Nicotine exerts antinociceptive effects by interacting with one or more of the subtypes of nicotinic acetylcholine receptors (nAChRs) that are present throughout the neuronal pathways that respond to pain. To identify the particular subunits involved in this process, we generated mice lacking the alpha4 subunit of the neuronal nAChR by homologous recombination techniques and studied these together with previously generated mutant mice lacking the beta2 nAChR subunit. Here we show that the homozygous alpha4-/- mice no longer express high-affinity [3H]nicotine and [3H]epibatidine binding sites throughout the brain. In addition, both types of mutant mice display a reduced antinociceptive effect of nicotine on the hot-plate test and diminished sensitivity to nicotine in the tail-flick test. Patch-clamp recordings further reveal that raphe magnus and thalamic neurons no longer respond to nicotine. The alpha4 nAChR subunit, possibly associated with the beta2 nAChR subunit, is therefore crucial for nicotine-elicited antinociception.

Olfactory and vomeronasal projections have been traditionally viewed as terminating in contiguous non-overlapping areas of the basal telencephalon. Original reports, however, described areas such as the anterior medial amygdala where both chemosensory afferents appeared to overlap. We addressed this issue by injecting dextran amines in the main or accessory olfactory bulbs of rats and the results were analyzed with light and electron microscopes. Simultaneous injections of different fluorescent dextran amines in the main and accessory olfactory bulbs were performed and the results were analyzed using confocal microscopy. Similar experiments with dextran amines in the olfactory bulbs plus FluoroGold in the bed nucleus of the stria terminalis indicate that neurons projecting through the stria terminalis could be integrating olfactory and vomeronasal inputs. Retrograde tracing experiments using FluoroGold or dextran amines confirm that areas of the rostral basal telencephalon receive inputs from both the main and accessory olfactory bulbs. While both inputs clearly converge in areas classically considered olfactory-recipient (nucleus of the lateral olfactory tract, anterior cortical amygdaloid nucleus, and cortex-amygdala transition zone) or vomeronasal-recipient (ventral anterior amygdala, bed nucleus of the accessory olfactory tract, and anteroventral medial amygdaloid nucleus), segregation is virtually complete at posterior levels such as the posteromedial and posterolateral cortical amygdalae. This provides evidence that areas so far considered receiving a single chemosensory modality are likely sites for convergent direct olfactory and vomeronasal inputs. Therefore, areas of the basal telencephalon should be reclassified as olfactory, vomeronasal, or mixed chemosensory structures, which could facilitate understanding of olfactory-vomeronasal interactions in functional studies.

The neurons of the locus ceruleus are responsible for most of the noradrenergic innervation in the brain and nicotine potentiates noradrenaline release from their terminals. Here we investigated the diversity and subcellular distribution of nicotinic acetylcholine receptors (nAChRs) in the locus ceruleus both somatically, by combining single-cell reverse transcription-PCR with electrophysiological characterization, and at the level of nerve terminals, by conducting noradrenaline efflux experiments. The proportion of neurons in the locus ceruleus expressing the nicotinic subunit mRNAs varied from 100% (␤2) to 3% (␣2). Yet, two populations of neurons could be distinguished on the basis of the pattern of expression of nAChR mRNAs and electrophysiological properties. One population (type A) of small cells systematically expressed ␣3 and ␤4 mRNAs (and often ␣6, ␤3, ␣5, ␣4), and nicotinic agonists elicited large currents with a potency order of cytisine > nicotine. Another population (type B) of cells with large soma did not contain ␣3 and ␤4 mRNAs but, systematically, ␣6 and ␤3 (and often ␣4) and responded to nicotinic agonists in the order of nicotine > cytisine. The nicotinic modulation of noradrenaline release in the hippocampus displayed an order of potency nicotine > cytisine, suggesting that noradrenergic terminals in the hippocampus originate largely from type B cells of the locus ceruleus. Accordingly, immunocytochemical labeling showed that ␤3 is present in hippocampal terminals. The ␣6␤3␤2(␣4) heterooligomer thus behaves as the main nicotinic regulator of the ceruleo-hippocampal pathway. Brain nicotinic acetylcholine receptors (nAChRs) are heteropentameric ligand-gated ion channels that result from the combinatorial assembly of up to nine different subunits (␣2-7, ␤2-4) (1). These subunits are differentially expressed throughout the central nervous system (2-6) and contribute to a wide diversity of brain functions and pathologies (7,8). The large number of possible subunit combinations allows the formation of many different subtypes of nAChRs. However, the small number of subunits expressed by a single neuron restrains the number of possible combinations of subunit. Furthermore, preferential association of subunits has been demonstrated in neurons containing a large number of nAChR subunits (9).Immunoprecipitation studies have demonstrated the existence of two main subtypes of nAChRs in the mammalian brain: ␣4␤2 heteromers (10) and ␣7 homomers (11). Several other associations have been demonstrated in chicken autonomic ganglia, such as ␣3␤4(␤2)␣5 (12), and in chicken retina, such as ␣4␤2␣5 (13) and ␣6␤4␤3 (14). Furthermore, only a limited number of pharmacological tools is available to discriminate between various forms of nAChR in the nervous system. However, at least three pharmacological classes of nAChRs may be distinguished on the basis of a subunit composition including ␣7, ␤2, or ␤4 (15, 16). Cytisine is a partial agonist in ␤2-containing nAChRs but it is more efficient than nicotine in ␤4-containing...