Α4 nAChR Subunits Research Articles

Acetylcholine and Alcohol Sensitivity of Neuronal Nicotinic Acetylcholine Receptors: Mutations in Transmembrane Domains

Background The effect of n-alcohols on glycine and γ-aminobutyric acid type A receptors depends on two specific amino acids (AAs) located in the transmembrane domains (TM) 2 and 3. Our aim was to assess whether the corresponding AAs in the neuronal nicotinic acetylcholine receptor (nAChR) also formed a binding pocket for alcohols. Methods We made single AA substitutions in the homologous sites in rat neuronal nAChR α2 and α4 (αL261 and αL283) and expressed them in Xenopus laevis oocytes in combination with β4 wild type. The effect of different n-alcohols was studied in α4(L261A)β4 and α4(L283A)β4 nAChRs. The effect of ethanol, propanol, and octanol on acetylcholine (ACh) responses was studied in α2(L261X)β4 and α2(L283X)β4 nAChRs. Results Most of the mutations in the α2 subunit, in either the 261 or the 283 position, induced changes in ACh sensitivity and increased alcohol action, but none was able to reduce ethanol potentiation. In α4(L283A)β4, enhancement of potentiation by short-chain alcohols was observed, as well as a change from inhibition to potentiation for long-chain alcohols. The exposure of the AAs was assessed through the action of a charged thiol-specific reagent on α2(L261C)β4 and α2(L283C)β4, and these experiments suggest that the AA in TM2 is located in a water-accessible position, whereas the AA in TM3 is inaccessible. However, a noncharged thiol-specific reagent did not affect either ACh responses or ethanol effect on α2(L261C)β4. Conclusions The AAs located at positions 261 and 283 of the α2 and α4 nAChR subunits do not seem to form a binding pocket for alcohols. Additional studies are required to determine whether alcohols act on a site near these AAs or on sites unrelated to the TM2-TM3 site found in glycine and γ-aminobutyric acid type A receptors.

Alpha-7 nicotinic receptor expression by two distinct cell types in the dorsal raphe nucleus and locus coeruleus of rat

The α7 nicotinic acetylcholine receptor (nAChR) subunit can be assembled to form a homomeric-pentamer with high permeability to calcium. Although the expression of the α7-nAChR has been demonstrated throughout the CNS, the neurochemical phenotype of neurons expressing α7 remains to a large extent unknown. Using an antibody against the α7 nAChR subunit, immunohistochemical staining was observed in rat dorsal raphe nucleus (DRN) and locus coeruleus (LC), serotonergic and noradrenergic brainstem nuclei, respectively. In both the DRN and LC, there appeared to be two histologically distinct α7-expressing cell types as distinguished by size, i.e. large versus small diameter. In rats treated with either a serotonergic (5,7-dihydroxytryptamine) or noradrenergic (anti-dopamine-β-hydroxylase saporin) neurotoxin, tryptophan hydroxylase and tyrosine hydroxylase immunostaining was abolished, respectively. Similarly, the α7-positive large-diameter cells were no longer detectable, suggesting that these cells were serotonergic DRN and noradrenergic LC neurons. Indeed, double-labeling experiments revealed in the large cell types coexpression of α7 with tryptophan hydroxylase in the DRN and with tyrosine hydroxylase in the LC of saline-treated rats. In contrast to the large-diameter cells, the α7-positive small-diameter cells were neither serotonergic nor adrenergic, and were still detected in both the DRN and LC of lesioned rats. Moreover, cell counts revealed an increase number of these cells in lesioned rats with expression of α7 in somal processes not seen in non-lesioned controls. Double labeling revealed coexpression of α7 and GABA within the majority, but not all, of the toxin-resistant cells. The results of these studies suggest that both serotonergic and noradrenergic neurons express α7 nAChRs. In addition, there appears to be a small-diameter cell-type in both the DRN and LC, possibly a GABAergic interneuron, expressing α7 that may be regulated by neurotoxic injury.

A Receptor-Mediated Mechanism of Nicotine Toxicity in Oral Keratinocytes

Smoking and smokeless tobacco cause morbidity that originates from the epithelium lining of the skin and upper digestive tract. Oral keratinocytes (OKC) express nicotinic acetylcholine receptors (nAChRs) that bind nicotine (Nic). We studied the mechanism of the receptor-mediated toxicity of tobacco products on OKC. Preincubation of normal human OKC with Nic altered the ligand-binding kinetics of their nAChRs, suggesting that the nAChRs underwent structural changes. This hypothesis was confirmed by the finding that exposure of OKC to Nic causes transcriptional and translational changes. Through RT-PCR and immunoblotting, we found a 1.5- to 2.9-fold increase in the mRNA and protein levels of α3, α5, α7, β2, and β4 nAChR subunits. Exposure of OKC to Nic also changed the mRNA and protein levels of the cell cycle and cell differentiation markers Ki-67, PCNA, p21, cyclin D1, p53, filaggrin, loricrin, and cytokeratins 1 and 10. The nicotinic antagonist mecamylamine prevented these changes, which indicates that the Nic-induced changes in the expression of both the nAChR and the cell cycle and cell differentiation genes resulted from pharmacologic stimulation of nAChRs with Nic. To establish the relevance of these findings to the pathobiologic effects of tobacco products in vivo, we studied the above parameters in the oral tissue of rats and mice after their exposure for 3 weeks to environmental cigarette smoke or drinking water containing equivalent concentrations of Nic that are pathophysiologically relevant. The changes of the nAChRs and the cell cycle and cell differentiation genes were similar to those found in vitro. The results of indirect immunofluorescence assay of tissue specimens validated these findings. Thus, some pathobiologic effects of tobacco products in oral tissues may stem from Nic-induced alterations of the structure and function of keratinocyte nAChRs responsible for the physiologic regulation of the cell cycle by the cytotransmitter acetylcholine.

Absence of α7-containing neuronal nicotinic acetylcholine receptors does not prevent nicotine-induced seizures

Nicotine is the primary addictive component in tobacco, and at relatively low doses it affects cardiovascular responses, locomotor activity, thermoregulation, learning, memory, and attention. At higher doses nicotine produces seizures. The mechanisms underlying the convulsive effects of nicotine are not known, but studies conducted on a number of inbred strains of mice have indicated a positive correlation between the number of α-bungarotoxin (α-BTX) binding sites in the hippocampus and the sensitivity to nicotine-induced seizures. Because α7-containing neuronal nicotinic acetylcholine receptors (nAChRs) represent the major binding site for α-BTX, mice lacking the α7 nAChR subunit were predicted to be less sensitive to the convulsive effects of nicotine. To test this hypothesis, we injected nicotine intraperitoneally in α7 mutant mice and found that the dose–response curve for nicotine-induced seizures was similar in the α7 +/+, α7 +/− and α7 −/− mice. The retained sensitivity to the convulsant effects of nicotine could not be explained by the presence of cholinergic compensatory mechanisms such as increases in mRNA levels for other nAChR subunits, or changes in binding levels or affinity for nicotinic ligands such as epibatidine and nicotine. These findings indicate that α7 may not be necessary for the mechanisms underlying nicotine-induced seizures.

Inefficient cell-surface expression of hybrid complexes formed by the co-assembly of neuronal nicotinic acetylcholine receptor and serotonin receptor subunits

Previous studies have demonstrated that relatively low levels of α4β2 neuronal nicotinic acetylcholine receptors (nAChRs) are expressed on the cell surface of transfected mammalian cell lines but that surface expression levels can be dramatically up-regulated by co-expression of these subunits with chimeric subunits containing the N-terminal portion of the neuronal nAChR α4 or β2 subunits together with the C-terminal domain of the 5-HT 3A subunit. Recent work has also suggested that the nAChR α4 subunit can co-assemble in a ‘promiscuous’ manner with the serotonin receptor 5-HT 3A subunit to form functional hybrid receptors. In this study we have examined whether co-assembly of either α4 or β2 with 5-HT 3A itself (rather than with the α4/5-HT 3A or β2/5-HT 3A subunit chimeras) can also facilitate cell surface expression of α4 and β2 subunits in transfected mammalian cells. Evidence has been obtained by immunoprecipitation, cell-surface antibody binding and radioligand binding which indicates that the 5-HT 3A can co-assemble with both the α4 and β2 nAChR subunits. We conclude, however, that co-assembly of 5-HT 3A with either α4 or β2 does not result in efficient cell surface expression of the nAChR subunits and that co-assembled hybrid (nAChR subunit + 5-HT 3R subunit) receptor complexes are largely retained within the cell.

Neuronal nicotinic acetylcholine receptor expression by O2A/oligodendrocyte progenitor cells

Oligodendrocyte precursor cells (O2A/OPC, A2B5+) were examined for expression of neuronal nicotinic acetylcholine receptors (nAChR). RT-PCR analysis and immunocytochemistry of O2A/OPCs purified from the rat corpus collusum revealed the expression of nAChR subunits α3, α4, α5, α7, β2, and β4. Immunoreactivity toward nAChR subunits was not detected in cells induced to differentiate into either oligodendrocytes or astrocytes. Approximately 65% of O2A/OPCs loaded with the calcium-responsive dye FURA-2 increased their intracellular free calcium in response to nicotine application. This response was sensitive to the nAChRα4/β2 antagonist, dihydro-β-erythroidine (DHβE), and the voltage-gated calcium channel antagonist, nifedipine. A subset of nicotine-responsive cells (37%) established DHβE or nifedipine-sensitive intracellular free calcium oscillations that continued in the presence of nicotine. Typical oscillations occurred at intervals of 20 to 30 s with progressively diminished amplitudes over a period of 2 to 3 min. In rare cases, oscillations persisted for as long as 10 min. O2A/OPCs exposed to carbachol or AMPA produced no oscillations despite robust increases in intracellular free calcium. The expression of nAChRs in non-neuronal glial precursor cells suggests an expanded role for this receptor system in the development of the mammalian brain. GLIA 33:306–313, 2001. Published 2001 Wiley-Liss, Inc.

Neuronal nicotinic acetylcholine receptor expression by O2A/oligodendrocyte progenitor cells

Oligodendrocyte precursor cells (O2A/OPC, A2B5(+)) were examined for expression of neuronal nicotinic acetylcholine receptors (nAChR). RT-PCR analysis and immunocytochemistry of O2A/OPCs purified from the rat corpus collusum revealed the expression of nAChR subunits alpha3, alpha4, alpha5, alpha7, beta2, and beta4. Immunoreactivity toward nAChR subunits was not detected in cells induced to differentiate into either oligodendrocytes or astrocytes. Approximately 65% of O2A/OPCs loaded with the calcium-responsive dye FURA-2 increased their intracellular free calcium in response to nicotine application. This response was sensitive to the nAChRalpha4/beta2 antagonist, dihydro-beta-erythroidine (DHbetaE), and the voltage-gated calcium channel antagonist, nifedipine. A subset of nicotine-responsive cells (37%) established DHbetaE or nifedipine-sensitive intracellular free calcium oscillations that continued in the presence of nicotine. Typical oscillations occurred at intervals of 20 to 30 s with progressively diminished amplitudes over a period of 2 to 3 min. In rare cases, oscillations persisted for as long as 10 min. O2A/OPCs exposed to carbachol or AMPA produced no oscillations despite robust increases in intracellular free calcium. The expression of nAChRs in non-neuronal glial precursor cells suggests an expanded role for this receptor system in the development of the mammalian brain. GLIA 33:306-313, 2001. Published 2001 Wiley-Liss, Inc.

Expression and function of striatal nAChRs differ in the Flinders sensitive (FSL) and resistant (FRL) rat lines

Rats of Flinders Sensitive (FSL) and Flinders Resistant lines (FRL) differ in their susceptibility to physiological and associated behavioral responses elicited by nicotine. In the present study, we measured dopamine (DA) content in striatal dialysates to investigate the sensitivity of FSL and FRL rats to nicotine delivered locally through a microdialysis probe placed in the striatum. We also measured the expression density of striatal high-affinity nicotinic acetylcholine receptors (nAChRs), and that of mRNAs encoding for α3, α4, α7 and β2 nAChR subunits in both lines. The DA content of dialysates was measured before and after a 1-min perfusion of nicotine (6, 10 or 20 nmoles/min) and the resulting DA increase was taken as a measure of the alkaloid's intrinsic activity for nAChRs involved in the release of DA. The nicotine-induced increase of striatal DA release was greater in FSL than in FRL rats for all concentrations of nicotine, suggesting that the intrinsic activity of nicotine was greater in the FSL than in the FRL rats. This was further supported by our finding that the density of high-affinity nAChRs in the striatum of FSL rats was 44% greater than in the FRL rats, whereas affinity ( K D) was virtually the same in the two lines of rats. Also the expression of mRNAs encoding for α 4, α 7, and β 2 subunits in the striatum was greater in FSL than in FRL rats (attomol/μg total RNA, α 4:98±10 vs 77±7; α 7:279±16 vs 184±16; β 2:310±19 vs 201±12). We hypothesize that the difference in nicotine-induced DA release in the striatum of FSL and FRL rats depends on the difference in nAChR subunit expression in the striatum between the two lines. The Flinders rats could be used as a model for nicotine self-administration studies to evaluate the susceptibilities of FSL and FRL rats to nicotine dependence.

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.

Rats exhibiting acute behavioural tolerance to nicotine have more [ 125I]α-bungarotoxin binding sites in brain than rats not exhibiting tolerance

Adult male Sprague-Dawley rats trained to discriminate nicotine (0.4 mg/kg, s.c. vs vehicle) using a two-lever food-reinforced operant discriminative stimulus (DS) paradigm were tested as to the ability of each subject to develop acute tolerance to nicotine. Nicotine (0.8 mg/kg, s.c.) was administered to nicotine-trained rats in their home cage and each rat tested as to its ability to detect a 2nd dose of nicotine (0.4 mg/kg, s.c.) injected at 30 min intervals thereafter (90–180 min). Tolerance was determined by evaluating nicotine-correct responding during a 2 min test session. The results of this experiment indicated that 8 out of 31 rats (26%) displayed acute tolerance (desensitizers); 18 rats (58%) did not exhibit acute tolerance (non-desensitzers) and five rats (16%) fell into a middle group and were designated as neither desensitizers or non-desensitizers. The mode time for acute tolerance was 150 min, with each desensitizer rat displaying a unique temporal profile which was replicable 4–5 weeks later. Receptor autoradiographic analysis indicated no significant differences in [ 3H]epibatidine binding sites in the brains of desensitizers and non-desensitizers. In contrast, [ 125I]α-bungarotoxin binding was significantly higher in a number of brain regions in desensitizers. In situ hybridization analysis revealed no difference in α7 nAChR subunit mRNA levels between desensitizers and non-desensitizers. These observations can be interpreted to suggest that the ability to display acute tolerance to nicotine is contingent upon the ability to upregulate α7 nAChRs. These data may also be central to understanding the variability of tobacco use in humans, which may be contingent on the ability of the receptors binding to α-bungarotoxin to be responsive to nicotine-induced desensitization.

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.

Subtype-selective inhibition of neuronal nicotinic acetylcholine receptors by cocaine is determined by the alpha4 and beta4 subunits.

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels of the central and peripheral nervous system that regulate synaptic activity from both pre- and postsynaptic sites. Nicotine binding to brain nAChRs is thought to underlie the induction of behavioral addiction to nicotine, probably as a result of desensitizing/inhibitory effects. Here, another commonly abused drug, cocaine, is shown to selectively inhibit particular nAChR subtypes with a potency in the low micromolar range by interacting with separate sites associated with the alpha4 and beta4 nAChR subunits. Chimeric receptor subunits and site-directed mutants were used to localize sequence determinants of cocaine affinity to: 1) a region of alpha4 located between residues 128 and 267 and 2) a site within the pore-lining M2 domain of beta4 that includes the 13' phenylalanine residue. The voltage dependence for inhibition associated with each site is consistent with these results. Analysis of the effects of incorporation of mutant and chimeric subunits also permitted identification of sequence elements important in receptor activation. For alpha3-containing receptors, a region or regions contained within the N-terminal extracellular domain of neuronal beta subunits influence the time course of responses to acetylcholine. Conversely, the 13' residue of the beta4 subunit M2 region is important in determining acetylcholine potency, indicating a role for this residue in agonist binding/gating processes. In summary, the present work describes sequence elements critical in both cocaine inhibition and acetylcholine activation of nAChRs and indicates that nAChRs may provide a site of interaction for the effects of nicotine and cocaine in the nervous system.

Choline acetyltransferase, acetylcholinesterase, and nicotinic acetylcholine receptors of human gingival and esophageal epithelia.

A non-neuronal cholinergic system that includes neuronal-like nicotinic acetylcholine receptors (nAChRs) has recently been described in epithelial cells that line the skin and the upper respiratory tract. Since the use of nicotine-containing products is associated with morbidity in the upper digestive tract, and since nicotine may alter cellular functions directly via nAChRs, we sought to identify and characterize a non-neuronal cholinergic system in the gingival and esophageal epithelia. mRNA transcripts for alpha3, alpha5, alpha7, and beta2 nAChR subunits, choline acetyltransferase, and the asymmetric and globular forms of acetylcholinesterase were amplified from gingival keratinocytes (KC) by means of polymerase chain-reactions. These proteins were visualized in the gingival and esophageal epithelia by means of specific antibodies. Variations in distribution and intensity of immunostaining were found, indicating that the repertoire of cholinergic enzymes and receptors expressed by the cells changes during epithelial maturation, and that an upward concentration gradient of free acetylcholine exists. Blocking of the nAChRs with mecamylamine resulted in reversible loss of cell-to-cell adhesion, and shrinking and rounding of cultured gingival KC. Activation of the receptors with acetylcholine or carbachol caused stretching and peripheral ruffling of the cytoplasmic aprons, and formation of new intercellular contacts. These results demonstrate that both the keratinizing epithelium of attached gingiva and the non-keratinizing epithelium lining the upper two-thirds of the esophageal mucosa possess a non-neuronal cholinergic system. The nAChRs expressed by these epithelia are coupled to regulation of cell adhesion and motility, and may provide a target for the deleterious effects of nicotine.

MTSEA potentiates 5-HT 3 receptors containing the nicotinic α4 subunit

In order to study the subunit composition of 5-HT 3 receptors (5-HT 3R), we report that (2-aminoethyl)methanethiosulfonate (MTSEA) can enhance the function of both nicotinic ACh receptors (nAChRs) comprised of α4/β2 subunits, and heteromeric channels assembled from serotonin 5-HT 3R and α4 nAChR subunits. MTSEA has no effect on homomeric 5-HT 3 receptor channels.

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.

Cloning and sequence of full-length cDNAs encoding the human neuronal nicotinic acetylcholine receptor (nAChR) subunits β3 and β4 and expression of seven nAChR subunits in the human neuroblastoma cell line SH-SY5Y and/or IMR-32

Using PCR-based techniques, we have isolated and sequenced the full-length cDNAs that encode the human neuronal nAChR subunits α3,4,5,7 and β2,3,4 in the neuroblastoma cell lines SH-SY5Y and/or IMR-32. The predicted nAChR β3- and β4-subunit proteins contain 458 and 498 amino acids, respectively. Except for the β2, all the other cloned cDNAs showed differences with the published sequences. Northern blots show expression of the nAChR subunits α3,7 and β2,4 in the human neuroblastoma cell line SH-SY5Y, with intensity of the hybridisation signal decreasing in the order α3>β4>β2>α7.

A sensitive technique for the detection of the α7 neuronal nicotinic acetylcholine receptor antagonist, methyllycaconitine, in rat plasma and brain

Methyllycaconitine (MLA) is the most potent and selective antagonist of the α-bungarotoxin sensitive neuronal nicotinic acetylcholine receptor (nAChR). In the present study, an accurate and reproducible technique for the extraction and analysis of MLA from rat plasma and brain is described. This study further sought to determine whether pharmacologically relevant concentrations of MLA could be achieved in brain following peripheral administration. The detection limits for MLA were 0.5 ng/ml for plasma samples and 1.0 ng/g for brain samples. The pharmacokinetic properties of MLA in rat are characterized by a short elimination half-life (19 min) following intravenous (i.v.) administration and poor bioavailability following oral (p.o.) administration. Remarkably, the elimination half-life is significantly longer following p.o. administration (408 min). To assess the extent to which MLA can penetrate into brain, brain and plasma levels of MLA were determined at different time points following intraperitoneal (i.p.) administration of a dose of MLA that produced no observable side effects. Maximal plasma and brain levels were 694 ± 106 ng/ml and 32 ± 3 ng/g, respectively. These concentrations are within a range previously reported to selectively block α7 nAChR mediated responses in vitro. Peripherally administered MLA may therefore be a useful tool to further probe the central nervous system functions of the α7 nAChR subunit in vivo.

Expression of Nicotinic Acetylcholine Receptors and Subunit mRNA Transcripts in Cultures of Neural Crest Cells

The expression of neuronal phenotypic characteristics by neural crest-derived neurons is dependent upon both environmental cues and intrinsic properties. Neuronal nicotinic acetylcholine receptors (nAChRs) are expressed by most neural crest-derived peripheral neurons; nevertheless, the factors that influence and modulate the early expression of nAChRs on neuronal precursor cells are unknown. We used RT-PCR to analyze the temporal sequence of the appearance of transcripts encoding α3, α5, α7, β2, and β4 nAChR subunits in cultures of quail neural crest cells. At 2 days in culture, mRNA encoding each of these nAChR subunits was detectable and, except for α7, remained so through 12 days. The appearance of NAPA 73 immunoreactivity during neutral crest cell migration suggested that some cells express neuronal characteristics early in development and that the expression of nAChR subunits may be cell autonomous for some neural crest-derived cells. Between 8 and 10 days in culture, a 2.0-kb transcript encoding α3 subunit appears in addition to the expected 3.5-kb transcript that is present at 8 days. nAChR protein was detected immunocytochemically at 7 days and was observed in immunoblots by 9 days. Wholecell patch clamp recordings demonstrated that functional nAChRs develop concurrently in a subset of neural crest-derived neurons. In addition, both neuronal and nonneuronal cells showed inward and delayed outward currents in response to step depolarization. The appearance of functional nAChRs on neural crest-derived cells, in vitro, occurs independently of innervation and of the components in growth medium. The data suggest that neural crest-derived cells express transcripts encoding nAChR subunits, possibly as an intrinsic property of some peripheral neurons, early in development.

Cellular expression of α4 subunit mRNA of the nicotinic acetylcholine receptor in the developing rat telencephalon

By contrast to adult brain, little is known on the development of nicotinic acetylcholine receptor (nAChR) expression. Using a digoxigenin-labeled riboprobe for in situ hybridization, α4 nAChR subunit mRNA expression was studied in embryonic and postnatal rat neocortex and hippocampus where it was transiently increased in neuronal subpopulations and preceded cholinergic fiber ingrowth. α4 expression was increased in neocortical layer VIb between E20 and P2 and, about birth, in dentate gyrus granule cells subsequently decreasing to adult levels. nAChR mmRNA expression is increased at the developing neuromuscular endplate preceding cholinergic innervation which triggers changes in non-α nAChR isoform expression. It has to be elucidated whether similar changes may occur in the telencephalon.

Characterization of nicotinic acetylcholine receptors expressed in primary cultures of cerebellar granule cells

Nicotinic acetylcholine receptors (nAChRs), like other calcium permeable channel receptors, may play a crucial role during neuronal development. We have characterized nAChRs in developing mouse cerebellar granule cells in primary culture. L-[3H]Nicotine, [3H]cytisine and [125I]alpha-bungarotoxin binding experiments revealed the presence of a single class of saturable and specific high affinity binding sites for each ligand. The expression of these nicotinic binding sites followed a developmental pattern reaching a maximum during the establishment of excitatory amino acid synaptic contacts. Immunolabeling with monoclonal antibodies to nAChR subunits revealed the presence of alpha 4 and beta 2 subunits in most neurons. Moreover, some neuronal cells displayed a somatic as well as a neuritic localization for the alpha 7 subunit as shown by [125I]alpha-bungarotoxin autoradiography. The reverse transcription-polymerase chain reaction (RT-PCR) detected the presence of mRNAs for alpha 3, alpha 4, alpha 5, alpha 7, beta 2 and beta 4 nAChR subunits. Non-neuronal cells did not express nAChRs, as shown by [3H]nicotine and [125I]alpha-bungarotoxin binding, immunocytochemistry and PCR. Maximum Ca2+ influx elicited by nicotine, and partly sensitive to alpha-bungarotoxin, was observed around 10-14 days after plating. This correlated with the time period at which the highest number of nicotine binding sites was detected. Sensitivity to several NMDA receptor antagonists as well as to removal of endogenous glutamate by pyruvate transaminase treatment revealed a glutamatergic component in the nicotine stimulated calcium influx. The time-dependent specific nAChR expression and the potential association between nAChRs and NMDA receptor activation suggest that nAChRs may regulate glutamatergic activity during synaptogenesis in cerebellar granule cells.