Do hippocampal CA1 neurones of the adult rat possess a slow inwardly-rectifying chloride conductance?

The presence of an inwardly-rectifying Cl- current was studied in hippocampal CA1 neurones using sharp intracellular microelectrodes. Following pharmacological block of the hyperpolarization-activated Ih current, a slow depolarizing sag of hyperpolarizing electrotonic potentials appeared when the microelectrode contained KCI or CsCl, but was absent with K acetate; the sag threshold was approximately 10 mV negative to rest. Under voltage clamp, slow inward current relaxations were observed on stepping to potentials between -80 and -130 mV; the activation time constant decreased with increasing hyperpolarization (>1 s at -130 mV). This conductance (termed G(Cl,slow)) was partially depressed by 100 microM Zn2+. We propose that G(Cl,slow) can contribute significantly to the resting conductance of adult hippocampal CA1 neurones only when they are loaded with Cl- via the recording electrode.

Developmental changes in localization of NMDA receptor subunits in primary cultures of cortical neurons.

Immunoblot analysis, using antibodies against distinct N-methyl-D-aspartic acid (NMDA) receptor subunits, illustrated that the NR2A and NR2B subunit proteins have developmental profiles in cultured cortical neurons similar to those seen in vivo. NR1 and NR2B subunits display high levels of expression within the first week. In contrast, the NR2A subunit is barely detectable at 7 days in vitro (DIV) and then gradually increased to mature levels at DIV21. Immunocytochemical analysis indicated that NMDA receptor subunits cluster in the dendrites and soma of cortical neurons. Clusters of NR1 and NR2B subunits were observed as early as DIV3, while NR2A clusters were rarely observed before DIV10. At DIV18, NR2B clusters partially co-localize with those of NR2A subunits, but NR2B clusters always co-localize with those of NR1 subunits. Synapse formation, as indicated by the presence of presynaptic synaptophysin staining, was observed as early as 48-72 h after plating. However, in several neurons at ages less than DIV5 where synapses were scarce, NR2B and NR1 clusters were abundant. Furthermore, while NR2B subunit clusters were seen both at synaptic and extrasynaptic sites, NR2A clusters occurred almost exclusively in front of synaptophysin-labelled boutons. This result was supported by electrophysiological recording of NMDA-mediated synaptic activity [NMDA-excitatory postsynaptic currents (EPSCs)] in developing neurons. At DIV6, but not at DIV12, CP101, 606, a NR1/NR2B receptor antagonist, antagonized spontaneously occurring NMDA-EPSCs. Our data indicate that excitatory synapse formation occurs when NMDA receptors comprise NR1 and NR2B subunits, and that NR2A subunits cluster preferentially at synaptic sites.

Increased contribution of NR2A subunit to synaptic NMDA receptors in developing rat cortical neurons.

1. Pharmacologically isolated miniature NMDA receptor-mediated excitatory postsynaptic currents (mN-EPSCs) were recorded in large visual cortical neurons in layer V of rat cortical slices. Haloperidol (100 microM) and CP101,606 (10 microM), two specific blockers of NMDA receptors comprising NR1/NR2B subunits, were tested on mN-EPSCs in rats at postnatal days 7 and 8 (P7-P8) and P13-P15. At both ages tested, no significant effects of these drugs were seen in the whole population of neurons, although in few neurons at both ages changes in amplitude were observed with haloperidol. Other dopamine receptor antagonists, spiperone and clozapine, failed to decrease mN-EPSCs in cortical neurons at P13-P15. 2. CP101,606 (10 microM) significantly decreased the amplitude of evoked N-EPSCs (eN-EPSCs) in visual cortical slices from rats at P3-P5, a developmental stage at which mRNA studies have indicated the virtual absence of NR2A mRNA. CP101,606 failed to significantly change evoked AMPA-mediated EPSCs at P5 and eN-EPSCs at P7-P8 and P13-P15. 3. NMDA receptor-mediated currents were also studied in somatic outside-out patches at P13-P15 with fast application of L-glutamate (1 mM). Haloperidol (50 microM) and CP101,606 (10 muM) blocked these currents in all patches tested. The effect of CP101,606 was concentration dependent. 4. We suggest that rather early in development synaptic receptors comprising NR1/NR2B subunits could be associated with other subunits so that blockade by haloperidol and CP101,606 is prevented. Moreover, the consistent blockade seen in outside out patches might be ascribed to the confinement of NR1/NR2B receptors to an extrasynaptic population.

The neuropeptide thyrotropin-releasing hormone modulates GABAergic synaptic transmission on pyramidal neurones of the rat hippocampal slice.

The modulatory action of thyrotropin-releasing hormone (TRH), an endogenously occurring neuropeptide, on synaptic potentials mediated by activation of GABAA or GABAB receptors was studied using intracellular recordings from CA1 pyramidal neurones of the rat hippocampal brain slice preparation. Bath-applied TRH (10 microM) produced a reversible depression of fast IPSPs (mediated by GABAA receptors) induced by electrical stimulation of the stratum lacunosum moleculare (LM) or stratum pyramidale (SP). This phenomenon was not associated with changes in the IPSP reversal potential, resting potential, or input resistance. GABAB receptor-mediated slow IPSPs elicited by SP stimulation were found insensitive to TRH whereas those induced by LM stimulation were attenuated by the peptide. AMPA receptor-mediated EPSPs and postsynaptic responses to isoguvacine or baclofen were unchanged by TRH. These data suggest that the action of TRH on GABAergic transmission was probably exerted at presynaptic level within the local circuitry comprising CA1 neurones. Such an effect of TRH represents an interesting example of transient downregulation of inhibitory processes by a physiological neuropeptide and is expected to augment excitability of pyramidal cells.

Enhancement of NMDA receptor mediated synaptic potentials of rat hippocampal neurones in vitro by thyrotropin releasing hormone.

The effects of thyrotropin releasing hormone (TRH) on excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of Schaffer collaterals on CA1 neurones of the adult rat hippocampal slice preparation were investigated using intracellular recording under current clamp conditions. At resting membrane potential and in the presence of extracellular Mg2+, TRH (10-20 microM) largely potentiated NMDA receptor-mediated EPSPs while leaving those mediated by non-NMDA receptors unaffected. This phenomenon had a brief duration (approximately 2 min) and was not accompanied by changes in resting membrane potential or input conductance. It is suggested that TRH provided a transient upregulation of synaptic responses due to NMDA receptor activation.

Electrophysiological interactions between 5-hydroxytryptamine and thyrotropin releasing hormone on rat hippocampal CA1 neurons.

Intracellular recording from CA1 neurons of the rat hippocampal slice preparation was used to examine the possibility of functional interactions between 5-hydroxytryptamine (5-HT) and thyrotropin releasing hormone (TRH), which act as cotransmitters in other areas of the central nervous system. 5-HT (30 microM) elicited complex effects consisting of biphasic changes in membrane potential and a strong depression of the afterhyperpolarization (AHP) following a spike burst. TRH (10 microM) did not alter membrane potential or input conductance but it produced a partial block of the AHP. Under single-electrode voltage clamp, 5-HT and TRH both reduced the amplitude of voltage-activated total K+ currents. When the two substances were co-applied, their actions were occluded. The voltage-activated K+ current remaining in Ca(2+)-free solution lost its sensitivity to 5-HT and TRH, suggesting that the K+ current modulated by TRH and 5-HT was Ca(2+)-dependent, although TRH itself did not depress high-threshold voltage-activated Ca2+ currents. When a relatively small concentration (5 microM) of 5-HT was co-applied with an equimolar amount of TRH, the degree of block of the spike AHP was the sum of the two individual effects of these drugs. It is suggested that in hippocampal pyramidal cells 5-HT and TRH influenced neuronal excitability by depressing a Ca(2+)-dependent K+ current, a phenomenon perhaps mediated through a common intracellular second messenger pathway.

Modulation by TRH of NMDA-elicited responses of CA1 neurones of the rat hippocampal slice preparation.

Intracellular recording from CA1 neurones of the rat hippocampal slice preparation was carried out to assess the ability of the endogenously-occurring neuropeptide thyrotropin-releasing hormone (TRH) to modulate responses elicited by the excitatory amino acid agonist N-methyl-D-aspartate (NMDA). TRH (5 microM) produced no change in resting membrane potential or input resistance but facilitated on-going synaptic activity. In the continuous presence of the peptide responses to NMDA were selectively enhanced for about 20 min. In approximately 50% of cells the potentiating effect of TRH persisted in tetrodotoxin (TTX) solution and was associated with removal of the apparent voltage-dependent increase in input resistance usually found during the NMDA-induced depolarization. It is suggested that TRH evoked a transient upregulation of NMDA responses which might account for the reported facilitation by this peptide of long term potentiation in the hippocampus.