Regulation of the sensitivity of acetylcholine receptors to nicotine in rat habenula neurons.

Time-dependent changes in nicotinic acetylcholine receptor (nAChR) function were studied in acutely isolated medial habenula neurons during whole-cell perfusion. The peak amplitude of inward currents induced by 1 s pulses of nicotinic agonists, applied at 30 s intervals, gradually increased over the first several minutes of whole-cell recording. The ratio of response amplitudes at 1 and 15 min (t15/t1) was 1.9. Run-up of responses occurred independently of channel activation and was specific to nAChRs. The channel blocker chlorisondamine (30 μM), co-applied with nicotine, was used to irreversibly block the majority (91 %) of the nAChRs that opened in the first 2 min of recording. Run-up in the remaining 9 % unblocked channels assessed at 15 min (t15/t2= 3.4) was similar to that in control cells not exposed to nicotine and chlorisondamine simultaneously, implying that run-up is not due to the incorporation of new receptors. A marked alteration in the sensitivity of nAChRs to extracellular Ca2+ was also observed during whole-cell perfusion. The ratio of current amplitudes obtained in 0.2 and 4.0 mM Ca2+ changed from 0.54 (t= 5 min) to 0.82 (t= 30 min). Inward rectification of nicotine-induced responses was reduced during internal dialysis. Voltages for half-maximal conductance were -23.0 and -13.8 mV at 2 and 15 min, respectively. Inclusion of either free Mg2+ (∼2 mM) or spermine (100 μM) in the internal solution counteracted the change in rectification, but did not prevent run-up. The period of run-up was followed by a use-dependent run-down phase. Little run-down in peak current amplitude was induced provided that agonist was applied infrequently (5 min intervals), whereas applications at 30 s intervals produced a loss of channel function after ∼15 min whole-cell perfusion. The time at which run-down began (∼5-30 min) was correlated with the initial rate of nAChR desensitization (∼200-4000 ms); slowly desensitizing nicotinic currents demonstrated delayed run-down. We suggest that run-up of nAChR-mediated responses does not require receptor activation and may result from a change in channel open probability. We also hypothesize that channel run-down reflects accumulation of nAChRs in long-lived desensitized/inactivated states. Neuronal nicotinic acetylcholine receptors (nAChRs) are a molecularly diverse group of ion channels localized to pre- and postsynaptic sites in the brain (Sargent, 1993; McGehee & Role, 1995; Lindstrom et al. 1996; Role & Berg, 1996; Colquhoun & Patrick, 1997; Wonnacott, 1997; Jones et al. 1999). In addition to conventional roles in synaptic transmission, nAChRs are implicated in certain forms of plasticity by virtue of their relatively high Ca2+ permeability (Mulle et al. 1992a; Vernino et al. 1992; Lena & Changeux, 1997; Radcliffe & Dani, 1998). nAChRs are themselves amenable to both short- and long-term cellular regulation, although the extent of such modulation is at present unclear (Swope et al. 1992; Role & Berg, 1996). For a complete understanding of the influence of nAChRs at synapses, knowledge of how the function of these channels is controlled will be essential. The extracellular environment, in particular the local concentration of Ca2+, has marked short-term effects on nAChR properties. Elevation of Ca2+ levels can increase the channel open probability (Popen) severalfold (Mulle et al. 1992b; Vernino et al. 1992) and can change the contribution of synaptically induced nicotinic currents to signalling in sympathetic neurons (Amador & Dani, 1995). The peptide transmitters, substance P and vasoactive intestinal peptide (VIP), affect the desensitization properties and increase peak currents through ganglionic nAChRs, respectively, via activation of various second messengers (Role, 1984; Downing & Role, 1987; Gurantz et al. 1994; Cuevas & Adams, 1996). The latter result may involve changes in the number of functional channels on the cell surface (Margiotta et al. 1987), and therefore provides a mechanism for long-term alteration of neuronal excitability. In the CNS, these types of receptor modulation may be relevant in cognitive processing (Levin & Simon, 1998) and could contribute to deficits in nAChR number and function that occur during nicotine addiction (Marks et al. 1983) and Alzheimer’s disease (Norberg, 1994). Extensive use of the whole-cell patch-clamp recording technique has led to a greater understanding of intracellular control of ion channel function (Horn & Korn, 1992). By allowing exchange of the contents of the patch pipette and the intracellular milieu, and by permitting the selective introduction of various molecules into the cytoplasm, there have been remarkable insights into the internal regulation of voltage-gated ion channels, e.g. Ca2+ channels (Chad & Eckert, 1986), and ligand-gated ion channels, e.g. N-methyl-D-aspartate (NMDA) receptors (MacDonald et al. 1989; Rosenmund & Westbrook, 1993) and neuronal nAChRs (Liu & Berg, 1999). The present study has examined the modulation of nAChRs during whole-cell and perforated-patch recordings from medial habenula (MHb) neurons (McCormick & Prince, 1987; Mulle & Changeux, 1990; Lester & Dani, 1995a). In the present study, we show that the intracellular environment is important for regulation of the function of nAChRs including their ability to sense changes to extracellular Ca2+. Preliminary reports of this work have appeared in abstract form (Lester & Dani, 1995b; Hicks & Lester, 1999).