Α4-containing nAChRs Research Articles

Altered baroreflex responses in α7 deficient mice

The autonomic nervous system controls and coordinates several cardiovascular functions, including heart rate, arterial pressure, blood flow and vasomotor tone. Neuronal nicotinic acetylcholine receptors (nAChRs) are the interface between the nervous system and the cardiovascular system, but it is not known which nAChR subtypes regulate autonomic function in vivo. Nicotinic AChRs containing the α7 subunit are a candidate subtype in autonomic ganglia. Stimulation of these nAChRs can increase neurotransmitter release via presynaptic mechanisms, as well as mediate fast synaptic transmission via postsynaptic mechanisms. To investigate the role of the α7 nAChR subunit in cardiac autonomic function, we measured baroreflex-mediated responses in α7 null mice. Here we show that the α7 null mice have impaired sympathetic responses to vasodilatation, as sodium nitroprusside infusion triggered a 48% heart rate increase in wild type mice but only a 21% increase in the α7 nulls ( P<0.001). The mutant mice developed supersensitivity to adrenergic agonists, although norepinephrine release from sympathetic nerve terminals could be elicited through mechanisms alternative to nAChR stimulation. Baroreflex-mediated parasympathetic responses were normal in α7 null mice. The decreased baroreflex-mediated tachycardia in α7 mutant mice indicates that α7-containing nAChRs participate in the autonomic reflex that maintains blood pressure homeostasis. The α7 mutant mice may serve as a model of baroreflex impairment arising from autonomic dysfunction.

Reduced nicotinic receptor-mediated antinociception following in vivo antisense knock-down in rat

Pharmacological activation of neuronal nicotinic acetylcholine receptors can produce non-opioid antinociception in rodents. However, multiple nAChR subtypes exist, the most abundant of which contain α4 and β2 subunits. The purpose of the present study was to investigate the role of α4-containing nAChRs in mediating nicotinic antinociception using an in vivo antisense strategy. Both i.c.v. infusion and repeated bolus injections into the cerebral aqueduct of an antisense oligonucleotide against the α4 subunit significantly attenuated the antinociceptive effects of the nAChR agonist A-85380 in the paw withdrawal test of acute thermal pain. Rats treated with a scrambled oligonucleotide displayed a full antinociceptive response to A-85380, while discontinuing antisense treatment restored the antinociceptive effects of the nicotinic agonist. Double immunohistochemical labeling revealed near-complete overlap of expression of the serotonin marker tryptophan hydroxylase and the α4 nAChR subunit in the dorsal raphe nucleus. The expression of α4-containing nAChRs by serotonergic neurons in the dorsal raphe offered a means to address nonspecific α4 knock-down, i.e., oligonucleotide-induced neurotoxicity. Immunohistochemical detection of α4 expression was reduced by nearly 50% in the dorsal raphe of antisense-treated rats as compared to either saline or missense-treated controls. In contrast, the expression of tryptophan hydroxylase, as well as, the α7 nAChR subunit in antisense-infused rats was similar to that observed in saline- and missense-treated controls. The results of these studies suggest that α4-containing nAChRs, possibly expressed by serotonergic neurons, are involved in nicotinic-mediated analgesia. However, these data do not eliminate the possibility that other nicotinic subunit combinations may also play a role in antinociception produced by nAChR activation.

Inhibition and disinhibition of pyramidal neurons by activation of nicotinic receptors on hippocampal interneurons.

Nicotinic acetylcholine receptors (nAChRs) are expressed in the hippocampus, and their functional roles are beginning to be delineated. The effect of nAChR activation on the activity of both interneurons and pyramidal neurons in the CA1 region was studied in rat hippocampal slices. In CA1 stratum radiatum with muscarinic receptors inhibited, local pressure application of acetylcholine (ACh) elicited a nicotinic current in 82% of the neurons. The majority of the ACh-induced currents were sensitive to methyllycaconitine, which is a specific inhibitor of alpha7-containing nAChRs. Methyllycaconitine-insensitive nicotinic currents also were present as detected by a nonspecific nAChR inhibitor. The ACh-sensitive neurons in the s. radiatum were identified as GABAergic interneurons by their electrophysiological properties. Pressure application of ACh induced firing of action potentials in approximately 70% of the interneurons. The ACh-induced excitation of interneurons could induce either inhibition or disinhibition of pyramidal neurons. The inhibition was recorded from the pyramidal neuron as a burst of GABAergic synaptic activity. That synaptic activity was sensitive to bicuculline, indicating that GABA(A) receptors mediated the ACh-induced synaptic currents. The disinhibition was recorded from the pyramidal neuron as a reduction of spontaneous GABAergic synaptic activity when ACh was delivered onto an interneuron. Both the inhibition and disinhibition were sensitive to either methyllycaconitine or mecamylamine, indicating that activation of nicotinic receptors on interneurons was necessary for the effects. These results show that nAChRs are capable of regulating hippocampal circuits by exciting interneurons and, subsequently, inhibiting or disinhibiting pyramidal neurons.

Expression of nicotinic acetylcholine receptor subunits in the cerebral cortex in Alzheimer’s disease: histotopographical correlation with amyloid plaques and hyperphosphorylated‐tau protein

Impairment of cholinergic transmission and decreased numbers of nicotinic binding sites are well-known features accompanying the cognitive dysfunction seen in Alzheimer's disease (AD). In order to elucidate the underlying cause of this cholinoceptive dysfunction, the expression of two pharmacologically different nicotinic acetylcholine receptor (nAChR) subunits (alpha4, alpha7) was studied in the cerebral cortex of Alzheimer patients as compared to controls. Patch-clamp recordings of 14 dissociated neurons of control cortices showed responses suggesting the existence of alpha4- and alpha7-containing functional nAChRs in the human cortex. In cortices of Alzheimer patients and controls, the pattern of distribution and the number of alpha4 and alpha7 mRNA-expressing neurons were similar, whereas at the protein level a decrease in the density of alpha4- and alpha7-expressing neurons of approximately 30% was observed in Alzheimer patients. The histotopographical correlation of nAChR expression with accompanying pathological changes, e.g. accumulation of hyperphosphorylated-tau (HP-tau) protein and beta-amyloid showed that neurons in the vicinity of beta-amyloid plaques bore both nAChR transcripts. Neurons heavily labelled for HP-tau, however, expressed little or no alpha4 and alpha7 mRNA. These results point to an impaired synthesis of nAChRs on the protein level as a possible cause of the cholinoceptive deficit in AD. Further investigations need to elucidate whether interactions of HP-tau with nAChR mRNA, or alterations in the quality of alpha4 and alpha7 transcripts give rise to decreased protein expression at the level of individual neurons.

Nicotinic Stimulation Produces Multiple Forms of Increased Glutamatergic Synaptic Transmission

Synaptic modulation and long-term synaptic changes are thought to be the cellular correlates for learning and memory (Madison et al., 1991; Aiba et al., 1994, Goda and Stevens, 1996). The hippocampus is a center for learning and memory that receives abundant cholinergic innervation and has a high density of nicotinic acetylcholine receptors (nAChRs) (Wada et al., 1989; Woolf, 1991). We report that stro ng, brief stimulation of nAChRs enhanced hippocampal glutamatergic synaptic transmission on two independent time scales and altered the relationship between consecutively evoked synaptic currents. The nicotinic synaptic enhancement required extracellular calcium and was produced by the activation of presynaptic alpha7-containing nAChRs. Although one form of glutamatergic enhancement lasted only for seconds, another form lasted for minutes after the nicotinic stimulation had ceased and the nicotinic agonist had been washed away. The synaptic enhancement lasting minutes suggests that nAChR activity can initiate calcium-dependent mechanisms that are known to induce glutamatergic synaptic plasticity. The results with evoked synaptic currents showed that nAChR activity can alter the relationship between the incoming presynaptic activity and outgoing postsynaptic signaling along glutamatergic fibers. Thus, the same information arriving along the same glutamatergic afferents will be processed differently when properly timed nicotinic activity converges onto the glutamatergic presynaptic terminals. Influencing information processing at glutamatergic synapses may be one way in which nicotinic cholinergic activity influences cognitive processes. Disruption of these nicotinic cholinergic mechanisms may contribute to the deficits associated with the degeneration of cholinergic functions during Alzheimer's disease.

Role of the Nucleus Raphe Magnus in Antinociception Produced by ABT-594: Immediate Early Gene Responses Possibly Linked to Neuronal Nicotinic Acetylcholine Receptors on Serotonergic Neurons

Recently, a novel cholinergic channel modulator, (R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594), was shown to produce potent analgesia in a variety of rodent pain models when administered either systemically or centrally into the nucleus raphe magnus (NRM). The purpose of the present study was to investigate the possible supraspinal contribution of ABT-594 by assessing its ability to induce expression of the immediate early gene c-fos, a biochemical marker of neuronal activation, in the NRM of rats. Putative serotonergic neurons in the NRM, a medullary nucleus proposed to be involved in descending antinociceptive pathways, were identified immunohistochemically using a monoclonal antibody (mAb) against tryptophan hydroxylase. ABT-594 (0.03-0.3 micromol/kg, i.p.) produced a dose-dependent induction of Fos protein that was blocked by the central nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine (5 micromol/kg, i.p.) but not by the peripheral nAChR antagonist hexamethonium (15 micromol/kg, i.p.). Immunohistological studies using mAb 299 revealed the expression of alpha4-containing nAChRs in the NRM. The alpha4 immunostaining was dramatically reduced by pretreating (30 d) animals with the serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), which was previously shown to substantially attenuate the antinociceptive actions of ABT-594. In a double immunohistochemical labeling experiment, coexpression of the serotonin marker tryptophan hxdroxylase and the alpha4 nAChR subunit in NRM neurons was observed. These results suggest that the analgesic mechanism of ABT-594 may in part involve the activation of the NRM, a site where alpha4-containing nAChRs are expressed by serotonergic neurons.

Functional contribution of the alpha7 subunit to multiple subtypes of nicotinic receptors in embryonic chick sympathetic neurones.

1. Many studies of the alpha7 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) family have demonstrated that this alpha-bungarotoxin (alpha-BgTx)-binding neuronal receptor can participate in ACh-gated channels. Heterologous expression studies reveal that alpha7 subunits form homomeric channels of unusually high Ca2+ permeability. However, the physiological role of the alpha7 subunit in native neuronal nAChR channels is less clear. 2. We present evidence that the alpha7 subunit contributes to the function of at least three subtypes of native nAChR expressed by embryonic chick sympathetic neurones. These subtypes are functionally distinct from heterologously expressed homomeric alpha7 nAChRs as well as homomeric-like currents described in studies of hippocampal and parasympathetic neurones. 3. The proposed nAChRs differ from one another and from homomeric alpha7 nAChRs in their sensitivity to block by alpha7 subunit-specific antagonists: alpha-BgTx and methyllycaconitine (MLA). While MLA blocks 60 % of the macroscopic ACh response, alpha-BgTx inhibits a small component of the macroscopic current described by slow-on and slow-off kinetics. 4. Functional deletion of the alpha7 subunit by antisense oligonucleotide treatment eliminates the susceptibility of the nAChRs to block by both MLA and alpha-BgTx. 5. Single channel recordings combined with pharmacological and antisense-mediated 'deletion' techniques reveal that alpha-BgTx-sensitive alpha7-containing nAChRs have a small unitary conductance (18 pS), brief open time kinetics and relatively low open probability (Po). MLA-sensitive alpha7 nAChRs are characterized by a conductance of approximately 35 pS, intermediate burst duration, and a relatively high Po. 6. The third nAChR subtype deleted by alpha7 antisense treatment is characterized by a unitary conductance of 50 pS and prolonged opening duration. 7. We propose that these three populations of native alpha7-containing nAChRs are distinct heteromeric complexes that include other alpha and/or beta nAChR subunits.

Acetylcholine Activates an α-Bungarotoxin-Sensitive Nicotinic Current in Rat Hippocampal Interneurons, But Not Pyramidal Cells

The effects of acetylcholine on both pyramidal neurons and interneurons in the area CA1 of the rat hippocampus were examined, using intracellular recording techniques in an in vitro slice preparation. In current-clamp mode, fast local application of acetylcholine (ACh) to the soma of inhibitory interneurons in stratum radiatum resulted in depolarization and rapid firing of action potentials. Under voltage-clamp, ACh produced fast, rapidly desensitizing inward currents that were insensitive to atropine but that were blocked by nanomolar concentrations of the nicotinic alpha7 receptor-selective antagonists alpha-bungarotoxin (alphaBgTx) and methyllycaconitine. Nicotinic receptor antagonists that are not selective for alpha7-containing receptors had little (mecamylamine) or no effect (dihydro-beta-erythroidine) on the ACh-induced currents. Glutamate receptor antagonists had no effect on the ACh-evoked response, indicating that the current was not mediated by presynaptic facilitation of glutamate release. However, the current could be desensitized almost completely by bath superfusion with 100 nM nicotine. In contrast to those actions on interneurons, application of ACh to the soma of CA1 pyramidal cells did not produce a detectable current. Radioligand-binding experiments with [125I]-alphaBgTx demonstrated that stratum radiatum interneurons express alpha7-containing nAChRs, and in situ hybridization revealed significant amounts of alpha7 mRNA. CA1 pyramidal cells did not show specific binding of [125I]-alphaBgTx and only low levels of alpha7 mRNA. These results suggest that, in addition to their proposed presynaptic role in modulating transmitter release, alpha7-containing nAChRs also may play a postsynaptic role in the excitation of hippocampal interneurons. By desensitizing these receptors, nicotine may disrupt this action and indirectly excite pyramidal neurons by reducing GABAergic inhibition.

Mice Deficient in the α7 Neuronal Nicotinic Acetylcholine Receptor Lack α-Bungarotoxin Binding Sites and Hippocampal Fast Nicotinic Currents

The alpha7 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) is abundantly expressed in hippocampus and is implicated in modulating neurotransmitter release and in binding alpha-bungarotoxin (alpha-BGT). A null mutation for the alpha7 subunit was prepared by deleting the last three exons of the gene. Mice homozygous for the null mutation lack detectable mRNA, but the mice are viable and anatomically normal. Neuropathological examination of the brain revealed normal structure and cell layering, including normal cortical barrel fields; histochemical assessment of the hippocampus was also normal. Autoradiography with [3H]nicotine revealed no detectable abnormalities of high-affinity nicotine binding sites, but there was an absence of high-affinity [125I]alpha-BGT sites. Null mice also lack rapidly desensitizing, methyllycaconitine-sensitive, nicotinic currents that are present in hippocampal neurons. The results of this study indicate that the alpha-BGT binding sites are equivalent to the alpha7-containing nAChRs that mediate fast, desensitizing nicotinic currents in the hippocampus. These mice demonstrate that the alpha7 subunit is not essential for normal development or for apparently normal neurological function, but the mice may prove to have subtle phenotypic abnormalities and will be valuable in defining the functional role of this gene product in vivo.

Immunocytochemical localization of nicotinic acetylcholine receptor in the rat cerebellar cortex

Although nicotinic acetylcholine receptors (nAChRs) have been reported to function in the cerebellar cortex, nicotinic acetylcholine receptor subtypes contributing to nicotinic currents in the cortex have not been well characterized at the subcellular level. This study deals with immunocytochemical localization of the nicotinic acetylcholine receptor α4 subunit using a monoclonal antibody against the α4 subunit. α4-LI (α4-like immunoreactivity) was detected in the cell bodies of molecular, Purkinje cell and granular layers. In particular, the cell bodies of Purkinje cells were extensively immunostained. In Purkinje cells, α4-LI was found in perikarya mainly associated with rough endoplasmic reticulum, plasma membrane, and cytoplasmic matrix. At higher magnification, the immunoreaction product was densely localized along with somatic plasma membranes at the axo-somatic synapse and the plasma membranes at extrasynaptic regions of cell bodies. α4-LI was also found in the axon terminals which form synapses with Purkinje cells. In the granular layer, somatic cell membranes of granular cells were immunostained. These morphological observations suggest that α4-containing nAChRs contribute nicotinic currents reported in Purkinje cells, and that presynaptic α4-containing nAChRs regulate the release of neurotransmitters on the axon terminals found near Purkinje cells.

α-Conotoxin MII Blocks Nicotine-Stimulated Dopamine Release in Rat Striatal Synaptosomes

Activation of presynaptic nicotinic acetylcholine receptors (nAChRs) can induce the release of neurotransmitters such as dopamine and norepinephrine in the CNS. Accumulating evidence suggests that distinct nAChR subtypes are involved; however, it has been difficult to determine the subunit composition of these receptors, in part because of the lack of a sufficient variety of selective nAChR ligands. We present experimental data that at least two different nAChR complexes are involved in dopamine release, one of which has an alpha3/beta2 subunit interface. The recently discovered peptide alpha-conotoxin MII is a potent and selective inhibitor of rat nAChRs containing an interface formed by alpha3 and beta2 subunits. We used this peptide to examine nicotine-stimulated release of dopamine from rat striatal synaptosomes and of norepinephrine from hippocampal synaptosomes. MII (100 nM) blocks 34-49% of the nicotine-stimulated dopamine release, but not dopamine release evoked by elevated [K+]. Furthermore, two peptides structurally related to alpha-conotoxin MII, namely alpha-conotoxin MI (selective for alpha1beta1gammadelta nAChRs) and alpha-conotoxin ImI (selective for alpha7-containing nAChRs), have no effect on nicotine-stimulated dopamine release. The results indicate that one third to half of the dopamine release in the striatal preparation is mediated by nAChRs with an alpha3/beta2 subunit interface. In contrast, </=10% of nicotine-stimulated release of norepinephrine from hippocampal synaptosomes is modulated by nAChRs with alpha3/beta2 subunit interfaces.