Beta-agkistrodotoxin inhibits large-conductance calcium-activated potassium channels in rat hippocampal CA1 pyramidal neurons

Effect of β-agkistrodotoxin (β-AgTx), a presynaptic neurotoxin purified from snake venom, on large-conductance calcium-activated potassium channels (BK_Ca) was studied in rat hippocampal CA1 pyramidal neurons using inside-out configuration of patch-clamp technique. The results showed that in equimolar K⁺ (150 mM) and 1 μM intracellular Ca²⁺ conditions, internal application of β-AgTx inhibited the activity of BK_Ca by reducing open probability (P_o) of the channels in a concentration-dependent manner. High concentration (74 nM) of β-AgTx completely eliminated opening of the channels. However, 37 nM β-AgTx (at −40 mV) decreased P_o from 0.49±0.07 to 0.03±0.03, switched two open time constants (0.51±0.32 and 8.77±1.63 ms) to be a single time constant of 0.46±0.40 ms. The results indicate that inhibition of BK_Ca by β-AgTx may account for the facilitatory phase of the toxin on acetylcholine release from nerve terminals.     

Effect of nilvadipine on the voltage-dependent Ca channels in rat hippocampal CA1 pyramidal neurons

Effects of nilvadipine on the low- and high-voltage activated Ca²⁺ currents (LVA and HVA I_Ca, respectively) were compared with other organic Ca²⁺ antagonists in acutely dissociated rat hippocampal CA1 pyramidal neurons. The inhibitory effects of nilvadipine, amlodipine and flunarizine on LVA I_Ca were concentration- and use-dependent. The apparent half-maximum inhibitory concentrations (IC₅₀s) at every 1- and 30-s stimulation were 6.3×10⁻⁷ M and 1.8×10⁻⁶ M for flunarizine, 1.9×10⁻⁶ M and 7.6×10⁻⁶ M for nilvadipine, and 4.0×10⁻⁶ M and 8.0×10⁻⁶ M for amlodipine, respectively. Thus, the strength of the use-dependence was in the sequence of nilvadipine>flunarizine>amlodipine. Nilvadipine also inhibited the HVA I_Ca in a concentration-dependent manner with an IC₅₀ of 1.5×10⁻⁷ M. The hippocampal CA1 neurons were observed to have five pharmacologically distinct HVA Ca²⁺ channel subtypes consisting of L-, N-, P-, Q- and R-types. Nilvadipine selectively inhibited the L-type Ca²⁺ channel current which comprised 34% of the total HVA I_Ca. On the other hand, amlodipine non-selectively inhibited the HVA Ca²⁺ channel subtypes. These results suggest that the inhibitory effect of nilvadipine on the neuronal Ca²⁺ influx through both LVA and HVA L-type Ca²⁺ channels, in combination with the cerebral vasodilatory action, may prevent neuronal damage during ischemia.     

Opioid- and GABA(A)-receptors are co-expressed by neurons in rat brain

Pharmacological data suggest that opioids exert their excitatory action in brain indirectly, by inhibiting release of the inhibitory neurotransmitter GABA. However, it is also possible that single neuron may interact with both opioids and GABA. In the present study, we investigated whether neurons in rat midbrain and medulla express both opioid and GABA(A) receptors. Coronal sections through rat brain were double-stained using antibodies against the alpha 1 subunit of GABA(A) receptor that were combined with antibodies either against the cloned mu-opioid receptor (MOR1) or the cloned kappa-opioid receptor (KOR1). Neurons double-labeled for GABA(A) receptors and either MOR1 or KOR1 were found in many brain regions including inferior colliculus, mesencephalic trigeminal nuclei, pontine reticular nuclei and raphe interpositus nucleus. Neurons double-labeled for GABA(A) and MOR1 were observed less frequently than those labeled for GABA(A) and KOR1. Our findings provide anatomical evidence that GABAergic and opioidergic systems are closely linked and activity of the same neuron may be regulated directly by both GABA and opioids.     

Action of vasopressin on CA1 pyramidal neurons in rat hippocampal slices

The effects of arginine⁸-vasopressin (AVP) on the excitability of 47 pyramidal cells of the CA1 region of the hippocampus were determined by using intracellular recording techniques in a submerged slice preparation. Addition of 10⁻⁶ M AVP to the bathing medium evoked an increase in spike discharge which was slow in onset and only gradually reversible. The discharge was accompanied by an increase in excitatory postsynaptic potentials without significant change of the resting input resistance. AVP-induced excitation was found in 81% of ventral and 29% of dorsal hippocampal CA1 pyramidal cells. In low Ca²⁺, high Mg²⁺ solution this excitatory action by AVP was blocked. Microiontophoretic application of AVP onto apical or basal dendrites or the cell body did not result in excitation. These observations suggest that the action of AVP on CA1 pyramidal cells is transsynaptic and is more pronounced in ventral than dorsal CA1.     

State-dependent cross-inhibition between anionic GABA(A) and glycine ionotropic receptors in rat hippocampal CA1 neurons

Using whole-cell patch-clamp recording of acutely isolated rat hippocampal CA1 neurons, we studied the state-dependent cross-inhibition between anionic GABA(A) and glycine (Gly) ionotropic receptors. Co-application of relatively high concentrations of GABA and Gly produced a total current (IGABA + Gly) much smaller than the linear summation of the two individual responses. The mutual inhibition between IGABA and IGly was voltage-independent. However, no current inhibition was observed with co-application of low concentrations of these two agonists. The hippocampal neurons lacking Gly response (IGly) showed no current inhibition with GABA current (IGABA), whereas the neurons losing GABA response (IGABA) (after complete rundown of IGABA) showed no current inhibition with IGly. The current inhibition was also absent in the presence of Gly or GABA(A) receptor antagonists, strychnine or bicuculline, respectively. The results indicate that cross-inhibition between GABA(A) and Gly receptors is state-dependent and a direct receptor-receptor interaction might enable the cross-inhibition to occur.     

Activity-mediated plasticity of GABA equilibrium potential in rat hippocampal CA1 neurons

The equilibrium potential (E_GABA-PSC) for γ-aminobutyric acid (GABA) A receptor mediated inhibitory postsynaptic currents (PSCs) in hippocampal CA1 pyramidal neurons shifts when theta-burst stimulation (four pulses at 100 Hz in each burst in a train consisting of five bursts with an inter-burst interval of 200 ms, the train repeated thrice at 30-s intervals) is applied to the input. E_GABA-PSC is regulated by K⁺/Cl⁻ co-transporter (KCC2). GABA_B receptors are implicated in modulating KCC2 levels. In the current study, the involvement of KCC2, as well as GABA_B receptors, in theta-burst-mediated shifts in E_GABA-PSC was examined. Whole-cell patch recordings were made from hippocampal CA1 pyramidal neurons (from 9 to 12 days old rats), in a slice preparation. Glutamatergic excitatory postsynaptic currents were blocked with dl-2-amino-5-phosphonovaleric acid (50 μM) and 6,7-dinitroquinoxaline-2,3-dione (20 μM). The PSC and the E_GABA-PSC were stable when stimulated at 0.05 Hz. However, both changed following a 30-min stimulation at 0.5 or 1 Hz. Furosemide (500 μM) and KCC2 anti-sense in the recording pipette but not bumetanide (20 or 100 μM) or KCC2 sense, blocked the changes, suggesting KCC2 involvement. Theta-burst stimulation induced a negative shift in E_GABA-PSC, which was prevented by KCC2 anti-sense; however, KCC2 sense had no effect. CGP55845 (2 μM), a GABA_B antagonist, applied in the superfusing medium, or GDP-β-S in the recording pipette, blocked the shift in E_GABA-PSC. These results indicate that activity-mediated plasticity in E_GABA-PSC occurs in hippocampal CA1 pyramidal neurons and theta-burst-induced negative shift in E_GABA-PSC requires KCC2, GABA_B receptors and G-protein activation.     

GAT-1 acts to limit a tonic GABA(A) current in rat CA3 pyramidal neurons at birth

Tonic activation of GABA(A) receptors takes place before the development of functional synapses in cortical structures. We studied whether inefficient GABA uptake might explain the presence of a tonic GABA(A)-mediated current (I(GABA-A)) in early postnatal hippocampal pyramidal neurons. The data show, however, that the tonic I(GABA-A) is enhanced by the specific blocker of GABA transporter-1 (GAT-1), NO-711 (1-[2-[[(Diphenylmethyleneimino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride), at birth in rat CA3 pyramidal neurons. NO-711 also prolonged the duration of GABA transients during endogenous hippocampal network events (known as giant depolarizing potentials) at postnatal day 0. The endogenous tonic I(GABA-A) was seen and it was enhanced by NO-711 in the presence of tetrodotoxin, which itself had only a minor effect on the holding current under control conditions. This indicates that the source of interstitial GABA is largely independent of action-potential activity. The tonic I(GABA-A) in neonatal CA3 pyramidal neurons was increased by zolpidem, indicating that at least a proportion of the underlying GABA(A) receptors contain gamma2 and alpha1-alpha3 subunits. The present data point to a significant role for GAT-1 in the control of the excitability of immature hippocampal neurons and networks.     

A plateau potential mediated by the activation of extrasynaptic NMDA receptors in rat hippocampal CA1 pyramidal neurons

Hippocampal pyramidal neurons express various extrasynaptic glutamate receptors. When glutamate spillover was facilitated by blocking glutamate uptake and fast synaptic transmission was blocked by antagonists of AMPA- and NMDA-type glutamate receptors and an ionotropic GABA receptor blocker, repetitive synaptic stimulation evoked a persistent membrane depolarization that consisted of an early Ca²⁺-independent component and a late Ca²⁺-dependent component. The early component, which we refer to as a plateau potential, had a half-width of 770 ± 160 ms and a steady peak level of −9.54 ± 3.50 mV. It was accompanied by an increase in membrane conductance, the I–V relationship of which showed a peak at −19.91 ± 2.18 mV and reversal of the current at −4.32 ± 2.13 mV, and was suppressed by high concentration of an NMDA receptor (NMDAR) antagonist d -APV, or an NMDAR glycine-binding site antagonist 5,7-dCK. After blocking synaptically located NMDARs using MK801, the potential was still evoked synaptically when spillover was facilitated. A sustained depolarization was evoked by iontophoretic application of glutamate in the presence or absence of a glutamate uptake blocker. This potential was not affected by Na⁺ or Ca²⁺ channel blockers, but was suppressed by 5,7-dCK, leaving an unspecified depolarizing potential. Iontophoresis of NMDA evoked a sustained depolarization that was blocked by a high concentration of d -APV or 5,7-dCK. The I–V relationship of the current during this potential was similar to that obtained during the synaptically induced plateau potentials. These results show that CA1 pyramidal neurons generate plateau potentials mediated most likely by activation of extrasynaptic NMDARs.     

Taurine activates GABA(A) but not GABA(B) receptors in rat hippocampal CA1 area

We investigated if taurine, an endogenous GABA analog, could mimic both hyperpolarizing and depolarizing GABA(A)-mediated responses as well as pre- and postsynaptic GABA(B)-mediated actions in the CA1 region of rat hippocampal slices. Taurine (10 mM) perfusion induced changes in membrane potential and input resistance that are compatible with GABA(A) receptor activation. Local pressure application of taurine and GABA from a double barrel pipette positioned along the dendritic shaft of pyramidal cells revealed that taurine evoked a very small change of membrane potential and resistance compared with the large changes induced by GABA in these parameters. Moreover, in the presence of GABA(A) antagonists, local application of GABA on the dendrites evoked a GABA(B)-mediated hyperpolarization while taurine did not induce any change. Taurine neither mimicked baclofen inhibitory actions on presynaptic release of glutamate and GABA as judging by the lack of taurine effect on paired-pulse facilitation ratio and slow inhibitory postsynaptic potentials, respectively. These results show that taurine mainly activates GABA(A) receptors located on the cell body, indicating therefore that if taurine has any action on the dendrites it will not be mediated by either GABA(A) or GABA(B) receptors activation.     

Argiotoxin-636 blocks excitatory synaptic transmission in rat hippocampal CA1 pyramidal neurons

Argiotoxin 636, (AR₆₃₆), a synaptic antagonist from orb weaver spider venom, is shown produce reversible blockade of excitatory transmission in CA1 pyramidal neurons of the in vitro rat hippocampus. Microtopical applicationof AR₆₃₆ (5–50 nM) resulted in a concentration-dependent suppression of the amplitude of the dendritic field EPSP recorded from stratum radiatum, and the amplitude of the population spike recorded from stratum pyramidale in response to stimulation of the Schaffer collaterals. The maximum effect of AR₆₃₆ occured at about 15–25 min. These effects were reversible after washing with toxin-free physiological solution with the rate of recovery having an inverse relationships to the concnetration of AR₆₃₆. In contrast to the effects observed with orthodromic stimulation, the amplitude of the antidromic spike was not affected by exposure to AR₆₃₆. The temporal pattern GABAergic paired-pulse inhibition was unaffected by exposure to AR₆₃₆. Neuronal discharge elicited by pressure injection of -glutamate was abolished by AR₆₃₆, whereas, responses to -aspartate were not significantlu affected. These data suggest that AR₆₃₆ functionsas a selective antagonist of glutamate-mediated synaptic transmission in rat hippocampus.     

The dendritic response to GABA in CA1 of the hippocampal slice

Application of GABA in the dendritic region of pyramidal cells elicits a depolarization which, in fact, is the sum of a hyperpolarizing and a depolarizing process. At the reversal potential of the depolarizing response (-42 mV) the GABA-induced current fluctuations do not have a minimum. Consequently, a conductance change to more than one ion is involved. Cl- is in part responsible, Ca2+ is not because Mn2+ and Mg2+ do not change the response. Whether Na+ is involved is uncertain. Substitution with choline had no effect but choline may permeate through the membrane during the depolarizing response. Nipecotic acid inhibits a Na+-GABA uptake mechanism but does not change the dendritic response.     

GABA mediated excitation in immature rat CA3 hippocampal neurons

Intracellular recordings from rat hippocampal neurons in vitro during the first postnatal week revealed the presence of spontaneous giant depolarizing potentials (GDPs). These were generated by the synchronous discharge of a population of neurons. GDPs reversed polarity at -27 and -51 mV when recorded with KCl or K-methylsulphate filled electrodes, respectively. GDPs were blocked by the GABAA receptor antagonist bicuculline (10 microM). Iontophoretic or bath applications of GABA (10-300 microM) in the presence of tetrodotoxin (1 microM), induced a membrane depolarization or in voltage clamp experiments an inward current which reversed polarity at the same potential as GDPs. The response to GABA was blocked in a non-competitive manner by bicuculline (10 microM) and did not desensitize. GABA mediated GDPs were presynaptically modulated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Their frequency was reduced or blocked by NMDA receptor antagonists and by the rather specific non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). The frequency of GDPs was enhanced by glycine and D-serine (10-30 microM) in a strychnine insensitive manner. This effect was blocked by AP-5, suggesting that it was mediated by the allosteric modulatory site of the NMDA receptor. These observations suggest that most of the 'excitatory' drive in immature neurons is mediated by GABA acting on GABAA receptors; furthermore excitatory amino acids modulate the release of GABA by a presynaptic action on GABAergic interneurons.     

Heterogeneous distribution of tetrodotoxin-sensitive calcium-conducting channels in rat hippocampal CA1 neurons

Voltage-dependent Ca2+ currents (ICas) in neurons can be classified into T-, N- and L-types. In the CA1 pyramidal neurons freshly isolated from rat hippocampus we found an additional tetrodotoxin (TTX)-sensitive Ca2+ current (termed 'TTX-ICa') which passed through the Na+ channel. The TTX-ICa showed a heterogeneous distribution in the dorsal site of Ca1 region.     

Ion channels activated by quinolinic acid in cultured rat hippocampal neurons

The membrane responses to quinolinic acid, an excitotoxic brain metabolite, were studied in cultured rat hippocampal neurons with the patch-clamp technique. In the whole-cell recording mode, pressure applications of quinolinic acid elicited inwardly directed membrane currents over a membrane potential range of −60 to −5 mV. The current response reversed at about 0 mV. The current-voltage (I–V) relation of the response had a negative slope conductance at membrane potentials more negative than −40 mV. On removal of Mg²⁺ from the extracellular solution, the current response showed no region of negative slope conductance at potentials more positive than −60 mV. In Mg²⁺-free solution applications of quinolinic acid elicited discrete pulse-like current flows through the outside-out membrane patch. The single channel conductance was 40–46 pS over a membrane potential range of −40 to −80 mV, and 50–55 pS at membrane potentials more positive than +30 mV, showing an outward rectification. These values of the single channel conductance were similar to those of the main conducting state of the channels activated by (NMDA). The responses to quinolinic acid were completely suppressed by the NMDA receptor antagonist (±)-2-amino-5-phosphonovaleric acid. The results indicate that quinolinic acid selectively activates NMDA receptors in the cultured rat hippocampal neurons.     

GABA(B) receptors inhibit backpropagating dendritic spikes in hippocampal CA1 pyramidal cells in vivo

Spike backpropagation has been proposed to enhance dendritic depolarization and synaptic plasticity. However, relatively little is known about the inhibitory control of spike backpropagation in vivo. In this study, the backpropagation of the antidromic spike into the dendrites of CA1 pyramidal cells was studied by extracellular recording in urethane-anesthetized rats. The population antidromic spike (pAS) in CA1 following stimulation of the alveus was recorded simultaneously with a 16-channel silicon probe and analyzed as current source density (CSD). The pAS current sink was shown to sequentially invade the soma and then the apical and basal dendrites. When the pAS was preceded <400 ms by a conditioning orthodromic CA3 stimulus, the apical and basal dendritic spike sinks were reduced and delayed. Dendritic spike suppression was large after a high-intensity CA3 conditioning stimulus that evoked a population spike, small after a low-intensity CA3 conditioning stimulus, and weak after conditioning by another pAS. The late (150-400 ms latency) inhibition of the backpropagating pAS at the apical and basal dendrites was partially relieved by a GABA(B) receptor antagonist, CGP35348 or CGP56999A, given intracerebroventricularly (icv). CGP35348 icv also decreased the latency of the antidromic spike sinks at all depths. A compartment cable model of a CA1 pyramidal cell with excitable dendrites, combined with a model of extracellular potential generation, confirms that GABA(B) receptor activation delays a backpropagating spike and blocks distal dendritic spikes. GABA(B) receptor-mediated conductance increase and hyperpolarization, amplified by removing dendritic I(A) inactivation, contribute to conditioned dendritic spike suppression. In addition, the model shows that slow Na(+) channel inactivation also participates in conditioned spike suppression, which may partly explain the small dendritic spike suppression after conditioning with a weak orthodromic stimulus or another antidromic spike. Thus, both theory and experiment confirm an important role of the GABA(B) receptors in controlling dendritic spike backpropagation.     

NMDA receptor-independent LTP in basal versus apical dendrites of CA1 pyramidal cells in rat hippocampal slice

The ability of hippocampal CA1 basal synapses to express N-methyl-D-aspartate (NMDA) receptor-independent long-term potentiation (non-NMDA LTP) was studied and compared to the simultaneously induced apical dendritic non-NMDA LTP. Non-NMDA LTP in basal and apical dendrites was induced using stimulation pattern similar to the sharp wave-associated CA3 bursts. Basal dendritic non-NMDA LTP was input-specific and displayed similar development and magnitude to the apical dendritic non-NMDA LTP. Both apical and basal dendritic non-NMDA potentiations were inhibited by the voltage-dependent calcium channel (VDCC) inhibitor verapamil and the tyrosine kinase inhibitors genistein and levandustin A. However, the difference in the degree and time course of these inhibitions suggests involvement of distinct mechanisms in the two dendritic subfields. Hippocampus 1998;8:373–379. © 1998 Wiley-Liss, Inc.     

Slow intrinsic spikes recorded in vivo in rat CA1-CA3 hippocampal pyramidal neurons

The membrane potential of 45 CA1-CA3 hippocampal pyramidal cells was recorded in curarized and urethanized rats. Two slow spike types were observed together with the usual Na+ type action potential. Slow spikes were termed HTS and LTS because they were essentially identical to the high and low threshold Ca2+ spikes observed in vitro and probably represent the same kinds of activities. LTS and HTS (22 and 29 cells, respectively) were triggered at potentials greater than or equal to 65 mV and less than or equal to 55 mV, had mean durations of 23.7 and 90.4 ms and mean amplitudes of 11.5 and 39.3 mV, respectively, and fired an overriding burst of the action potentials. LTS and HTS were sometimes present in the same neuron (n = 16). Depolarizing pulses triggered rhythmic HTS at rates that increased with depolarization up to a 5 impulses/s maximum. The same limit was found with imposed membrane potential sine currents at frequencies within the theta rhythm range. With spontaneous or imposed hyperpolarizations LTS were evoked by depolarizing, at the break of hyperpolarizing pulses, or spontaneously. They were also evoked at the depolarizing, or recovery, slopes of inhibitory postsynaptic potentials. HTS and LTS supply pyramidal neurons with different firing patterns which enhance the system's integrative possibilities. Evidence is provided that theta is not exclusively generated by network properties, since rhythmic HTS may participate.     

Dendritic action potentials activated by NMDA receptor-mediated EPSPs in CA1 hippocampal pyramidal cells

Intradendritic recordings were obtained in rat CA1 hippocampal pyramidal cells. Repetitive stimulation produced substantial short-term potentiation of the dendritic excitatory postsynaptic potential (EPSP) which was partly attributable to activation ofn-methyl-d-aspartate receptors. Accompanying the potentiated synaptic response were Na⁺-mediated spikes which appeared to originate at multiple sites in the dendritic arbor. These discrete dendritic action potentials are rarely distinguishable in somatic recordings but may contribute to the subthreshold response at the pyramidal cell body. In addition, dendritic spikes may interact with other voltage-dependent dendritic conductances.     

Enhancement of GABA neurotransmission after cerebral ischemia in the rat reduces loss of hippocampal CA1 pyramidal cells

Increased excitation may be involved in the development of delayed CA1 pyramidal cell death in hippocampus after global cerebral ischemia. Therefore we investigated the possible neuroprotective effect of the GABA uptake inhibitor, R-(-)-1-(4,4-(3-methyl-2-thienyl)-3-butenyl)-3-piperidine carboxylic acid (No-328), in a rat cerebral ischemia model of delayed CA1 pyramidal cell death. No-328 in doses of 36 mg/kg given 30 min before, and 1, 24, 48 and 72 h after ischemia significantly reduced the CA1 neuron loss. Doses of 50 mg/kg of No-328 given immediately before, 24 h and 48 h after ischemia, also reduced the CA1 neuron loss significantly. Furthermore, we demonstrated that postischemic treatment with diazepam (4 x 15 mg/kg) significantly reduced the CA1 neuron loss. However, postischemic treatment with several doses (5 x 12 mg/kg) of the GABA analog, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), offered no CA1 neuron protection when given alone, but when administrated together with diazepam (4 x 15 mg/kg) it significantly reduced the CA1 neuron loss. We conclude that enhancement of postischemic GABA neurotransmission, during the first 2-3 days after ischemia, may reduce the ischemic CA1 damage through a continuous increase in hippocampal GABA extracellular levels (No-328), or through an increase in sensitivity to GABA neurotransmission (diazepam).     

Spontaneous release from mossy fiber terminals inhibits Ni2+-sensitive T-type Ca2+ channels of CA3 pyramidal neurons in the rat organotypic hippocampal slice

Mossy fibers (axons arising from dentate granule cells) form large synaptic contacts exclusively onto the proximal apical dendrites of CA3 pyramidal neurons. They can generate large synaptic currents that occur in close proximity to the soma. These properties mean that active conductance in the proximal apical dendrite could have a disproportionate influence on CA3 pyramidal neuron excitability. Ni(2+)-sensitive T-type Ca(2+) channels are important modulators of dendritic excitability. Here, we use an optical approach to determine the contribution of Ni(2+) (100 microM)-sensitive Ca(2+) channels to action potential (AP) elicited Ca(2+) flux in the soma, proximal apical and distal apical dendrites. At resting membrane potentials Ni(2+)-sensitive Ca(2+) channels do not contribute to the Ca(2+) signal in the proximal apical dendrite, but do contribute in the other cell regions. Spontaneous release from mossy fiber terminals acting on 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-sensitive postsynaptic channels underlies a tonic inhibition of Ni(2+)-sensitive channels. Chelating Zn(2+) with CaEDTA blocks CNQX-sensitive changes in Ca(2+) flux implicating a mechanistic role of this ion in T-type Ca(2+) channel block. To test if this inhibition influenced excitability, progressively larger depolarizing pulses were delivered to CA3 pyramidal neurons. CNQX significantly reduced the size of the depolarizing step required to generate APs and increased the absolute number of APs per depolarizing step. This change in AP firing was completely reversed by the addition of Ni(2+). This mechanism may reduce the impact of T-type Ca(2+) channels in a region where large synaptic events are common.