Activation of ryanodine receptors is required for PKA‐mediated downregulation of A‐type K+ channels in rat hippocampal neurons

A‐type K⁺ channels (I_A channels) contribute to learning and memory mechanisms by regulating neuronal excitabilities in the CNS, and their expression level is targeted by Ca²⁺ influx via synaptic NMDA receptors (NMDARs) during long‐term potentiation (LTP). However, it is not clear how local synaptic Ca²⁺ changes induce I_A downregulation throughout the neuron, extending from the active synapse to the soma. In this study, we tested if two major receptors of endoplasmic reticulum (ER), ryanodine (RyRs), and IP₃ (IP₃R) receptors, are involved in Ca²⁺‐mediated I_A downregulation in cultured hippocampal neurons of rats. The downregulation of I_A channels was induced by doubling the Ca²⁺ concentration in culture media (3.6 mM for 24 hrs) or treating with glycine (200 μM for 3 min) to induce chemical LTP (cLTP), and the changes in I_A peaks were measured electrophysiologically by a whole‐cell patch. We confirmed that Ca²⁺ or glycine treatment significantly reduced I_A peaks and that their effects were abolished by blocking NMDARs or voltage‐dependent Ca²⁺ channels (VDCCs). In this cellular processing, blocking RyRs (by ryanodine, 10 μM) but not IP₃Rs (by 2APB, 100 μM) completely abolished I_A downregulation, and the LTP observed in hippocampal slices was more diminished by ryanodine rather than 2APB. Furthermore, blocking RyRs also reduced Ca²⁺‐mediated PKA activation, indicating that sequential signaling cascades, including the ER and PKA, are involved in regulating I_A downregulation. These results strongly suggest a possibility that RyR contribution and mediated I_A downregulation are required to regulate membrane excitability as well as synaptic plasticity in CA3‐CA1 connections of the hippocampus. © 2017 Wiley Periodicals, Inc.     

Astrocytic IP3Rs: Contribution to Ca2+ signalling and hippocampal LTP

Astrocytes regulate hippocampal synaptic plasticity by the Ca²⁺ dependent release of the N‐methyl d ‐aspartate receptor (NMDAR) co‐agonist d ‐serine. Previous evidence indicated that d ‐serine release would be regulated by the intracellular Ca²⁺ release channel IP₃ receptor (IP₃R), however, genetic deletion of IP₃R2, the putative astrocytic IP₃R subtype, had no impact on synaptic plasticity or transmission. Although IP₃R2 is widely believed to be the only functional IP₃R in astrocytes, three IP₃R subtypes (1, 2, and 3) have been identified in vertebrates. Therefore, to better understand gliotransmission, we investigated the functionality of IP₃R and the contribution of the three IP₃R subtypes to Ca²⁺ signalling. As a proxy for gliotransmission, we found that long‐term potentiation (LTP) was impaired by dialyzing astrocytes with the broad IP₃R blocker heparin, and rescued by exogenous d ‐serine, indicating that astrocytic IP₃Rs regulate d ‐serine release. To explore which IP₃R subtypes are functional in astrocytes, we used pharmacology and two‐photon Ca²⁺ imaging of hippocampal slices from transgenic mice (IP₃R2^?/? and IP₃R2^?/?;3^?/?). This approach revealed that underneath IP₃R2‐mediated global Ca²⁺ events are an overlooked class of IP₃R‐mediated local events, occurring in astroglial processes. Notably, multiple IP₃Rs were recruited by high frequency stimulation of the Schaffer collaterals, a classical LTP induction protocol. Together, these findings show the dependence of LTP and gliotransmission on Ca²⁺ release by astrocytic IP₃Rs. GLIA 2017;65:502–513     

Apamin induces plastic changes in hippocampal neurons in senile Sprague-Dawley rats

Apamin is a neurotoxin extracted from honey bee venom and is a selective blocker of small‐conductance Ca²⁺‐activated K⁺ channels (SK). Several behavioral and electrophysiological studies indicate that SK‐blockade by apamin may enhance neuron excitability, synaptic plasticity, and long‐term potentiation in the CA1 hippocampal region, and, for that reason, apamin has been proposed as a therapeutic agent in Alzheimer's disease treatment. However, the dendritic morphological mechanisms implied in such enhancement are unknown. In the present work, Golgi–Cox stain protocol and Sholl analysis were used to study the effect of apamin on the dendritic morphology of pyramidal neurons from hippocampus and the prefrontal cortex as well as on the medium spiny neurons from the nucleus accumbens and granule cells from the dentate gyrus (DG) of the hippocampus. We found that only granule cells from the DG and pyramidal neurons from dorsal and ventral hippocampus were altered in senile rats injected with apamin. Our research suggests that apamin may increase the dendritic morphology in the hippocampus, which could be related to the neuronal excitability and synaptic plasticity enhancement induced by apamin. Synapse 2011. © 2011 Wiley‐Liss, Inc.     

Different characteristics of cell volume and intracellular calcium ion concentration dynamics between the hippocampal CA1 and lateral cerebral cortex of male mouse brain slices during exposure to hypotonic stress

The mechanism of brain edema is complex and still remains unclear. Our aim was to investigate the regional differences of cell volume and intracellular Ca²⁺ concentration ([Ca²⁺]_i) dynamics during hypotonic stress in male mouse hemi‐brain slices. Brain slices were loaded with the fluorescence Ca²⁺ indicator fura‐2, and cell volume and [Ca²⁺]_i in the lateral cerebral cortex (LCC) and hippocampal CA1 (CA1) region were measured simultaneously during exposure to hypotonic stress using Ca²⁺ insensitive (F360) and Ca²⁺ sensitive fluorescence (F380), respectively. Brain cell swelling induced by hypotonic stress was followed by a regulatory volume change that coincided with an increase in [Ca²⁺]_i. The degrees of change in cell volume and [Ca²⁺]_i were significantly different between the LCC and CA1. The increase in cell volume and [Ca²⁺]_i in the LCC, but not in the CA1, was decreased by the transient receptor potential channel blockers LaCl₃ and GdCl₃. The increase in [Ca²⁺]_i in both the LCC and CA1, was significantly decreased by the intracellular Ca²⁺ modulators thapsigargin and xestospongin C. The K⁺ channel activator isoflurane and Cl^‐ channel blocker NPPB significantly decreased [Ca²⁺]_i in the LCC. This study demonstrated that, between cells located in the LCC and in the CA1, the characteristics of brain edema induced by hypotonic stress are different. This can be ascribed to the different contribution of volume sensitive G‐protein coupled receptor and stretch sensitive Ca²⁺ channels.     

Hyperforin modulates dendritic spine morphology in hippocampal pyramidal neurons by activating Ca2+‐permeable TRPC6 channels

The standardized extract of the St. John's wort plant (Hypericum perforatum ) is commonly used to treat mild to moderate depression. Its active constituent is hyperforin, a phloroglucinol derivative that reduces the reuptake of serotonin and norepinephrine by increasing intracellular Na⁺ concentration through the activation of nonselective cationic TRPC6 channels. TRPC6 channels are also Ca²⁺‐permeable, resulting in intracellular Ca²⁺ elevations. Indeed, hyperforin activates TRPC6‐mediated currents and Ca²⁺ transients in rat PC12 cells, which induce their differentiation, mimicking the neurotrophic effect of nerve growth factor. Here, we show that hyperforin modulates dendritic spine morphology in CA1 and CA3 pyramidal neurons of hippocampal slice cultures through the activation of TRPC6 channels. Hyperforin also evoked intracellular Ca²⁺ transients and depolarizing inward currents sensitive to the TRPC channel blocker La³⁺, thus resembling the actions of the neurotrophin brain‐derived neurotrophic factor (BDNF) in hippocampal pyramidal neurons. These results suggest that the antidepressant actions of St. John's wort are mediated by a mechanism similar to that engaged by BDNF. © 2012 Wiley Periodicals, Inc.     

Enhancement in activities of large conductance calcium-activated potassium channels in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia

It has been reported previously that the neuronal excitability persistently suppresses and the amplitude of fast afterhyperpolarization (fAHP) increases in CA1 pyramidal cells of rat hippocampus following transient forebrain ischemia. To understand the conductance mechanisms underlying these post-ischemic electrophysiological alterations, we compared differences in activities of large conductance Ca²⁺-activated potassium (BK_Ca) channels in CA1 pyramidal cells acutely dissociated from hippocampus before and after ischemia by using inside-out configuration of patch clamp techniques. (1) The unitary conductance of BK_Ca channels in post-ischemic neurons (295 pS) was higher than that in control neurons (245 pS) in symmetrical 140/140 mM K⁺ in inside-out patch; (2) the membrane depolarization for an e-fold increase in open probability (P_o) showed no significant differences between two groups while the membrane potential required to produce one-half of the maximum P_o was more negative after ischemia, indicating no obvious changes in channel voltage dependence; (3) the [Ca²⁺]_i required to half activate BK_Ca channels was only 1 μM in post-ischemic whereas 2 μM in control neurons, indicating an increase in [Ca²⁺]_i sensitivity after ischemia; and (4) BK_Ca channels had a longer open time and a shorter closed time after ischemia without significant differences in open frequency as compared to control. The present results indicate that enhanced activity of BK_Ca channels in CA1 pyramidal neurons after ischemia may partially contribute to the post-ischemic decrease in neuronal excitability and increase in fAHP.     

Non‐classical mechanism of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor channel block by fluoxetine

Antidepressants have many targets in the central nervous system. A growing body of data demonstrates the influence of antidepressants on glutamatergic neurotransmission. In the present work, we studied the inhibition of native Ca²⁺‐permeable and Ca²⁺‐impermeable α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors in rat brain neurons by fluoxetine. The Ca²⁺‐impermeable AMPA receptors in CA1 hippocampal pyramidal neurons were weakly affected. The IC₅₀ value for the inhibition of Ca²⁺‐permeable AMPA receptors in giant striatal interneurons was 43 ± 7 μm . The inhibition of Ca²⁺‐permeable AMPA receptors was voltage dependent, suggesting deep binding in the pore. However, the use dependence of fluoxetine action differed markedly from that of classical AMPA receptor open‐channel blockers. Moreover, fluoxetine did not compete with other channel blockers. In contrast to fluoxetine, its membrane‐impermeant quaternary analog demonstrated all of the features of channel inhibition typical for open‐channel blockers. It is suggested that fluoxetine reaches the binding site through a hydrophobic access pathway. Such a mechanism of block is described for ligands of sodium and calcium channels, but was never found in AMPA receptors. Molecular modeling suggests binding of fluoxetine in the subunit interface; analogous binding was proposed for local anesthetics in closed sodium channels and for benzothiazepines in calcium channels.     

IP3 mobilization and diffusion determine the timing window of Ca2+ release by synaptic stimulation and a spike in rat CA1 pyramidal cells

Synaptically activated calcium release from internal stores in CA1 pyramidal neurons is generated via metabotropic glutamate receptors by mobilizing IP₃. Ca²⁺ release spreads as a large amplitude wave in a restricted region of the apical dendrites of these cells. These Ca²⁺ waves have been shown to induce certain forms of synaptic potentiation and have been hypothesized to affect other forms of plasticity. Pairing a single backpropagating action potential (bAP) with repetitive synaptic stimulation evokes Ca²⁺ release when synaptic stimulation alone is subthreshold for generating release. We examined the timing window for this synergistic effect under conditions favoring Ca²⁺ release. The window, measured from the end of the train, lasted 250–500 ms depending on the duration of stimulation tetanus. The window appears to correspond to the time when both IP₃ concentration and [Ca²⁺]_i are elevated at the site of the IP₃ receptor. Detailed analysis of the mechanisms determining the duration of the window, including experiments using different forms of caged IP₃ instead of synaptic stimulation, suggest that the most significant processes are the time for IP₃ to diffuse away from the site of generation and the time course of IP₃ production initiated by activation of mGluRs. IP₃ breakdown, desensitization of the IP₃ receptor, and the kinetics of IP₃ unbinding from the receptor may affect the duration of the window but are less significant. The timing window is short but does not appear to be short enough to suggest that this form of coincidence detection contributes to conventional spike timing‐dependent synaptic plasticity in these cells. © 2009 Wiley‐Liss, Inc.     

Bradykinin regulates the expression of claudin‐5 in brain microvascular endothelial cells via calcium‐induced calcium release

To investigate the mechanism underlying the regulation of claudin‐5, a tight junction protein that participates primarily in the constitution of the blood–brain barrier by bradykinin (BK), we established a primary culture of rat brain microvascular endothelial cells (BMECs). BMECs were treated with 10^?5 M BK, and changes in the intracellular Ca²⁺ levels were measured by using the sensitive fluorescent dye fluo‐3; the expression and distribution of claudin‐5 were investigated by immunocytochemistry and Western blot analyses. We did not detect any expression of bradykinin B2 receptors in the BMECs or freshly isolated rat brain microvessels. We found that 10^?5 M BK triggered Ca²⁺ transients in BMECs, and further investigations revealed that inositol 1,4,5‐trisphosphate receptors (IP₃Rs) and ryanodine receptors (RyRs) on the endoplasmic reticulum (ER) were responsible for the Ca²⁺ fluctuation. Consequently, these intracellular Ca²⁺ changes that occur in response to BK application were identified as Ca²⁺‐induced Ca²⁺ release (CICR). Immunocytochemistry and Western blot results demonstrated that 10^?5 M BK could cause the internalization and a decrease in the expression of claudin‐5; agonists of IP₃Rs and RyRs, such as IP₃ and caffeine, enhanced the BK‐induced downregulation of claudin‐5, whereas antagonists of IP₃Rs and RyRs, such as 2‐APB and ryanodine, abrogated BK's effect on claudin‐5. In conclusion, the BK‐induced CICR in primary culture BMECs might be the mechanism by which BK modulates claudin‐5. © 2014 Wiley Periodicals, Inc.     

Correlation of anoxic neuronal responses and calbindin-D28k localization in stratum pyramidale of rat hippocampus

Immunohistochemical staining for the calcium-binding protein calbindin-D_28k (CaBP) was combined with Lucifer Yellow (LY) identification and intracellular recording of changes in membrane parameters of pyramidal neurons in CA2, CA1, and the sebiculum of rat hippocampal slices during brief exposure (4.0 ± 0.19 min) to N₂. Anoxia evoked either a depolarization or hyperpolarization of membrane potential (V_M) (+21.5 ± 2.79 mV above V_M = ?70.5 ± 1.50 mV, n = 30 and ?7.2 ± 0.72 mV below V_M = ?68.2 ± 1.34 mV, n = 24, respectively) and a fall in membrane resistance of =20%. Differences in the response could be correlated with the presence or absence of CaBP and the localization of neurons in different layers of stratum pyramidale and sectors of the hippocampus. For neurons immunopositive for calbindin (CaBP(⁺)), depolarization was observed more frequently (83%) than hyperpolarization (17%); in contrast, 44% of responses of calbindin-negative (CaBP(^?)) neurons were depolarizing and 56% were hyperpolarizing. Depolarizations of CaBP(⁺) neurons were more gradual in slope, and more rapidly reached a plateau in comparison with those recorded in CaBP(^?) neurons. Responses of neurons in the superficial layer of stratum pyramidale (in which 79% of CaBP(⁺) pyramidal neurons were situated) were mainly depolarizing (91%), while for those in the deep layer (which contained 89% of the CaBP(^?) cells) such responses were observed less often (45%). Depolarization was also more common than hyperpolarization for cells located in CA2/CA1c/CA1b (63%) than in the CA1a/subicular region (37%). The depolarizing response of the majority of pyramidal neurons which are CaBP(⁺), superficial, and closer to CA3 may reflect an efficient buffering of intracellular Ca²⁺, which maintains a low [Ca²⁺]_i, steep gradient for Ca²⁺ influx and may facilitate the movement of Ca²⁺ away from points of entry. The neurons which are CaBP(^?), deep, and closer to subiculum and in which N₂ evokes hyperpolarization, on the other hand, may have a sustained elevation/accumulation of cytosolic Ca²⁺ which could activate K⁺ conductance, inhibit Ca²⁺ influx, and stabilize the membrane potential. These experiments provide a functional correlate for CaBP and suggest that it may have a significant role in Ca²⁺ homeostasis and the determination of selective neuronal vulnerability. © 1995 Wiley-Liss, Inc.     

Slow-decaying presynaptic calcium dynamics gate long-lasting asynchronous release at the hippocampal mossy fiber to CA3 pyramidal cell synapse

Action potentials trigger two modes of neurotransmitter release, with a fast synchronous component and a temporally delayed asynchronous release. Asynchronous release contributes to information transfer at synapses, including at the hippocampal mossy fiber (MF) to CA3 pyramidal cell synapse where it controls the timing of postsynaptic CA3 pyramidal neuron firing. Here, we identified and characterized the main determinants of asynchronous release at the MF–CA3 synapse. We found that asynchronous release at MF–CA3 synapses can last on the order of seconds following repetitive MF stimulation. Elevating the stimulation frequency or the external Ca²⁺ concentration increased the rate of asynchronous release, thus, arguing that presynaptic Ca²⁺ dynamics is the major determinant of asynchronous release rate. Direct MF bouton Ca²⁺ imaging revealed slow Ca²⁺ decay kinetics of action potential (AP) burst-evoked Ca²⁺ transients. Finally, we observed that asynchronous release was preferentially mediated by Ca²⁺ influx through P/Q-type voltage-gated Ca²⁺ channels, while the contribution of N-type VGCCs was limited. Overall, our results uncover the determinants of long-lasting asynchronous release from MF terminals and suggest that asynchronous release could influence CA3 pyramidal cell firing up to seconds following termination of granule cell bursting.     

Plasma membrane insertion of TRPC5 channels contributes to the cholinergic plateau potential in hippocampal CA1 pyramidal neurons

In cultured hippocampal neurons, transient receptor potential 5 (TRPC5) channels are translocated and inserted into plasma membranes of hippocampal neurons to generate nonselective cation (NSC) currents. We investigated whether TRPC5 channel translocation also contributes to the generation of NSC currents underlying the afterdepolarizations and plateau potentials (PPs) in hippocampal pyramidal cells that are induced by muscarinic receptor activation. Using a biotinylation assay to quantify the change in surface membrane proteins in acute hippocampal slices, we found that muscarinic stimulation significantly enhanced the levels of TRPC5 protein on the membrane surface but not those of TRPC1 or TRPC4 channels. We then investigated the pharmacological sensitivity of the cation current observed during muscarinic stimulation to determine if a component could be due to TRPC5 channels. The TRPC channel antagonists 2‐APB and SKF96365 strongly depressed the generation of PPs, the underlying tail currents (I_tail) and the associated dendritic Ca²⁺ influx induced by muscarinic receptor activation in pyramidal neurons. High intracellular concentrations of ATP, which specifically inhibit TRPC5 channels, depressed I_tail. In addition, pretreatment with the calmodulin (CaM) inhibitor W‐7, which depresses recombinant TRPC5 currents, inhibited both the cation current (I_tail) and the surface insertion of TRPC5 channels. Finally, the phosphatidylinositide 3‐kinase (PI₃K) inhibitor wortmannin, which blocks translocation of TRPC5 channels in cell culture, also inhibited both the I_tail and the surface insertion of TRPC5 channels. Therefore, we conclude that insertion of TRPC5 channels contributes to the generation of the prolonged afterdepolarizations following muscarinic stimulation. This altered plasma membrane expression of TRPC5 channels in pyramidal neurons may play an important role in the generation of prolonged neuronal depolarization and bursting during the epileptiform seizure discharges of epilepsy. © 2010 Wiley‐Liss, Inc.     

Effect of charybdotoxin and leiurotoxon I on potassium currents in bullfrog sympathetic ganglion and hippocampal neurons

The effects of charybdotoxin and leiurotoxin I were examined on several classes of K⁺ currents in bullfrog sympathetic ganglion and hippocampal CA1 pyramidal neurons. Highly purified preparations of charybdotoxin selectively blocked a large voltage- and Ca²⁺-dependent K⁺ current (I_c) responsible for action potential repolarization (IC₅₀ = 6 nM) while leiurotoxin I selectively blocked a small Ca²⁺-dependent K⁺ conductance (I_AHP) responsible for the slow afterhyperpolarization following an action potential (IC₅₀ = 7.5 nM) in bullfrog sympathetic ganglion neurons. Neither of the toxins had a significant effects on other K⁺ currents (M-current [I_M], A-current [I_A] and the delayed rectifier [I_KD] present in these cells. Leiurotoxin I at a concentration of 20 nM had no detectable effect on currents in hippocampal CA1 pyramidal neurons. This lack of effect on I_AHP in central neurons suggests that the channels underlying slow AHPs in those neurons are pharmacologically distinct from analogous channels in peripheral neurons.     

Ca2+‐dependent release of ATP from astrocytes affects herpes simplex virus type 1 infection of neurons

Astrocytes provide metabolic support for neurons and modulate their functions by releasing a plethora of neuroactive molecules diffusing to neighboring cells. Here we report that astrocytes also play a role in cortical neurons' vulnerability to Herpes simplex virus type‐1 (HSV‐1) infection through the release of extracellular ATP. We found that the interaction of HSV‐1 with heparan sulfate proteoglycans expressed on the plasma membrane of astrocytes triggered phospholipase C‐mediated IP₃‐dependent intracellular Ca²⁺ transients causing extracellular release of ATP. ATP binds membrane purinergic P2 receptors (P2Rs) of both neurons and astrocytes causing an increase in intracellular Ca²⁺ concentration that activates the Glycogen Synthase Kinase (GSK)‐3β, whose action is necessary for HSV‐1 entry/replication in these cells. Indeed, in co‐cultures of neurons and astrocytes HSV‐1‐infected neurons were only found in proximity of infected astrocytes releasing ATP, whereas in the presence of fluorocitrate, an inhibitor of astrocyte metabolism, switching‐off the HSV‐1‐induced ATP release, very few neurons were infected. The addition of exogenous ATP, mimicking that released by astrocytes after HSV‐1 challenge, restored the ability of HSV‐1 to infect neurons co‐cultured with metabolically‐inhibited astrocytes. The ATP‐activated, P2R‐mediated, and GSK‐3‐dependent molecular pathway underlying HSV‐1 infection is likely shared by neurons and astrocytes, given that the blockade of either P2Rs or GSK‐3 activation inhibited infection of both cell types. These results add a new layer of information to our understanding of the critical role played by astrocytes in regulating neuronal functions and their response to noxious stimuli including microbial agents via Ca²⁺‐dependent release of neuroactive molecules.     

Plasticity of L‐type Ca2+ channels after cocaine withdrawal

Increased reactivity of certain frontal cortical brain regions to cocaine re‐exposure or drug‐associated cues in cocaine‐abstinent human addicts is linked to drug craving. Similarly, in rats tested after withdrawal from repeated cocaine exposure, cocaine or other strong excitatory stimuli produce greater activation of pyramidal neurons in the medial prefrontal cortex (mPFC). Our recent findings indicate that the increased mPFC neuronal activation depends primarily upon enhanced voltage‐sensitive Ca²⁺ influx, most likely through high‐voltage activated (HVA) L‐type Ca²⁺ channels, but the mechanism underlying the enhanced Ca²⁺ currents is unknown. In this study, we used a protein crosslinking assay to show that repeated cocaine injections, resulting in behavioral sensitization, increased total protein levels and cell surface expression of HVA‐Ca_v1.2 L‐type channels in pyramidal neurons in deep layers of the mPFC. These changes in Ca_v1.2 L‐channels were time dependent and subtype specific (i.e., differed from those observed for Ca_v1.3 L‐channels). Furthermore, we found enhanced PKA activity in the mPFC of cocaine‐sensitized rats that persisted for 21 days after withdrawal. PKA phosphorylation of L‐channels increases their activity, so Ca²⁺ currents after cocaine withdrawal could be enhanced as a result of both increased activity and number of HVA‐Ca_v1.2 L‐channels on the cell surface. By increasing the suprafiring threshold excitability of mPFC pyramidal neurons, excessive upregulation of HVA L‐channel activity and number may contribute to the cortical hyper‐responsiveness that enhances vulnerability to cocaine craving and relapse. More generally, our results are the first to demonstrate that repeated cocaine exposure alters the membrane trafficking of a voltage‐sensitive ion channel. Synapse 63:690–697, 2009. © 2009 Wiley‐Liss, Inc.     

Epileptiform activity induced by low Mg in cultured rat hippocampal slices

Organotypic cultured slices of the rat hippocampus undergo synaptic reorganization. Besides the establishment of reciprocal connections between area CA1 and the dentate gyrus (DG), collateral excitatory connections between granule cells are formed which are similar to those appearing in several epilepsy models and in the DG from patients with temporal lobe epilepsy. We studied the characteristics of epileptiform activity induced by low Mg²⁺ perfusion in cultured hippocampal slices using extra- and intracellular recordings. With low Mg²⁺ perfusion synchronous seizure like events (SLEs) were readily observed in the DG and areas CA3 and CA1. Also, the isolated DG was able to display seizure like activity. Intracellular recordings revealed long lasting depolarization shifts in granule cells of the DG and pyramidal cells of areas CA3 and CA1. The SLEs, lasting 2–3 s, could be recorded for at least 3 h in areas CA1 and CA3. However, approximately an hour after perfusion with low Mg²⁺, the epileptiform activity disappeared in the DG and responses to single pulse hilar stimulation progressively deteriorated. These responses returned to control values 1 week after reincubating the cultures. Interestingly, no deterioration of stimulus induced responses was observed in the isolated DG after exposure to low Mg²⁺.     

Preferential inhibition of L- and N-type calcium channels in the rat hippocampal neurons by cilnidipine

The effect of a dihydropyridine Ca²⁺ antagonist, cilnidipine, on voltage-dependent Ca²⁺ channels was studied in acutely dissociated rat CA1 pyramidal neurons using the nystatin-perforated patch recording configuration under voltage-clamp conditions. Cilnidipine had no effect on low-voltage-activated (LVA) Ca²⁺ channels at the low concentrations under 10⁻⁶ M. On the other hand, cilnidipine inhibited the high-voltage-activated (HVA) Ca²⁺ current (I_Ca) in a concentration-dependent manner and the inhibition curve showed a step-wise pattern; cilnidipine selectively reduced only L-type HVA I_Ca at the low concentrations under 10⁻⁷ and 10⁻⁶ M cilnidipine blocked not only L- but also N-type HVA I_Ca. At the high concentration over 10⁻⁶ M cilnidipine non-selectively blocked the T-type LVA and P/Q- and R-type HVA Ca²⁺ channels. This is the first report that cilnidipine at lower concentration of 10⁻⁶ M blocks both L- and N-type HVA I_Ca in the hippocampal neurons.     

Psychosine blocks quisqualate-induced glutamate excitotoxicity in hippocampal CA sector neurons

Quisqualate is a potent specific agonist for Group 1 metabotropic glutamate receptors (mGluR's), that activate G protein-coupled phospholipase C (PLC) in a molecular signal-transduction mechanism that raises cytoplasmic Ca²⁺ and, when excessive, damages hippocampal neurons. Psychosine (β-galactosylsphingosine), a cationic lysosphingolipid occurring naturally in nervous tissues, dose-dependently inhibited PLC activation induced by metabotropic α₁-adrenergic receptor signaling in cultured rat brain astrocytes in vitro. In the present study, we have tested neuroprotective efficacy of psychosine in vivo, in a rat model of glutamate excitotoxicity induced by intracerebroventricular (i.c.v.) administration of quisqualate. A sublethal i.c.v. dose of quisqualate caused episodes of prolonged akinesia and convulsions, and major damage to pyramidal neurons of the hippocampal CA1 and CA3 sector, but not to granule cell neurons of the dentate gyrus. Prior infusion of psychosine greatly attenuated quisqualate-induced behaviors, and fully prevented destruction by quisqualate of vulnerable hippocampal neurons. Psychosine may prove useful in prophylaxis of neurodegenerative disorders that arise from intensive hippocampal Group 1 mGluR stimulation.