Role of zinc influx via AMPA/kainate receptor activation in metabotropic glutamate receptor-mediated calcium release

The uptake of free zinc into CA3 pyramidal cells and its significance was examined in rat hippocampal slices with ZnAF-2DA, a membrane-permeable zinc indicator. Intracellular ZnAF-2 signal in the CA3 pyramidal cell layer was increased during delivery of tetanic stimuli to the dentate granule cell layer. This increase was completely blocked in the presence of CNQX, an AMPA/kainate receptor antagonist. These results suggest that free zinc is taken up into CA3 pyramidal cells via activation of AMPA/kainate receptors. The effect of free zinc levels in the CA3 pyramidal cells on the increase in intracellular calcium via Group I metabotropic glutamate receptors was examined by regional delivery of tADA, a Group I metabotropic glutamate receptor agonist, to the stratum lucidum after blockade of AMPA/kainate receptor-mediated calcium and zinc influx. Intracellular calcium orange signal in the CA3 pyramidal cell layer was increased by tADA, whereas intracellular ZnAF-2 signal was not increased even in the presence of 100 muM zinc, suggesting that tADA induces calcium release from internal stores in CA3 pyramidal cells and is not involved in zinc uptake. The increase in calcium orange signal by tADA was enhanced by perfusion with pyrithione, a zinc ionophore that decreased basal ZnAF-2 signal in the CA3 pyramidal cell layer. It was blocked by perfusion with pyrithione and zinc that increased basal ZnAF-2 signal. The present study indicates that the increase in free calcium levels via the metabotropic glutamate receptor pathway is inversely related to free zinc levels in CA3 pyramidal cells.     

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.     

Associative mossy fibre LTP induced by pairing presynaptic stimulation with postsynaptic hyperpolarization of CA3 neurons in rat hippocampal slice

Whole cell recordings of excitatory postsynaptic potentials/currents (EPSPs/EPSCs) evoked by minimal stimulation of commissural-associative (CF) and mossy fibre (MF) inputs were performed in CA3 pyramidal neurons. Paired responses (at 50 ms intervals) were recorded before, during and after hyperpolarization of the postsynaptic membrane (20-30 mV for 15-35 min). Membrane hyperpolarization produced a supralinear increase of EPSPs/EPSCs amplitude in MF-inputs. Synaptic responses remained potentiated for the rest of the recording period (up to 40 min) after resetting the membrane potential to control level (221 +/- 60%, n = 15 and 219 +/- 61%, n = 11 for MF-EPSP and MF-EPSC, respectively). We shall refer to this effect as hyperpolarization-induced LTP (HI-LTP). In the absence of afferent stimulation, membrane hyperpolarization was unable to produce HI-LTP. In contrast to MF-EPSPs, the mean amplitude of CF-EPSPs did not increase significantly after hyperpolarization relative to controls (138 +/- 29%, n = 22). HI-LTP was associated with modifications of classical indices of presynaptic release: paired-pulse facilitation, failures rate, coefficient of variation of EPSP amplitudes and quantal content. The induction of HI-LTP was NMDA independent but was dependent on metabotropic glutamate receptors (mGluRs) activation and calcium release from inositol 1,4,5-triphosphate (IP3)-sensitive intracellular stores: it was prevented by mGluR antagonist, intracellular heparin and BAPTA. We conclude that while the induction of HI-LTP was postsynaptic, its expression was presynaptic.     

Postnatal development of temporal integration,spike timing and spike threshold regulation by a dendrotoxin‐sensitive K+ current in rat CA1 hippocampal cells

Spike timing and network synchronization are important for plasticity, development and maturation of brain circuits. Spike delays and timing can be strongly modulated by a low‐threshold, slowly inactivating, voltage‐gated potassium current called D‐current (I_D). I_D can delay the onset of spiking, cause temporal integration of multiple inputs, and regulate spike threshold and network synchrony. Recent data indicate that I_D can also undergo activity‐dependent, homeostatic regulation. Therefore, we have studied the postnatal development of I_D‐dependent mechanisms in CA1 pyramidal cells in hippocampal slices from young rats (P7–27), using somatic whole‐cell recordings. At P21–27, these neurons showed long spike delays and pronounced temporal integration in response to a series of brief depolarizing current pulses or a single long pulse, whereas younger cells (P7–20) showed shorter discharge delays and weak temporal integration, although the spike threshold became increasingly negative with maturation. Application of α‐dendrotoxin (α‐DTX), which blocks I_D, reduced the spiking latency and temporal integration most strongly in mature cells, while shifting the spike threshold most strongly in a depolarizing direction in these cells. Voltage‐clamp analysis revealed an α‐DTX‐sensitive outward current (I_D) that increased in amplitude during development. In contrast to P21–23, I_D in the youngest group (P7–9) showed smaller peri‐threshold amplitude. This may explain why long discharge delays and robust temporal integration only appear later, 3 weeks postnatally. We conclude that I_D properties and I_D‐dependent functions develop postnatally in rat CA1 pyramidal cells, and I_D may modulate network activity and plasticity through its effects on synaptic integration, spike threshold, timing and synchrony.     

Dopamine decreases the calcium-activated afterhyperpolarization in hippocampal CA1 pyramidal cells

The effect of dopamine (DA) on the calcium-activated potassium conductance underlying the slow afterhyperpolarization (AHP) which follows a train of action potentials in hippocampal pyramidal cells was studied utilizing the in vitro hippocampal slice preparation. Bath-applied DA (1-100 microM) significantly reduced the AHP in a reversible, dose-dependent manner. Neither the amount of current injected to elicit the AHP nor its initial amplitude had an effect on the reduction of the AHP by DA. DA did not depress calcium spikes, suggesting that the blockade of the AHP likely occurs at a step subsequent to the entry of calcium. Since DA's actions on the AHP closely mimicked those of norepinephrine, we examined the effect of beta-adrenergic antagonists on DA's actions. At concentrations which in other systems have been shown not to block DA stimulated adenylate cyclase, beta-adrenergic antagonists completely inhibited the reduction of the AHP by DA. In some cells DA also elicited small hyperpolarizations which were not blocked by application of dopamine receptor antagonists. These findings strongly suggest that a major electrophysiological action of DA in the hippocampus (i.e. blockade of the AHP) is due to its cross reactivity with beta-adrenergic receptors and that rigid pharmacologic criteria must be used before attributing an action of DA unambiguously to its interaction with DA receptors.     

Increased proximal bifurcation of CA1 pyramidal apical dendrites in sema3A mutant mice

Semaphorin‐3A (Sema3A) is an attractive guidance molecule for cortical apical dendrites. To elucidate the role of Sema3A in hippocampal dendritic formation, we examined the Sema3A expression pattern in the perinatal hippocampal formation and analyzed hippocampal dendrites of the brains from young adult sema3A mutant mice. Sema3A protein was predominantly expressed in the hippocampal plate and the inner marginal zone at the initial period of apical dendritic growth. Neuropilin‐1 and plexin‐A, the receptor components for Sema3A, were also localized in the same regions. The Golgi impregnation method revealed that in wildtype mice more than 90% of hippocampal CA1 pyramidal neurons extended a single trunk or apical trunks bifurcated in stratum radiatum. Seven percent of the pyramidal neurons showed proximal bifurcation of apical trunks in stratum pyramidale or at the border of the stratum pyramidale and stratum radiatum. In sema3A mutant mice, proximally bifurcated apical dendrites were increased to 32%, while the single apical dendritic pyramidal neurons were decreased. We designate this phenotype in sema3A mutant mice as “proximal bifurcation.” In the dissociated culture system, approximately half of the hippocampal neurons from wildtype mice resembled pyramidal neurons, which possess a long, thick, and tapered dendrite. In contrast, only 30% of the neurons from sema3A mutants exhibited pyramidal‐like morphology. Proximal bifurcation of CA1 pyramidal neurons was also increased in the mutant mice of p35, an activator of cyclin‐dependent kinase 5 (Cdk5). Thus, Sema3A may facilitate the initial growth of CA1 apical dendrites via the activation of p35/Cdk5, which may in turn signal hippocampal development. J. Comp. Neurol. 516:360–375, 2009. © 2009 Wiley‐Liss, Inc.     

The effect of nicotine on spiking activity and Ca2+ dynamics of dendritic spines in rat CA1 pyramidal neurons

Nicotinic acetylcholine receptors (nAChRs) of the hippocampus have been thought to contribute to cognitive enhancement by cigarette smoking. Although positive modulation on cognitive functions is linked to the smoked, low-dose nicotine, the cellular correlate behind this modulation is unknown. It has been accepted that cellular mechanisms underlying plastic effects on memory involve the association of backpropagating action potentials (bAPs) with synaptic activity in the hippocampus. Here, we show the effects of low-dose (1 microM) nicotine on bAP-evoked Ca2+ transients in basal dendrites and spines of pyramidal neurons in rat hippocampal slices. Although nicotine application failed to have any direct effect in low concentration, it could significantly enhance bAP-evoked Ca2+ transients through presynaptic nAChRs located on axon terminals innervating pyramidal cells. The activation of these receptors is known to release neurotransmitters and induce postsynaptic currents. High-dose (250-500 microM) nicotine could induce firing and Ca2+ accumulation in spines. Large amplitude currents were observed occasionally (8 out of 18 cells) in voltage clamp recordings in response to pressure application of high-dose nicotine. This may explain the relatively low incidence of nicotine-induced firing (7 out of 27 cells) under current clamp. These data indicate that (i) activation of presynaptic nAChRs can modulate backreporting in dendrites of pyramidal neurons and (ii) there is a group of pyramidal neurons with higher nicotine-sensitivity, producing firing at strong stimulations. Our data revealed a subcellular effect of nicotine through regulation of Ca2+ levels in the computational units of pyramidal neurons.     

The chemokine CCL2 modulates Ca2+ dynamics and electrophysiological properties of cultured cerebellar Purkinje neurons

The chemokine CCL2 is produced at high levels in the central nervous system (CNS) during infection, injury, neuroinflammation and other pathological conditions. Cells of the CNS including neurons and glia express receptors for CCL2 and these receptors may contribute to a signaling system through which pathologic conditions in the CNS are communicated. However, our understanding of the consequences of activation of chemokine signaling in the CNS is limited, especially for neurons. In many cell types, chemokine signaling alters intracellular Ca(2+) dynamics. Therefore, we investigated the potential involvement of this mechanism in neuronal signaling activated by CCL2. In addition, we examined the effects of CCL2 on neuronal excitability. The studies focused on the rat cerebellar Purkinje neuron, an identified CNS neuronal type reported to express both CCL2 and its receptor, CCR2. Immunohistochemical studies of Purkinje neurons in situ confirmed that they express CCR2 and CCL2. The effect of exogenous application on Purkinje neurons was studied in a cerebellar culture preparation. CCL2 was tested by micropressure or bath application, at high concentrations (13-100 nm) to simulate conditions during a pathologic state. Results show that Purkinje neurons express receptors for CCL2 and that activation of these receptors alters several neuronal properties. CCL2 increased resting Ca(2+) levels, enhanced the Ca(2+) response evoked by activation of metabotropic glutamate receptor 1 and depressed action potential generation in the cultured Purkinje neurons. Passive membrane properties were unaltered. These modulatory effects of CCL2 on neuronal properties are likely to contribute to the altered CNS function associated with CNS disease and injury.     

Dynamics of Ca(2+) and Na(+) in the dendrites of mouse cerebellar Purkinje cells evoked by parallel fibre stimulation

Ca2+ and Na+ play important roles in neurons, such as in synaptic plasticity. Their concentrations in neurons change dynamically in response to synaptic inputs, but their kinetics have not been compared directly. Here, we show the mechanisms and dynamics of Ca2+ and Na+ transients by simultaneous monitoring in Purkinje cell dendrites in mouse cerebellar slices. High frequency parallel fibre stimulation (50 Hz, 3-50-times) depolarized Purkinje cells, and Ca2+ transients were observed at the anatomically expected sites. The magnitude of the Ca2+ transients increased linearly with increasing numbers of parallel fibre inputs. With 50 stimuli, Ca2+ transients lasted for seconds, and the peak [Ca2+] reached approximately 100 microm, which was much higher than that reported previously, although it was still confined to a part of the dendrite. In contrast, Na+ transients were sustained for tens of seconds and diffused away from the stimulated site. Pharmacological interventions revealed that Na+ influx through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and Ca2+ influx through P-type Ca channels were essential players, that AMPA receptors did not operate as a Ca2+ influx pathway and that Ca2+ release from intracellular stores through inositol trisphosphate receptors or ryanodine receptors did not contribute greatly to the large Ca2+ transients.     

Regulation of synaptic currents by mGluR2 at reciprocal synapses in the mouse accessory olfactory bulb

The throughput of information from the accessory olfactory bulb (AOB) to downstream structures is controlled by reciprocal dendrodendritic inhibition of mitral cells by granule cells. Given the high expression levels of mGluR2, a metabotropic glutamate receptor, in the AOB and the fact that the activation of mGluR2 permits the formation of a specific olfactory memory, we reasoned that mGluR2 might play an important role in regulating dendrodendritic inhibition. To test this hypothesis, we examined the effects of pharmacological and genetic manipulations of mGluR2 on synaptic responses measured from mitral or granule cells in slice preparations from 23‐ to 36‐day‐old Balb/c mice. To evoke dendrodendritic inhibition, a depolarizing voltage step from –70 to 0 mV or a threshold current stimulus adjusted to elicit action potential(s) was applied to a mitral cell using either a nystatin‐perforated or conventional whole‐cell configuration. We found that an agonist for group II metabotropic glutamate receptors (mGluR2/mGluR3), DCG‐IV [(2S,1′R,2′R,3′R)‐2‐(2,3‐dicarboxycyclopropyl)glycine], suppressed, whereas the mGluR2/mGluR3 antagonist LY341495 [(αS)‐α‐amino‐α‐[(1S,2S)‐2‐carboxycyclopropyl]‐9H‐xanthine‐9‐propanoic acid] enhanced dendrodendritic inhibition. Genetic ablation of mGluR2 markedly impaired the effects of DCG‐IV and LY341495 on dendrodendritic inhibition. DCG‐IV reduced both the frequency and the amplitude of spontaneous miniature excitatory postsynaptic currents recorded from granule cells. Additionally, DCG‐IV inhibited high‐voltage‐activated calcium currents in both mitral and granule cells. These results suggest that mGluR2 reduces dendrodendritic inhibition by inhibiting synaptic transmission between mitral cells and granule cells in the AOB.     

Long-term potentiation of transmitter exocytosis expressed by Ca2+-induced Ca2+ release from thapsigargin-sensitive Ca2+ stores in preganglionic nerve terminals

We have studied whether Ca(2+)-induced Ca(2+) release (CICR) is involved in the mechanism of long-term potentiation (LTP) at nicotinic synapses of bullfrog sympathetic ganglia. Fast excitatory postsynaptic potentials (fast EPSPs) were recorded in a low-Ca(2+), high-Mg(2+) solution and quantal analysis was applied. The conditioning stimulation of the B-type preganglionic nerve at 20 Hz for 4 min consistently enhanced the amplitude and quantal content of fast EPSP for > 2 h, but only sometimes enhanced the quantal size. The LTP of quantal content produced by the conditioning tetanus was blocked by thapsigargin, a blocker of Ca(2+) pumps at Ca(2+) stores, applied before or after the conditioning tetanus, and by Xestospongin C, a blocker of inositoltrisphosphate (IP(3)) receptors, applied before the tetanus. It was not, however, blocked by ryanodine, a blocker and/or activator of ryanodine receptors, or by propranolol, a blocker of beta-adrenergic receptors. Thus the long-lasting activity of the preganglionic nerve at a high frequency causes the LTP of impulse-evoked transmitter release by the activation of CICR from thapsigargin-sensitive Ca(2+) stores in the nerve terminals. It is likely that a large Ca(2+) entry into the nerve terminals during tetanic activity primes ryanodine-insensitive Ca(2+) release channels for activation.     

Effect of protein malnutrition on CA3 hippocampal pyramidal cells in rats of three ages

Prenatal and postnatal protein deprivation effect on CA3 hippocamapal pyramidal pyramidal cells were investigated in 30-, and 90- and 220-day old rats Female rats were fed either a 6% or a 25% casein diet 5 wk before conception and the litters were maintained on their respective diet until sacrificed. In 216 rapid Golgi-impregnatd cells, we measured somal size, length and diameter of apical dendrite, number of apical dendrites intersecting 10 concentric rings 38 μm apart, thorny excrescence area and length, head diameter and density of synaptic spines on 50-μm segments of apical dendrite. The present experiments showed that malnutrition produced significant reductions of somal size in animals at 220 days of age. There were significant reductions of apical dendrite diameters in animals of 30 and 90 days, and of density and head diameter of synaptic spines at the three ages studied, and significant decrease of the thorny excrescence area at 220 days of age. At this latter age, dendritic branching was significantly decreased in the last four rings representing the area into which the perforant pathway projects. In 30-day malnourished rats, dendritic branching showed a significant increase in rings 4–6 representing the area in which the Schaffer collaterals synapse. The location of the deficit spines corresponds to the sites where mossy fibers synapse on the apical dendrites of CA3 neurons. Age-related changes normally observed in control rats (e.g., the 30-day-old control group showed the smallest somal size and 220-day-old controls the largest size) failed to occur in the malnourished rats. The deficits in spine density and dendritic branching (in animals of 220 days old) were similar to those found in our previous studies on fascia dentata.     

Sensitivity of postsynaptic GABAB receptors on hippocampal CA1 and CA3 pyramidal neurons to ethanol

Baclofen-induced hyperpolarization of hippocampal CA1 and CA3 pyramidal neurons was examined to assess the impact of ethanol on postsynaptic GABA_B receptors. These receptors activate outward K⁺ currents via a pertussis toxin-sensitive G protein cascade to reduce membrane potential during the slow inhibitory postsynaptic potential. This inhibitory action may play a role in ethanol intoxication and withdrawal excitability. In both types of pyramidal neurons, baclofen applied consecutively in increasing concentrations caused concentration dependent hyperpolarization. There were no significant differences in resting membrane potential, input resistance, maximum baclofen-induced hyperpolarization or EC₅₀ between CA1 and CA3 neurons, although slope values were significantly smaller in the former neurons. These parameters were not significantly changed in the presence of ethanol 10–100 mM. Chronic ethanol treatment (12 days) sufficient to induce physical dependence also did not shift sensitivity or maximum response to baclofen in CA1 neurons. These results suggest that GABA_B receptors in this model are essentially insensitive to ethanol and do not confirm our earlier preliminary observation of a possible down-regulation of postsynaptic GABA_B receptor function by chronic ethanol treatment.     

Carnosine and β-alanine release is stimulated by glutamatergic receptors in cultured rat oligodendrocytes

Oligodendrocytes obtained from rat brain 0-2 A progenitor cells and differentiated in culture take up β-alanine and synthesize carnosine (β-Ala-His). The present study was designed to determine whether carnosine and β-alanine are released from such cultures in response to some stimuli. An evoked release of these substances was not observed when the cells were incubated with 1mM glutamate or 0.3 mM kainate. Addition of 0.1 mM cyclothiazide (CTZ) to the corresponding stimulus was accompanied by a distinct peak of release consisting of both carnosine and β-alanine. The efflux was blocked completely in the case of kainate and to 80% in the case of glutamate when 50 μM 6,7- dinitroquinoxaline-2,3 (1H,4H)-dion (DNQX) was added to the cells at the same time as the receptor agonist. An increase of the efflux was observed in the presence of Zn²⁺. This effect was concentration- dependent. Total substitution of NaCl in the efflux medium by LiCl caused only a partial reduction of the release. GABA or 55 mM KCl showed only negligible effect. A large release of carnosine and β-alanine was observed when oligodendrocyte cultures were treated with Ca²⁺ ionophore A 23187. These results suggest that oligodendrocytes exhibit a glutamate receptor-mediated release of carnosine and β-alanine. The release is dependent on elevated intracellular Ca²⁺ concentration. GLIA 24:346–351, 1998. © 1998 Wiley-Liss, Inc.     

Differential contributions of Ca2+‐activated K+ channels and Na+/K+‐ATPases to the generation of the slow afterhyperpolarization in CA1 pyramidal cells

In many types of CNS neurons, repetitive spiking produces a slow afterhyperpolarization (sAHP), providing sustained, intrinsically generated negative feedback to neuronal excitation. Changes in the sAHP have been implicated in learning behaviors, in cognitive decline in aging, and in epileptogenesis. Despite its importance in brain function, the mechanisms generating the sAHP are still controversial. Here we have addressed the roles of M‐type K⁺ current (I_M), Ca²⁺‐gated K⁺ currents (I_Ca(K)'s) and Na⁺/K⁺‐ATPases (NKAs) current to sAHP generation in adult rat CA1 pyramidal cells maintained at near‐physiological temperature (35 °C). No evidence for I_M contribution to the sAHP was found in these neurons. Both I_Ca(K)'s and NKA current contributed to sAHP generation, the latter being the predominant generator of the sAHP, particularly when evoked with short trains of spikes. Of the different NKA isoenzymes, α₁‐NKA played the key role, endowing the sAHP a steep voltage‐dependence. Thus normal and pathological changes in α₁‐NKA expression or function may affect cognitive processes by modulating the inhibitory efficacy of the sAHP.     

Difference in time course of modulation of synaptic transmission by group II versus group III metabotropic glutamate receptors in region CA1 of the hippocampus

We investigated the time course of modulation of synaptic transmission by group II and group III metabotropic glutamate receptors in region CA1 of the hippocampus. In the presence of 50 microM picrotoxin, pressure pulse application of 1 mM glutamate resulted in a fast onset of suppression of synaptic transmission in stratum lacunosum moleculare and a slower onset of suppression in stratum radiatum, with both effects returning to baseline over the course of several minutes. Application of 50 microM of the group II agonist (2R,4R)-APDC in stratum lacunosum moleculare resulted in the same fast onset of suppression while having no effect in stratum radiatum. Pressure pulse application of 100 microM DL-AP4 in stratum lacunosum moleculare and stratum radiatum resulted in a much slower onset of suppression of synaptic transmission than (2R,4R)-APDC. Suppression by (2R,4R)-APDC was accompanied by a rapid enhancement of paired pulse facilitation, indicative of a presynaptic mechanism. This demonstrates that activation of group II mGluRs in the hippocampus causes a fast onset of suppression in stratum lacunosum moleculare, while activation of group III mGluRs causes a slower onset of suppression. The difference in time course for group II vs. group III mGluRs suggests a different functional role, with group II playing a potential role in making synapses act as low pass filters.     

Enkephalin inhibition of inhibitory input to CA1 and CA3 pyramidal neurons in the hippocampus

Enkephalin-induced excitation in the hippocampus has been attributed to the attenuation of inhibitory input as well as to augmentation of excitatory input to pyramidal neurons. We have further examined these possible mechanisms of enkephalin action, as well as the possibility that enkephalins may be affecting intrinsic membrane properties, by recording intracellularly from CA1 and CA3 pyramidal cells in the guinea pig hippocampal brain slice preparation. It was observed that the inhibitory synaptic potential was significantly decreased in the presence of leucine enkephalin and D-alanine, D-leucine-enkephalin (DADL), whereas the excitatory synaptic potential, revealed by block of the inhibitory postsynaptic potential (IPSP) by bicuculline, was unaltered. In addition, the response of pyramidal cells to pressure-applied GABA was unaffected by enkephalin, as were the voltage-dependent membrane conductances. The increase in excitability which was observed in both field potential and intracellular recordings to drop application of DADL must, then, be due to a purely presynaptic block of inhibitory interneurons in both the CA1 and CA3 areas of the hippocampus.