nAChR Subunit mRNA Expression Research Articles

Cellular protein and mRNA expression of β1 nicotinic acetylcholine receptor (nAChR) subunit in brain, skeletal muscle and placenta

The β1 nicotinic acetylcholine receptor (nAChR) subunit is a muscle type subunit of this family and as such, is found predominantly in muscle. Recent reports document its expression in other tissues and cell lines including adrenal glands, carcinomas, lung and brain. However, the majority of studies were of tissue lysates, thus the cellular distribution was not determined. This study aimed to determine the cellular distribution of the β1 nAChR subunit in the brain, at both the mRNA and protein levels, using non-radioactive in situ hybridization (ISH) and immunohistochemistry (IHC), respectively, and to compare it to two muscle tissue types, skeletal and placenta. Tissue was formalin fixed and paraffin embedded (all tissue types) and frozen (placenta) from humans. Additional control tissue from the piglet and mouse brain were also studied, as was mRNA for the α3 nAChR and N-methyl-d-aspartate receptor 1 (NR1) subunit. We found no β1 nAChR subunit mRNA expression in the human and piglet brain despite strong protein expression. Some signal was seen in the mouse brain but considered inconclusive given the probes designed were not of 100% homology to the mouse. In the skeletal muscle and placenta tissues, β1 nAChR subunit mRNA expression was prominent and mirrored protein expression. No α3 nAChR or NR1 mRNA was seen in the skeletal muscle, as expected, although both subunit mRNAs were present in the placenta. This study concludes that further experiments are required to conclusively state that the β1 nAChR subunit is expressed in the human, piglet and mouse brain.

Aerobic training attenuates nicotinic acethylcholine receptor changes in the diaphragm muscle during heart failure.

Heart failure (HF) is a progressive myopathy, with clinical signs of fatigue and limb weakness that can damage the nerve-muscle interaction, altering synaptic transmission and nicotinic acetylcholine receptors (nAChR) in neuromuscular junctions (NMJs). The diaphragm is composed of a mixed proportion of muscle fibres, and during HF, this muscle becomes slower and can alter its function. As exercise training is an accepted practice to minimise abnormalities of skeletal muscle during HF, in this study, we evaluated the hypothesis that aerobic training attenuates alterations in the expression of nAChR subunits in NMJs diaphragm during heart failure. The aim of this study was to evaluate the distribution and expression of nAChR subunits in the diaphragm muscle fibres of rats subjected to an aerobic training programme during HF. Control (Sham), control training (ShamTR), aortic stenosis (AS) and aortic stenosis training (ASTR) groups were evaluated. The expression of nAChR subunits (γ, α1, ε, β1 and δ) was determined by qRT-PCR, and NMJs were analysed using confocal microscopy. We observed increased expression of the γ, α1 and β1 subunits in the AS group compared with the ASTR group. The distribution of NMJs was modulated in these groups. HF alters the mRNA expression of nAChR subunits and the structural characteristics of diaphragm NMJs. In addition, aerobic training did not alter NMJs morphology but attenuated the alterations in heart structure and function and in nAChR subunit mRNA expression. Our findings demonstrate the beneficial effects of aerobic exercise training in maintaining the integrity of the neuromuscular system in the diaphragm muscle during HF and may be critical for non-pharmacological therapy to improve the quality of life for patients with this syndrome.

The duration of nicotine withdrawal-associated deficits in contextual fear conditioning parallels changes in hippocampal high affinity nicotinic acetylcholine receptor upregulation

A predominant symptom of nicotine withdrawal is cognitive deficits, yet understanding of the neural basis for these deficits is limited. Withdrawal from chronic nicotine disrupts contextual learning in mice and this deficit is mediated by direct effects of nicotine in the hippocampus. Chronic nicotine treatment upregulates nicotinic acetylcholine receptors (nAChR); however, it is unknown whether upregulation is related to the observed withdrawal-induced cognitive deficits. If a relationship between altered learning and nAChR levels exists, changes in nAChR levels after cessation of nicotine treatment should match the duration of learning deficits. To test this hypothesis, mice were chronically administered 6.3mg/kg/day (freebase) nicotine for 12 days and trained in contextual fear conditioning on day 11 or between 1 to 16 days after withdrawal of treatment. Changes in [125I]-epibatidine binding at cytisine-sensitive and cytisine-resistant nAChRs and chronic nicotine-related changes in α4, α7, and β2 nAChR subunit mRNA expression were assessed. Chronic nicotine had no behavioral effect but withdrawal produced deficits in contextual fear conditioning that lasted 4 days. Nicotine withdrawal did not disrupt cued fear conditioning. Chronic nicotine upregulated hippocampal cytisine-sensitive nAChR binding; upregulation continued after cessation of nicotine administration and the duration of upregulation during withdrawal paralleled the duration of behavioral changes. Changes in binding in cortex and cerebellum did not match behavioral changes. No changes in α4, α7, and β2 subunit mRNA expression were seen with chronic nicotine. Thus, nicotine withdrawal-related deficits in contextual learning are time-limited changes that are associated with temporal changes in upregulation of high-affinity nAChR binding.

Pharmacological and immunochemical characterization of α2* nicotinic acetylcholine receptors (nAChRs) in mouse brain

alpha2 nAChR subunit mRNA expression in mice is most intense in the olfactory bulbs and interpeduncular nucleus. We aimed to investigate the properties of alpha2* nAChRs in these mouse brain regions. alpha2 nAChR subunit-null mutant mice were engineered. Pharmacological and immunoprecipitation studies were used to determine the composition of alpha2 subunit-containing (alpha2*) nAChRs in these two regions. [(125)I]Epibatidine (200 pmol/L) autoradiography and saturation binding demonstrated that alpha2 deletion reduces nAChR expression in both olfactory bulbs and interpeduncular nucleus (by 4.8+/-1.7 and 92+/-26 fmol mg(-1) protein, respectively). Pharmacological characterization using the beta2-selective drug A85380 to inhibit [(125)I]epibatidine binding proved inconclusive, so immunoprecipitation methods were used to further characterize alpha2* nAChRs. Protocols were established to immunoprecipitate beta2 and beta4 nAChRs. Immunoprecipitation specificity was ascertained using tissue from beta2- and beta4-null mutant mice, and efficacy was good (>90% of beta2* and >80% of beta4* nAChRs were routinely recovered). Immunoprecipitation experiments indicated that interpeduncular nucleus alpha2* nAChRs predominantly contain beta2 subunits, while those in olfactory bulbs contain mainly beta4 subunits. In addition, the immunoprecipitation evidence indicated that both nuclei, but especially the interpeduncular nucleus, express nAChR complexes containing both beta2 and beta4 subunits.

Nicotinic acetylcholine receptor subunit mRNA expression in adult and developing rat medullary catecholamine neurons

Nicotinic acetylcholine receptors (nAChRs) mediate numerous visceral functions via medullary catecholamine (CA) neurons found in the nucleus tractus solitarius (NTS), dorsal motor nucleus of the vagus (DMV), and ventrolateral medulla (VLM). However, the nAChR subtypes involved are not known. We have therefore characterized expression of nine nAChR subunit mRNAs in adult and developing rat medullary CA nuclei using combined isotopic/nonisotopic in situ hybridization. Tyrosine hydroxylase (TH) mRNA, the CA-synthesizing enzyme, was used as a marker for CA neurons, because these nuclei consist of heterogeneous populations of cells. Subunit mRNA expression varied within and between nuclei, along the rostrocaudal axis, between cell types, and across development. All CA neurons expressed beta2 mRNA, whereas alpha2 mRNA was completely absent. alpha6 And beta3 mRNA expression were restricted mainly to the VLM. alpha4, alpha5, And alpha7 mRNA expression was significantly greater in the rostral than in the caudal VLM. alpha3 And beta4 mRNAs were highly expressed in the dorsal region of the NTS, whereas dense alpha7 mRNA expression was restricted to the DMV and ventral NTS. The remaining subunit mRNAs were detected to some degree in both DMV and NTS. Except for alpha4 mRNA, which peaked prenatally, expression levels of subunit transcripts in the NTS and DMV were lower during development compared with adults. In the VLM, alpha3, alpha4, and alpha5 mRNAs expression peaked perinatally, whereas alpha6 and beta3 levels increased with age. These variations in nAChR subunit mRNA expression suggest that different receptor subtypes may produce function-specific regulation of medullary CA systems.

Maternal nicotine exposure increases oxidative stress in the offspring

Fetal and neonatal nicotine exposure causes β-cell apoptosis and loss of β-cell mass, but the underlying mechanisms are unknown. The goal of this study was to determine whether maternally derived nicotine can act via the pancreatic nicotinic acetylcholine receptor (nAChR) during fetal and neonatal development to induce oxidative stress in the pancreas. Female Wistar rats were given saline or nicotine (1 mg/kg/day) via subcutaneous injection for 2 weeks prior to mating until weaning (postnatal day 21). In male offspring, nAChR subunit mRNA expression was characterized in the developing pancreas and various oxidative stress markers were measured at weaning following saline and nicotine exposure. The nAChR subunits α2-α4, α6, α7, and β2–β4 were present in the pancreas during development. Fetal and neonatal exposure to nicotine significantly increased pancreatic GPx-1 and MnSOD protein expression, as well as islet ROS production. Furthermore, protein carbonyl formation was higher in nicotine-exposed offspring relative to controls, particularly within the mitochondrial fraction. There was also a nonsignificant trend toward higher serum 8-isoPG levels. These data suggest that β-cell apoptosis in the fetal and neonatal pancreas may be the result of a direct effect of nicotine via its receptor and that this effect may be mediated through increased oxidative stress.

Gene expression of nAChR, SNAP-25 and GAP-43 in skeletal muscles following botulinum toxin A injection: a study in rats

Purpose: Botulinum toxin A (BoNT-A) is used to manage spasticity in cerebral palsy. BoNT-A cleaves SNAP-25 protein, blocking acetylcholine release and weakening the muscle. Nicotinic acetylcholine receptors (nAChR) including alpha, beta, delta, gamma, and epsilon subunits, and GAP-43 protein are associated with functional recovery of neuromuscular junctions (NMJ) following BoNT-A. To better understand the mechanism behind this functional recovery, this study attempted to (1) document changes in NMJ morphometry following BoNT-A, and (2) determine the gene expression of nAChR subunits, SNAP-25, and GAP-43 protein. Methods: In this rat study (46 rats), 6 units/kg body weight of BoNT-A was injected into the gastrocnimus. NMJ morphometry and the time course of gene expression of nAChR subunits, SNAP-25, and GAP-43 were evaluated up to 1 year post-injection. Results: NMJ morphometry: gutter depth was reduced vs. the control side at two months, and normalizing by 6 months following BoNT. nAChR alpha mRNA and gamma mRNA increased by 3 days, peaked at 7 days and returned to control levels; delta mRNA peaked at 3 days. Epsilon mRNA peaked by 7 days. SNAP-25 mRNA increased from 60 to 90 days, returning to control levels by 6 months. GAP-43 mRNA was unchanged. Conclusions: Specific nAChR subunit mRNA expression up-regulates and then returns to normal within two weeks, preceding changes in NMJ morphometry. Although GAP-43 participates in nerve sprouting, no increase of GAP-43 mRNA occurred following BoNT-A. Delayed up-regulation of SNAP-25 mRNA might be associated with muscle functional recovery.

Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons.

Many behavioral effects of nicotine result from activation of nigrostriatal and mesolimbic dopaminergic systems. Nicotine regulates dopamine release not only by stimulation of nicotinic acetylcholine receptors (nAChRs) on dopamine cell bodies within the substantia nigra and ventral tegmental area (SN/VTA), but also on presynaptic nAChRs located on striatal terminals. The nAChR subtype(s) present on both cell bodies and terminals is still a matter of controversy. The purpose of this study was to use double-labeling in situ hybridization to identify nAChR subunit mRNAs expressed within dopamine neurons of the SN/VTA, by using a digoxigenin-labeled riboprobe for tyrosine hydroxylase as the dopamine cell marker and (35)S-labeled riboprobes for nAChR subunits. The results reveal a heterogeneous population of nAChR subunit mRNAs within midbrain dopamine neurons. Within the SN, almost all dopamine neurons express alpha2, alpha4, alpha5, alpha6, beta2, and beta3 nAChR mRNAs, with more than half also expressing alpha3 and alpha7 mRNAs. In contrast, less than 10% express beta4 mRNA. Within the VTA, a similar pattern of nAChR subunit mRNA expression is observed except that most subunits are expressed in a slightly lower percentage of dopamine neurons than in the SN. Within the SN, alpha4, beta2, alpha7, and beta4 mRNAs are also expressed in a significant number of nondopaminergic neurons, whereas within the VTA this only occurs for beta4. The heterogeneity in the expression of nAChR subunits within the SN/VTA may indicate the formation of a variety of different nAChR subtypes on cell bodies and terminals of the nigrostriatal and mesolimbic pathways.

Nicotinic acetylcholine receptor subunit mRNA expression and channel function in medial habenula neurons

Relationships between nicotinic acetylcholine receptor (nAChR) channel function and nAChR subunit mRNA expression were explored in acutely isolated rat medial habenula (MHb) neurons using a combination of whole-cell recording and single cell RT–PCR techniques. Following amplification using subunit-specific primers, subunits could be categorized in one of three ways: (i) present in 95–100% cells: α3, α4, α5, β2 and β4; (ii) never present: α2; and (iii) sometimes present (≈40% cells): α6, α7 and β3. These data imply that α2 subunits do not participate in nAChRs on MHb cells, that α6, α7 and β3 subunits are not necessary for functional channels but may contribute in some cells, and that nAChRs may require combinations of all or subsets of α3, α4, α5, β2 and β4 subunits. Little difference in the patterns of subunit expression between nicotine-sensitive and insensitive cells were revealed based on this qualitative analysis, implying that gene transcription per se may be an insufficient determinant of nAChR channel function. Normalization of nAChR subunit levels to the amount of actin mRNA, however, revealed that cells with functional channels were associated with high levels (>0.78 relative to actin; 11/12 cells) of all of the category (i) subunits: α3, α4, α5, β2 and β4. Conversely, one or more of these subunits was always low (<0.40 relative to actin) in all cells with no detectable response to nicotine. Thus the formation of functional nAChR channels on MHb cells may require critical levels of several subunit mRNAs.

Nicotinic receptor subunit mRNA in the thalamus of the rat: relevance to schizophrenia?

Recent evidence suggests that aberrant nicotinic receptor (nAChR) expression plays an important role in schizophrenia. The present study sought to examine the distribution of nAChRs within the thalamus and associated cholinergic structures by examining nAChR subunit mRNA expression using in situ hybridization histochemistry. Transcripts for alpha4 and beta2 subunits were found in high levels in all thalamic nuclei and at lower levels in cholinergic nuclei (PPTg and MS/VDB). Distribution of mRNA encoding for additional subunits was restricted; lower alpha3 subunit transcript levels were detected in the anterior thalamic nuclei and portions of the lateral and posterior thalamic nuclei, with alpha7 transcripts being detected in cholinergic nuclei, the IMD and very low levels in the RTN. Low levels of alpha6 and beta3 transcript were found only within the RTN.

Diminution of nicotinic receptor alpha 3 subunit mRNA expression in aged rat brain

Losses in nicotinic acetylcholine receptors (nAChRs) have been linked to a decline in cognitive function in patients with neurodegenerative diseases, but the impact of normal aging on the different neuronal nicotinic receptor subunits has yet to be fully characterized. The expression pattern of nine nAChR subunits mRNA (α2–7 and β2–4) was investigated in this study in young and aged rat brains, 5 weeks and 30 months old, respectively. Microtissue samples were dissected from brain slices and nAChR subunit mRNA expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR) from eight different brain areas. In several regions, a loss of PCR signal was found for the α3, and to a lesser extent, for α2 subunit mRNA in aged rat brain. A relative quantification of α3 and α4 mRNA expression was then carried out in four of these brain regions. A significant diminution of α3 expression level was observed in all regions tested while, in comparison, much less modification in α4 mRNA was detected. This decrease in α3 subunit mRNA may represent a selective degradation of neurons expressing the α3 subunit or a diminution of α3-containing nAChR subtypes in those neurons during aging.

Identification of the subunits of the nicotinic cholinergic receptors in the rat cochlea using RT–PCR and in situ hybridization

There are two tissues in the adult mammalian cochlea that are post-synaptic to cholinergic efferent fibers: The outer hair cells (OHCs) and the dendrites of the afferent fibers of the type I spiral ganglion cells. The unusual nicotinic-like pharmacology of cochlear cholinergic responses and the unique embryonic development of cochlear tissues suggest that the inner-ear nicotinic cholinergic receptor (nAChR) may be different from nAChRs described previously at synapses in the mammalian brain, autonomic ganglia, or skeletal muscle. In this study, we determined the mRNA expression of the α2–7, α9, and β2–4 subunits of the nicotinic acetylcholine receptor (nAChR) family in the rat cochlea. In micro-dissected tissue from the organ of Corti, spiral ganglion, and the membranous lateral wall, we found mRNA expression of the α7 and α9 subunits in the organ of Corti and α5–7, and β2 and β3 in the spiral ganglion using RT–PCR. Employing in situ hybridization with 35S-riboprobes, we localized α9 in hair cells regions and α6, α7 and β2 in the type I cells of the spiral ganglion. No evidence of nAChR subunit mRNA expression was found in supporting cells, but β2 was expressed in type II spiral ganglion cells, which are neither cholinergic nor cholinoceptive.

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.