Impaired passive avoidance learning in mice lacking central neuronal nicotinic acetylcholine receptors.

The nicotinic cholinergic system influences cognition, anxiety, locomotion, and addiction by acting upon nicotinic acetylcholine receptors (nAChRs). To date, there are 12 known neuronal mammalian nAChR subunits leading to a rich pharmacological diversity that is difficult to attribute to specific subunits. We generated alpha7-beta2 nAChR double mutant mice by breeding to investigate the effect of a minimal number of nAChRs in the CNS. These mice have been used to determine the role these receptor subunits play in a variety of behaviors. A battery of behavioral tests was used to determine the effect of the mutation in anxiety, locomotor activity, startle response, pre-pulse inhibition, motor coordination and learning and memory. Mice lacking both the alpha7 and the beta2 nAChR subunits displayed impaired learning and memory performance in a passive avoidance test and showed enhanced motor performance on the rotarod.

Effects of nicotine in the dopaminergic system of mice lacking the alpha4 subunit of neuronal nicotinic acetylcholine receptors.

The mesostriatal dopaminergic system influences locomotor activity and the reinforcing properties of many drugs of abuse including nicotine. Here we investigate the role of the alpha4 nicotinic acetylcholine receptor (nAChR) subunit in mediating the effects of nicotine in the mesolimbic dopamine system in mice lacking the alpha4 subunit. We show that there are two distinct populations of receptors in the substantia nigra and striatum by using autoradiographic labelling with 125I alpha-conotoxin MII. These receptors are comprised of the alpha4, beta2 and alpha6 nAChR subunits and non-alpha4, beta2, and alpha6 nAChR subunits. Non-alpha4 subunit-containing nAChRs are located on dopaminergic neurons, are functional and respond to nicotine as demonstrated by patch clamp recordings. In vivo microdialysis performed in awake, freely moving mice reveal that mutant mice have basal striatal dopamine levels which are twice as high as those observed in wild-type mice. Despite the fact that both wild-type and alpha4 null mutant mice show a similar increase in dopamine release in response to intrastriatal KCl perfusion, a nicotine-elicited increase in dopamine levels is not observed in mutant mice. Locomotor activity experiments show that there is no difference between wild-type and mutant mice in basal activity in both habituated and non-habituated environments. Interestingly, mutant mice sustain an increase in cocaine-elicited locomotor activity longer than wild-type mice. In addition, mutant mice recover from depressant locomotor activity in response to nicotine at a faster rate. Our results indicate that alpha4-containing nAChRs exert a tonic control on striatal basal dopamine release, which is mediated by a heterogeneous population of nAChRs.

Requirement of the nicotinic acetylcholine receptor beta 2 subunit for the anatomical and functional development of the visual system.

In the mammalian visual system the formation of eye-specific layers at the thalamic level depends on retinal waves of spontaneous activity, which rely on nicotinic acetylcholine receptor activation. We found that in mutant mice lacking the beta2 subunit of the neuronal nicotinic receptor, but not in mice lacking the alpha4 subunit, retinofugal projections do not segregate into eye-specific areas, both in the dorso-lateral geniculate nucleus and in the superior colliculus. Moreover, beta2-/- mice show an expansion of the binocular subfield of the primary visual cortex and a decrease in visual acuity at the cortical level but not in the retina. We conclude that the beta2 subunit of the nicotinic acetylcholine receptor is necessary for the anatomical and functional development of the visual system.

Altered neuroadaptation in opiate dependence and neurogenic inflammatory nociception in alpha CGRP-deficient mice.

The neuropeptide alpha CGRP (calcitonin gene-related peptide) is involved in the complex process of pain signaling, but the precise contribution of alpha CGRP remains unclear. Here we show that mice lacking alpha CGRP display an attenuated response to chemical pain and inflammation. Furthermore, alpha CGRP(-/-) mice do not show changes in heroin self-administration or morphine tolerance, but display a marked decrease in morphine withdrawal signs, suggesting an important contribution of alpha CGRP to opiate withdrawal.

Nicotinic receptor function: new perspectives from knockout mice.

Knockout mice, in which one or more genes of interest are silenced, provide unique opportunities to analyse diverse aspects of gene function in vivo. In particular, the contribution of the encoded protein(s) in complex behaviours can be assessed. Since the first targeted disruption in 1995 of the gene encoding the beta2-subunit of the nicotinic acetylcholine receptor (nAChR), all but a few of the mammalian nAChR subunits have been disrupted (i.e. alpha7, alpha4, alpha3, alpha9, beta4 and beta3). Recent advances brought by genetically modified mice to our understanding of the endogenous composition and role of nAChRs in the nervous system, and of the diverse pharmacological actions of nicotine regarding learning, analgesia, reinforcement, development and aging in the brain will be discussed.

Nicotinic acetylcholine receptor knockout mice as animal models for studying receptor function.

Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels, which are involved in a wide range of neuronal functions. During the past decade, a large number of nicotinic acetylcholine receptor subunits have been cloned and showed a discreet yet overlapping distribution pattern. Recently, several groups have produced mutant mice lacking specific nicotinic acetylcholine receptor subunits. In this review, we focus on how the study of these knockout mouse models has advanced our understanding of the role individual nicotinic acetylcholine receptor subunits play in the function and composition of endogenous receptors and the diverse pharmacological actions of nicotine in the mammalian nervous system.

Ultrastructural localization of the alpha4-subunit of the neuronal acetylcholine nicotinic receptor in the rat substantia nigra.

The distribution of the alpha4-subunit of the neuronal nicotinic acetylcholine receptor (nAChR) in the rat brain was examined at light and electron microscopy levels using immunohistochemical staining. In the present study we demonstrate the specificity, in both tissue homogenates and brain sections, of a polyclonal antibody raised against the rat nAChR alpha4-subunit. The characterization of this antibody involved: (1) Western blot analysis of rat brain homogenates and membrane extracts from cells previously transfected with diverse combinations of neuronal nAChR subunits, and (2) immunohistochemistry using transfected cells and rat brain tissue. At the light microscope level, the alpha4-subunit-like-immunoreactivity (LI) was widely distributed in the rat brain and matched the distribution of the alpha4-subunit transcripts observed previously by in situ hybridization. Strong immunohistochemical labeling was detected in the mesencephalic dopaminergic nuclei. The nAChRs in this region are thought to be responsible for the modulation of dopaminergic transmission. The neurotransmitter identity of alpha4-immunolabeled neurons in the substantia nigra pars compacta (SNpc) and the ventral tegmental area was thus assessed by investigating the possible colocalization of the nAChR alpha4-subunit with tyrosine hydroxylase using confocal microscopy. The double labeling experiments unambiguously indicated that the alpha4-subunit-LI is present in dopaminergic neurons. At the electron microscope level, the neurons in the SNpc exhibited alpha4-subunit-LI in association with a minority of postsynaptic densities, suggesting that the alpha4-subunit may be a component of functional nAChRs mediating synaptic transmission between midbrain cholinergic neurons and mesencephalic dopaminergic neurons.

Reduced antinociception in mice lacking neuronal nicotinic receptor subunits.

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.

Acetylcholine receptors containing the beta2 subunit are involved in the reinforcing properties of nicotine.

Release of the neurotransmitter dopamine in the mesolimbic system of the brain mediates the reinforcing properties of several drugs of abuse, including nicotine. Here we investigate the contribution of the high-affinity neuronal nicotinic acetylcholine receptor to the effects of nicotine on the mesolimbic dopamine system in mice lacking the beta2 subunit of this receptor. We found that nicotine stimulates dopamine release in the ventral striatum of wild-type mice but not in the ventral striatum of beta2-mutant mice. Using patch-clamp recording, we show that mesencephalic dopaminergic neurons from mice without the beta2 subunit no longer respond to nicotine, and that self-administration of nicotine is attenuated in these mutant mice. Our results strongly support the idea that the beta2-containing neuronal nicotinic acetylcholine receptor is involved in mediating the reinforcing properties of nicotine.

Isoform expression of the voltage-dependent calcium channel alpha 1E.

The alpha 1E voltage-dependent calcium channel has not been clearly identified with a specific neuronal calcium current. To help identify the role of alpha 1E, we examined differential expression of alpha 1E splice variants in mouse brain and cultured cell lines and examined the gene structure of the region encoding the amino terminal. Three splice variants were analyzed by a ribonuclease protection assay, and a fourth variant reported previously in a fetal human alpha 1E sequence was also detected in mouse brain and a pituitary cell line. Whole brain, telencephalon, and olfactory bulb contained predominantly the splice variant corresponding to alpha 1E-1 although other known variants could be detected. Neuroendocrine cells in vitro (beta TC3 insulinoma cells and AtT-20 pituitary cell lines) expressed predominantly one alpha 1E isoform. The existence of a 5' exon accounting for the origin of variant 5' ends reported in different species was suggested by the sequence of the mouse alpha 1E gene in the region encoding the amino terminal.

Structure and functional characterization of neuronal alpha 1E calcium channel subtypes.

We have cloned overlapping cDNAs encoding alpha 1E Ca2+ channel subunits from mouse and human brain. We observed that these alpha 1E transcripts were widely distributed in the central nervous system. We also demonstrated the existence of two variants of the human alpha 1E subunit. Comparison of the sequence of these alpha 1E subunits to those from other species suggests that at least four alternatively spliced variants of alpha 1E exist. Expression of human alpha 1E in HEK293 cells and Xenopus oocytes produced high voltage-activated Ca2+ currents that inactivated rapidly (tau approximately 20 ms at 0 mV). The size of the currents obtained were enhanced approximately 40-fold by co-expression with human neuronal alpha 2 and beta Ca2+ channel subunits. alpha 1E currents were insensitive to the drugs and toxins previously used to define other classes of voltage-activated Ca2+ channels. Thus, alpha 1E-mediated Ca2+ channels appear to be a pharmacologically distinct class of voltage-activated Ca2+ channels.

Spinal origin of sympathetic preganglionic neurons in the rat.

The segmental distribution of sympathetic preganglionic neurons (SPNs) and dorsal root ganglion cells (DRGs) was studied after Fluoro-gold injections into the major sympathetic ganglia and adrenal gland in rats. A quantitative assessment of the segmental and nuclear locations was made. Four general patterns of innervation were apparent: (1) a large number of SPNs (1000-2000/ganglion) innervate the sympathetic ganglia which control head or thoracic organs and a relatively small number of SPNs (100-400/ganglion) innervate the sympathetic ganglia controlling the gut, kidney, and pelvic organs; this difference in density of innervation probably relates to the level of fine control that can occur in these end organs by the SPNs; (2) the reverse pattern is seen in the DRG labeling where a large number of DRGs were labeled after Fluoro-gold injections into the preaortic ganglia (celiac, superior, and inferior mesenteric) and a small number were labeled after injections into the cervical sympathetic ganglia; (3) the intermediolateral cell column is the main source of SPNs except for the inferior mesenteric ganglion which is innervated predominantly by SPNs originating in the central autonomic nucleus (75%); the lateral funiculus is a source of SPNs mainly for the cervical sympathetic ganglia; and (4) each sympathetic ganglion and the adrenal gland receives a multisegmental SPN and DRG input with one segment being the predominant source of the innervation. The adrenal gland shows an intermediate position in terms of the density of SPN input (approximately 800 cells) and dorsal root input (approximately 300 cells); it has a widespread segmental input (T4-T12) with the T8 segment being the major source.