E. Cherubini

Giant synaptic potentials in immature rat CA3 hippocampal neurones.

1. Intracellular recordings were made from rat CA3 hippocampal neurones in vitro during the first eighteen days of postnatal life. The cells had resting membrane potentials more negative than ‐51 mV, action potentials greater than 55 mV and membrane input resistances of 117 +/‐ 12 M omega. An unusual characteristic of these cells was the presence of spontaneous giant depolarizing potentials (GDPs) which were observed during the first eight postnatal (P) days in over 85% of neurones. They were less frequent between P9 and P12 (48%) and disappeared after P12. 2. The GDPs were synchronously generated by a population of neurones; they reversed polarity at ‐27 mV when recorded with KCl‐containing electrodes and at ‐51 mV with potassium acetate‐ or potassium methylsulphate‐filled electrodes. 3. The GDPs were blocked by bath application of bicuculline (10 microM) or picrotoxin (100‐200 microM). Exogenously applied gamma‐aminobutyric acid (GABA; 0.2‐1 mM) induced at resting membrane potential a bicuculline‐sensitive membrane depolarization which reversed polarity at ‐25 and ‐51 mV when recorded with KCl‐ or potassium methylsulphate‐filled electrodes respectively. 4. The GDPs were reduced in frequency or blocked by the N‐methyl‐D‐aspartate (NMDA) receptor antagonists DL‐2‐amino‐7‐phosphonoheptanoate (AP‐7; 50 microM), D(‐)2‐amino‐5‐phosphonovalerate (AP‐5, 10‐50 microM) and (+‐)3‐(2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid (CPP, 10‐50 microM) or NMDA channel blockers phencyclidine (2 microM) and ketamine (20 microM). 5. Stimulation of the hilus during the first week of life evoked a GDP followed by a hyperpolarization. The GDPs were generated by a population of synchronized neurones and reversed polarity at ‐27 mV with KCl‐filled electrodes and at ‐52 mV with potassium acetate‐ or potassium methylsulphate‐containing electrodes. 6. Bath application of bicuculline (1‐10 microM) or picrotoxin (100‐200 microM) reversibly blocked the evoked GDPs in the majority of cells. The NMDA receptor antagonists AP‐5 (50 microM), AP‐7 (50 microM) and CPP (30 microM) usually reduced the amplitude and the duration of the evoked GDPs. In neurones in which evoked GDPs were blocked by bicuculline, a NMDA‐mediated component was revealed by increasing the strength or the frequency of stimulation. 7. During the second week of postnatal life, when spontaneous GDPs were extremely rare or absent, superfusion with bicuculline (10 microM) induced, as in adult slices, interictal discharges. These reversed polarity near 0 mV with KCl‐ or potassium acetate‐containing electrodes and were reduced in amplitude and duration by AP‐5 (50 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Strychnine-sensitive glycine responses of neonatal rat hippocampal neurones.

1. Intracellular recordings employing current and voltage clamp techniques were used to study the effects of glycine on rat CA3 hippocampal neurones during the first 3 weeks of postnatal (P) life. 2. Glycine (0.3‐1 mM) depolarized neurones from rats less than 4 days old (P4). Neurones from older neonates (P5‐P7) were hyperpolarized by glycine, whereas adult neurones were unaffected. 3. Both depolarizing and hyperpolarizing responses were associated with large conductance increases; they reversed polarity at a potential which changed with the extracellular chloride concentration. The responses persisted in tetrodotoxin (1 microM) or in a solution with a much reduced calcium concentration. 4. Strychnine (1 microM) but not bicuculline (10‐50 microM) antagonized the effects of glycine. The action of strychnine was apparently competitive with a dissociation constant of 350 nM. 5. In voltage clamp experiments, glycine elicited a non‐desensitizing outward current at ‐60 mV. When a maximal concentration of glycine was applied at the same time as gamma‐aminobutyric acid (GABA), the conductance increase induced by the two agonists was additive, suggesting the activation of different populations of channels. 6. Concentrations of glycine lower than 100 microM did not affect membrane potential. However, at 30‐50 microM glycine increased the frequency of spontaneous GABA‐mediated synaptic responses; this action was not blocked by strychnine. 7. It is concluded that during the first 2 weeks of life glycine acts at strychnine‐sensitive receptors to open chloride channels.

Blockade of excitatory synaptic transmission by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in the hippocampus in vitro.

Superfusion of hippocampal slices with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 2-5 microM) reversibly blocked the Schaffer collateral and mossy fibre excitatory postsynaptic potential (EPSP), while sparing the fast and slow gamma-aminobutyric acid (GABA)-mediated inhibition. Membrane potential, input resistance and spike accommodation were not altered. Inward currents induced by quisqualate were reduced to a greater extent by CNQX than those induced by kainate or N-methyl-D-aspartate. We suggest that CNQX may be a useful antagonist to study excitatory amino acid-mediated synaptic transmission.

Epileptiform bursts elicited in CA3 hippocampal neurons by a variety of convulsants are not blocked by N-methyl-d-aspartate antagonists

Intracellular and extracellular recordings from CA3 hippocampal neurons in vitro were used to study the ability of several NMDA (N-methyl-D-aspartate) receptor antagonists to suppress epileptiform bursts induced by NMDA and convulsants not thought to act at NMDA receptors. The antagonists, APV (D-2-amino-5-phosphonovalerate), AP-7 (D,L-2-amino-7-phosphonohepatanoate) and CPP (D,L-3[(+/-)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid), blocked the spontaneous and evoked bursts induced by NMDA. CPP, but not APV or AP-7, prevented the development of bursts induced by Mg-free medium. The NMDA antagonists failed to block bursting induced by kainate, 7 mM K+, mast cell degranulating peptide, anoxia or spontaneous bursting. In some cases the NMDA antagonists induced spontaneous bursts or enhanced burst frequency, a proconvulsant effect. It is concluded that activation of NMDA receptors is sufficient but not necessary for burst generation in the CA3 region.

Kainate reduces two voltage-dependent potassium conductances in rat hippocampal neurons in vitro

The mechanisms of action of kainate were studied in CA1 hippocampal neurons using the single electrode voltage-clamp technique in vitro. Kainate (100-200 nM) reduced the potassium current which is responsible for the anomalous rectification (IQ). In 30% of the cells the drug reduced the calcium-dependent potassium current (IC) which is responsible for the afterhyperpolarization that follows calcium action potentials. The reduction of IC will contribute to the enhancement of the neural excitability by this drug.

l-Homocysteic acid mediates synaptic excitation at NMDA receptors in the hippocampus

beta-p-Chlorophenylglutamate (Chlorpheg), a specific L-homocysteate (L-HC) uptake blocker, was tested on the L-HC- and L-glutamate-induced currents and on the excitatory postsynaptic potentials (EPSPs) evoked in CA1 rat hippocampal neurons by Schaffer collaterals stimulation. In the presence of tetrodotoxin (TTX; 1 microM), Chlorpheg (0.5-2 mM) potentiated L-HC- but not L-glutamate-induced currents. In normal magnesium containing medium and at resting membrane potential, Chlorpheg (1.5-1 mM) increased the amplitude and duration of the EPSPs evoked by Schaffer collaterals stimulation. This effect was prevented by bath application of the N-methyl-D-aspartate (NMDA) receptor antagonist CPP (20 microM). Chlorpheg enhanced also the NMDA component of the EPSP, evoked in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM), bicuculline (20 microM) and glycine (100 microM). This effect was blocked by CPP (20 microM). It is concluded that L-HC is an endogenous NMDA agonist at the Schaffer collateral-CA1 synapse.

Modulation of GABA-mediated Synaptic Potentials by Glutamatergic Agonists in Neonatal CA3 Rat Hippocampal Neurons.

Intracellular recordings were made from slices of adult and neonatal hippocampal neurons. During the first 2 weeks of life the majority of pyramidal cells exhibited spontaneous gamma‐aminobutyric acid (GABA)‐mediated synaptic potentials, which were depolarizing at birth and became hyperpolarizing by the end of the first postnatal week. These synaptic potentials were reduced in frequency or blocked by the N‐methyl‐d‐aspartate (NMDA) receptor antagonist d(‐)2‐amino‐5‐phosphonovalerate (AP‐5, 50 μM) (13/15 cells). The non‐NMDA antagonist, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 5–10μM) abolished the GABA‐mediated synaptic potentials in all the cells tested (n= 12), Superfusion of l‐glutamate (up to 100 μM) increased the frequency of both depolarizing and hyperpolarizing GABA‐mediated synaptic potentials. This effect was reduced by AP‐5 or dl‐2‐amino‐7‐phosphonoheptanoate (AP‐7, 50 μM) and fully blocked by concomitant application of AP‐5 (50 μM) and CNQX (5–10 μM). NMDA (0.5–2 μM) increased the frequency of the GABA‐mediated synaptic potentials. These effects were blocked by AP‐5 (50 μM) and by bicuculline (10 μM). Quisqualate (100–300 nM), (RS)‐alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐izopropionate (AMPA, 100–300 nM) and kainate (100 nM) also increased the frequency of the GABA‐mediated synaptic potentials. These effects were blocked by CNQX (5–10 μM) and by bicuculline (10 μM) but not by AP‐5 (50 μM). In the presence of tetrodotoxin (TTX, 1 μM), quisqualate (up to 300 nM), AMPA (up to 500 nM) and kainate (100 nM) had no effect on membrane potential or input resistance. In conclusion, our experiments suggest that, in early postnatal life, NMDA and non‐NMDA receptors located on GABAergic interneurons modulate GABAergic synaptic potentials.

Somatic and dendritic actions of γ-aminobutyric acid agonists and uptake blockers in the hippocampusin vivo

Abstract In rats under urethane anaesthesia γ-aminobutyric acid agonists and uptake blockers were microiontophoretically applied in the pyramidal layer of CA1 and in the apical dendrites using a twin set of multibarrelled micropipettes. Thus, the somatic and dendritic field potentials elicited by commissural stimulation were recorded simultaneously and the effects of iontophoretic applications at either site studied. Somatic applications of γ-aminobutyric acid, isoguvacine or muscimol produced an inhibition of the somatic population spike; this showed rapid fade and was followed by an “off” response i.e. an enhancement of the population spike discharge and the occurrence of a second (and occasionally third) spike. The order of potency with regard to the “off” response was muscimol > isoguvacine ≫γ-aminobutyric acid. In contrast, the inhibition of the population spike produced by 4, 5, 6, 7-tetrahydroisoxazolo(5, 4-C) pyridin 3-OL showed little fade and no prominent “off” response. The fade and “off” response were not associated with significant changes in the dendritic field excitatory postsynaptic potential concommittantly recorded and were exclusively restricted to the immediate vicinity of the pyramidal layer. Ejection of γ-aminobutyric acid and its agonists in the stratum radiatum produced a reduction of the field excitatory postsynaptic potential and the somatic spike, this effect however showed no fade (even during prolonged applications of high doses) and no “off” response. Somatic applications of the uptake blockers nipecotic acid or guvacine consistently produced: (a) an increase in the effectiveness of the inhibition produced by γ-aminobutyric acid and its analogues; (b) a decrease in the latency to peak of the inhibition and an increase in the time to recovery; (c) a full blockade of the fade and the “off” response. All of these effects were rapid and fully reversible without significant changes in either the field excitatory postsynaptic potential or the (control) somatic spikes. The more specific glial uptake blocker, 4, 5, 6, 7-tetrahydroisoxazolo(4, 5-C) pyridin 3-OL occasionally blocked the “off” response, however it was less potent and also tended to reduce the spike amplitude. Dendritic applications of the uptake blockers reduced the excitatory postsynaptic potential and the somatic spike but failed to produce prominent changes in the action of γ-aminobutyric acid and its analogues. Somatic, but not dendritic, applications of nipecotic acid or guvacine effectively reduced the enhancement of the spikes which is easily evoked in this preparation by an increase of the frequency of stimulation. These observations suggest that removal of γ-aminobutyric acid in the pyramidal layer is highly efficient; the possibility that this plays a role in the susceptibility of the hippocampus to epileptogenic procedures is discussed.

Antagonism of spontaneous and evoked bursts by 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) in the CA3 region of the in vitro hippocampus

Superfusion of hippocampal slices with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) antagonized kainate-induced bursts and bursts of unknown origin in the CA3 region. CNQX also increased the latency of and eventually blocked evoked bursts which persist following kainate washout. In contrast, D-(-)-2-amino-7-phosphonoheptanoic acid did not alter burst latency or block bursts unless applied subsequent to CNQX. We conclude that the quisqualate type receptor has a prominent role in burst generation with a smaller contribution from N-methyl-D-aspartate receptors.

Endogenous and network bursts induced by N-methyl-d-aspartate and magnesium free medium in the CA3 region of the hippocampal slice

The epileptogenic properties of N-methyl-D-aspartate and magnesium-free medium were investigated in the CA3 region of the hippocampal slice preparation in the rat. Bath application of N-methyl-D-aspartate (5-10 microM) or magnesium-free medium induced both spontaneous and stimulus-evoked bursts. Both endogenous and network bursts were generated, the former always preceding the latter. The paroxysmal depolarizing shift underlying the network bursts generated by N-methyl-D-aspartate and magnesium-free medium resembled a giant excitatory postsynaptic potential with a reversal potential near 0 mV and a synaptic input in the apical dendrites above the mossy fibre zone. In the presence of N-methyl-D-aspartate or magnesium-free medium, population bursts were synchronized by activating single CA3 neurons. N-methyl-D-aspartate receptor antagonists prevented the development of N-methyl-D-aspartate-induced spontaneous and stimulus-evoked bursts. However, the only N-methyl-D-aspartate receptor antagonist effective in preventing such bursts in magnesium-free medium was DL-3-[(+/-)-2-carboxypiperazin-4-yl-]-propyl-1-phosphonic acid. Endogenous bursting in the CA3 region has not been observed with other convulsants and thus may reflect the novel voltage dependence of the N-methyl-D-aspartate receptor gated ionic channel. N-methyl-D-aspartate receptors may also partially contribute to the excitatory interaction between CA3 neurons and thereby account for the synchronization of the population observed when activating single CA3 neurons.