifer yellow was loaded into turtle neurons during normoxic, anoxic, 0 calcium, hypo-osmotic, cold shock, (+)-isoproterenol, nitric oxide donor S-nitoso-acetyl penicillamine, and 8-bromo-guanosine 3',5'-cyclic monophosphate aCSF perfusion. Dye propagation was only observed in 3 of 20 cold shock experiments (4 degrees C). We conclude that gap junctions are not involved in the acute reduction in G(w) previously observed during anoxia and that our results support the hypothesis that ion channel arrest is involved.