Paired-pulse potentiation of alpha7-containing nAChRs in rat hippocampal CA1 stratum radiatum interneurones

Abstract

Diverse subtypes of nicotinic acetylcholine receptors (nAChRs), including fast-desensitizing alpha7-containing receptors, are expressed in the CNS. While nAChRs appear to regulate cognitive processing and synaptic plasticity, little is known to date about how this regulation occurs, particularly in brain regions known to be important for cognition. By combining patch-clamp electrophysiology with local photolysis of caged carbachol to rapidly activate the alpha7-containing nAChRs in rat hippocampal CA1 stratum radiatum interneurones in slices, we describe a novel transient up-regulation of channel function. The nAChRs were activated using a paired-pulse uncaging protocol, where the duration of the UV laser pulses (5-25 ms) and the interval between pulses (200 ms to 30 s) were varied. At relatively long interpulse intervals, we observed a strong (> 75%) decrease in the amplitude of the second response due to desensitization. However, when two pulses were applied at a 200 ms interval, a > 3-fold increase in the amplitude of the second response was observed, a phenomenon referred to here as paired-pulse potentiation. Interestingly, this potentiation appeared to be regulated by [Ca2+]i, and/or Ca2+-dependent processes, as it was significantly enhanced by dialysing cells with either the Ca2+ chelator BAPTA, or with peptide inhibitors of either calcineurin or PKC, and was attenuated by dialysing cells with the CaMKII inhibitor KN-93. No potentiation was observed using caged GABA or glutamate, indicating some specificity for nAChRs. Thus, rat hippocampal alpha7-containing nAChRs possess a newly described phenomenon of paired-pulse potentiation that may be involved in regulating synaptic plasticity in the hippocampus.

Figure 1. Paired-pulse activation of nAChRs in hippocampal interneurones is potentiated at a 200 ms pulse interval

A, representative traces of α7-containing nAChR responses from a single CA1 hippocampal interneurone evoked by photolysis of cCarb (500 μm) by 15 ms UV laser pulses at varying intervals. The interpulse intervals are shown above the traces; for the bottom traces, the interval between the first pulse (shown on the left) and second are indicated above the hash marks. Scale bars are 50 pA and 250 ms. B, plot of the ratio of the amplitude of the response evoked by the second uncaging pulse to the first uncaging pulse (Resp2/Resp1) versus pulse interval. Data are means ± s.e.m. from 7–10 interneurones for each data point.

Figure 2. Paired-pulse potentiation of nAChRs in hippocampal interneurones is dependent on pulse duration

A, representative traces of α7-containing nAChR responses from a single CA1 hippocampal interneurone evoked by photolysis of cCarb (500 μm) while varying the UV laser pulse durations (5–25 ms) and maintaining a constant 200 ms interval. Scale bars are 100 pA and 100 ms. B, plot of Resp2/Resp1 ratio versus pusle duration. Data are means ± s.e.m. from 19–23 interneurones.

Figure 3. Paired-pulse activation of GABA and glutamate receptors

Representative traces of interneurones responding to photolysis of 500 μm cGlu (A) or 2 mm cGABA (B). Varying paired-pulse durations (1–25 ms, as indicated above traces) were delivered at a 200 ms interval (n≥ 4 for each transmitter). Scale bars are 50 pA and 100 ms for cGlutamate, and 50 pA and 250 ms for cGABA.

Figure 4. Paired-pulse potentitation of nAChRs in hippocampal interneurones is regulated by [Ca²⁺]_I

A, representative traces illustrating paired-pulse potentiation evoked by a 5 ms pulse duration at a 200 ms pulse interval in a control cell (left) and in another cell dialysed with 20 mm BAPTA in the patch pipette (right). Scale bars are 50 pA and 200 ms. B, plot of Resp2/Resp1 ratio versus pulse duration using a 200 ms interval (left) or versus pulse interval using a 15 ms duration (right) for comparison of control (▪) and BAPTA-dialysed cells (•). Data are mean ± s.e.m. from 10 interneurones for BAPTA. Significant differences are indicated by an asterisk.

Figure 5. Paired-pulse potentitation of nAChRs is regulated by intracellular signalling pathways

Plot of Resp2/Resp1 ratio versus pulse duration for control, or in the presence of either the calcineurin autoinhibitory peptide (CaN-AIP; 300 μm), or the PKC inhibitory peptide fragment 19-31 (PKC-I_19–31; 200 μm), or a CaMKII inhibitor (KN-93; 5 μm). Data are means ± s.e.m. from 10 interneurones for CaN-AIP and PKC-I_19–31, and 8 interneurones for KN-93.