Identification of four classes of brain nicotinic receptors using beta2 mutant mice

Abstract

Although the expression patterns of the neuronal nicotinic acetylcholine receptor (nAChR) subunits thus far described are known, the subunit composition of functional receptors in different brain areas is an ongoing question. Mice lacking the beta2 subunit of the nAChR were used for receptor autoradiography studies and patch-clamp recording in thin brain slices. Four distinct types of nAChRs were identified, expanding on an existing classification [Alkondon M, Albuquerque EX (1993) Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. I. Pharmacological and functional evidence for distinct structural subtypes. J Pharmacol Exp Ther 265:1455-1473.], and tentatively identifying the subunit composition of nAChRs in different brain regions. Type 1 nAChRs bind alpha-bungarotoxin, are not altered in beta2 -/- mice, and contain the alpha7 subunit. Type 2 nAChRs contain the beta2 subunit because they are absent in beta2 -/- mice, bind all nicotinic agonists used with high affinity (excluding alpha-bungarotoxin), have an order of potency for nicotine >> cytisine in electrophysiological experiments, and are likely to be composed of alpha4 beta2 in most brain regions, with other alpha subunits contributing in specific areas. Type 3 nAChRs bind epibatidine with high affinity in equilibrium binding experiments and show that cytisine is as effective as nicotine in electrophysiological experiments; their distribution and persistence in beta2 -/- mice strongly suggest a subunit composition of alpha3 beta4. Type 4 nAChRs bind cytisine and epibatidine with high affinity in equilibrium binding experiments and persist in beta2 -/- mice; cytisine = nicotine in electrophysiological experiments. Type 4 nAChRs also exhibit faster desensitization than type 3 nAChRs at high doses of nicotine. Knock-out animals lacking individual alpha subunits should allow a further dissection of nAChR subclasses.

Fig. 1.

Film autoradiograms of ³H-nicotine,³H-cytisine, and ³H-epibatidine binding at bregma level −1.5 mm of β2 +/+, +/−, and −/− mice. Thearrow indicates the medial habenula. Both³H-cytisine and ³H-epibatidine binding persist in the medial habenula of β2 −/− mice. However,³H-epibatidine is distributed homogeneously in the medial habenula, whereas ³H-cytisine binding is markedly more concentrated in the lateral than in the medial portion of this nucleus.CYT, Cytisine; EPI, epibatidine;NIC, nicotine.

Fig. 2.

Film autoradiograms of ³H-nicotine,³H-cytisine, and ³H-epibatidine binding at bregma level −3.4 mm of β2 +/+, +/−, and −/− mice. Thearrow and double arrow indicate the ventral and dorsal interpeduncular nucleus, respectively. Both³H-cytisine and ³H-epibatidine binding persist in the interpeduncular nucleus of β2 −/− mice. However,³H-epibatidine is distributed homogeneously in the interpeduncular nucleus, whereas ³H-cytisine binding is markedly more concentrated in the dorsal than in the ventral portion of this nucleus. For abbreviations, see the legend to Figure 1.

Fig. 3.

Film autoradiograms of³H-methylcarbamylcholine and ³H-acetylcholine binding at bregma level −3.4 mm of β2 +/+, +/−, and −/− mice. Thearrow and double arrow indicate the ventral and dorsal interpeduncular nucleus, respectively. Both ligands show persisting binding in the dorsal portion of the interpeduncular nucleus of β2 −/− mice. ACH, Acetylcholine;MCC, methylcarbamylcholine.

Fig. 4.

Film autoradiograms of ³H-nicotine,³H-cytisine, and ³H-epibatidine binding at bregma level −5.3 mm of β2 +/+, +/−, and −/− mice. Thearrow indicates the dorsal cortex of the inferior colliculus. Both ³H-cytisine and ³H-epibatidine binding persist in this area. For abbreviations, see the legend to Figure 1.

Fig. 5.

Film autoradiograms of ³H-nicotine,³H-cytisine, and ³H-epibatidine binding at bregma level −7.5 mm of β2 +/+, +/−, and −/− mice. Thearrow and double arrow indicate the dorsal motor nucleus of the vagus nerve and the area postrema, respectively. Only ³H-epibatidine binding persists in these nuclei of β2 −/− mice. For abbreviations, see the legend to Figure1.

Fig. 6.

Quantitative analysis of³H-epibatidine (EPI in A),³H-cytisine (CYT in B), and¹²⁵I-α-bungarotoxin (BTX inC) binding in β2 +/− and −/− mice. The results are expressed as the mean percentage values ± SEM of the respective +/+ mean value. Statistical analysis according to Mann–WhitneyU test; *p < 0.01 versus β2 +/+ mean value. For abbreviations, see the legend to Table 1 plus the following: CA1, CA3, hippocampal fields CA1 and CA3;DG, dentate gyrus; Hyp, hypothalamus;IC, inferior colliculus; IO, inferior olive; IPnl, interpeduncular nucleus, lateral part;PAG, periaqueductal gray; R, red nucleus;Sp5, spinal trigeminal nucleus; VCo, ventral cochlear nucleus.

Fig. 7.

Film autoradiograms of ³H-cytisine and³H-epibatidine binding and α3, α5, and β4 mRNA levels at bregma level −13.8 mm of adult rats. The arrow anddouble arrow indicate the dorsal motor nucleus of the vagus nerve and the area postrema, respectively. For abbreviations, see the legend to Figure 1.

Fig. 8.

Film autoradiograms of³H-cytisine and ³H-epibatidine binding and α3, α5, and β4 mRNA levels at bregma level −7.5 mm of adult mice. The arrow and double arrow indicate the dorsal motor nucleus of the vagus nerve and the area postrema, respectively. For abbreviations, see the legend to Figure 1.

Fig. 9.

Dose–response curve for epibatidine, nicotine, cytisine, and DMPP in the brain of β2 +/+ and β2 −/− mice.A, Antero-ventral (AV) thalamus of β2 +/+ mice.B–D, Other brain regions of β2 −/− mice: ventromedial MHb (B), (antero-) dorsal IPn (C), and DMnX (D). Eachpoint is the mean ± SEM of 2–12 measures. Normalization between different cells was performed with responses to 10 μm DMPP in the thalamus and to 10 μmnicotine in the other structures. Then the normalized currents were multiplied by the average amplitude of the responses to 10 μm nicotine and DMPP. Note the smaller amplitude of responses in the thalamus as compared with other structures. We have recorded 51 cells in the thalamus, 31 cells in the MHb, 13 cells in the DMnX, and 40 cells in the IPn. The average number of different conditions tested per cell is 4.5.

Fig. 10.

Differential sensitivity to the nicotinic antagonists methyllycaconitine (MLA), dihydro-β-erythroidine (DHβE), and mecamylamine (MCA) of the nicotinic responses in the thalamus of β2 +/+ mice and in the MHb and IPn of β2 −/− mice. The antagonists were applied 5–10 min before (and during) the application of nicotine. MLA (5 nm) had little effect on the nicotinic responses, whereas 10 μm mecamylamine totally abolished the nicotinic responses to 10 μm nicotine. The responses were totally inhibited by 1 μm DHβE in the wild-type thalamus, partially inhibited in the mutant interpeduncular nucleus (IPn), and not inhibited in the mutant medial habenula (MHb). (**), MCA application and MLA and DHbE applications were performed in two different thalamic neurons with similar control response to 10 μm DMPP.

Fig. 11.

Desensitization of nicotinic responses in the IPn and MHb of β2 −/− mice. The responses to 100 μmnicotine in the medial habenula and in the interpeduncular nucleus have been normalized. The decay of the responses was fit by a single exponential function (dashed line). In the cells shown, the desensitization time constant of the responses was 30.0 sec (MHb) and 3.95 sec (IPn).