Expression Of Functional nAChRs Research Articles

Functional α7 Nicotinic ACh Receptors on Astrocytes in Rat Hippocampal CA1 Slices

Although much is known about the functional expression of the neuronal nicotinic acetylcholine receptors (nAChRs) in various neuronal populations in the brain and elsewhere, much less is known about their expression and functional relevance in glial cells. The expression of functional nAChRs has been reported for cultured astrocytes; however, previous work has failed to detect nAChR-mediated responses in astrocytes in acute slices. In the current study, functional α7 nAChRs on astrocytes in the CA1 region of the rat hippocampus were studied in situ using whole-cell patch-clamp recording and two-photon calcium imaging techniques in acute slices. We found that astrocytes and the chondroitin sulfate proteoglycan NG2-expressing (i.e., NG2) cells did express functional α7 nAChRs. Although the amplitudes of the responses were small, they could be enhanced by the α7-selective positive allosteric modulator PNU-120596. Under these conditions, we found that in comparing the properties of these responses between astrocytes, NG2, and interneurons, there were differences in the kinetics and increases in intracellular calcium levels. This is the first demonstration of functional α7 nAChR-mediated current responses in astrocytes in acute hippocampal slices, data which may shed light on the role of α7 nAChRs in neuroprotection.

Age-dependent changes in the functional expression of two nicotinic receptor subtypes in CA1 stratum radiatum interneurons in the rat hippocampus

Protein density measurements and mRNA analysis have provided valuable information on age-dependent changes in the distribution of different nicotinic receptor (nAChR) subtypes in various areas of the rat brain, including the hippocampus. However, very little is known regarding the functional expression of nAChRs in individual neuron types at various ages. Likewise, there is paucity of information regarding the functional and pharmacological profile of nAChRs in the mature rat hippocampus. To address these issues, we used the whole-cell patch-clamp technique to record nicotinic responses from CA1 stratum radiatum (SR) interneurons in hippocampal slices from rat pups (5–19 days old) and adult rats (2–5 months old). As previously observed in the hippocampus of rat pups, CA1 SR interneurons in the hippocampus of adult rats responded to choline (10 mM, 12 s) with whole-cell currents that decayed to the baseline within the agonist pulse, were sensitive to inhibition by methyllycaconitine (10 nM) or α-bungarotoxin (50 nM), and were, therefore, mediated by α7* 1 1 According to the nomenclature for nAChRs and their subunits [Lukas RJ, Changeux JP, Le Novere N, Albuquerque, EX, Balfour DJ, Berg, DK et al. International Union of Pharmacology. XX. Current status of the nomenclature for nicotinic acetylcholine receptors and their subunits. Pharmacol Rev 1999; 51: 397–401.], the asterisk next to nAChR subunits throughout text is meant to indicate that the exact subunit composition is not known. [1] nAChRs. Likewise, as previously observed in the hippocampus of young rats, in the adult rat hippocampus excitatory postsynaptic currents (EPSCs) were recorded from SR interneurons in response to a pulse of ACh (0.1 mM, 12 s) applied in the presence of the GABA A receptor antagonist bicuculline. ACh-triggered EPSCs were inhibited by mecamylamine (1 μM) or choline (1 mM) and were, therefore, likely to have resulted from activation of α3β4β2* nAChR. The magnitude of α7* nAChR-mediated responses increased with the age of the animals. In contrast, the magnitude of α3β4β2* nAChR-mediated responses was highest at the second postnatal week. The distinct age dependency of functional expression of α7* and α3β4β2* nAChRs strongly suggests that the excitability of CA1 SR interneurons is differentially regulated by the nicotinic cholinergic system in the hippocampus of rat pups and adult rats.

Functional somato‐dendritic α7‐containing nicotinic acetylcholine receptors in the rat basolateral amygdala complex

Multiple subtypes of nicotinic acetylcholine receptors (nAChRs) are expressed in the CNS. The amygdala complex, the limbic structure important for emotional memory formation, receives cholinergic innervation from the basal forebrain. Although cholinergic drugs have been shown to regulate passive avoidance performance via the amygdala, the neuronal subtypes and circuits involved in this regulation are unknown. In the present study, whole-cell patch-clamp electrophysiological techniques were used to identify and characterize the presence of functional somato-dendritic nAChRs within the basolateral complex of the amygdala. Pressure-application of acetylcholine (ACh; 2 mm) evoked inward current responses in a subset of neurons from both the lateral (49%) and basolateral nuclei (72%). All responses displayed rapid activation kinetics, and were blocked by the alpha7-selective antagonist methyllycaconitine. In addition, the alpha7-selective agonist choline induced inward current responses that were similar to ACh-evoked responses. Spiking patterns were consistent with pyramidal class I neurons (the major neuronal type in the basolateral complex); however, there was no correlation between firing frequency and the response to ACh. The local photolysis of caged carbachol demonstrated that the functional expression of nAChRs is located both on the soma and dendrites. This is the first report demonstrating the presence of functional nAChR-mediated current responses from rat amygdala slices, where they may be playing a significant role in fear and aversively motivated memory.

Acetylcholine increases intracellular Ca2+ via nicotinic receptors in cultured PDF-containing clock neurons of Drosophila.

Light entrains the biological clock both in adult and larval Drosophila melanogaster. The Bolwig organ photoreceptors most likely constitute one substrate for this light entrainment in larvae. Acetylcholine (ACh) has been suggested as the neurotransmitter in these photoreceptors, but there is no evidence that ACh signaling is involved in photic input onto circadian pacemaker neurons. Here we demonstrate that the putative targets of the Bolwig photoreceptors, the PDF-containing clock neurons (LNs), in the larval brain express functional ACh receptors (AChRs). With the use of GAL4-UAS-driven expression of green fluorescent protein (GFP), we were able to identify LNs in dissociated cell culture. After loading with the Ca(2+)-sensitive dye fura-2, we monitored changes in intracellular Ca(2+) levels ([Ca(2+)](i)) in GFP-marked LNs while applying candidate neurotransmitters. ACh induced transient increases in [Ca(2+)](i) at physiological concentrations. These increases were dependent on extracellular Ca(2+) and Na(+) and were likely caused by activation of voltage-dependent Ca(2+) channels. Application of nicotinic and muscarinic agonists and antagonists showed that the AChRs on cultured LNs have a nicotinic pharmacology. Antibodies to several subunits of nicotinic AChRs (nAChRs) labeled the putative contact site of the Bolwig organ axon terminals with the dendrites of LNs, as well as dissociated LNs in culture. Our findings support a role of ACh as input factor onto the LNs and suggest that Ca(2+) is used as a second messenger mediating cholinergic input within the LNs. Experiments using a more general GAL4-UAS-driven expression of GFP showed that functional expression of nAChRs is a widespread phenomenon in peptidergic neurons.

A motif present in the main cytoplasmic loop of nicotinic acetylcholine receptors and catalases

A motif containing five conserved amino acids (RXPXTH(X)14P) was detected in 111 proteins, including 82 nicotinic acetylcholine receptor (nAChR) subunits and 20 catalases. To explore possible functional roles of this motif in nAChRs two approaches were used: first, the motif sequences in nAChR subunits and catalases were analysed and compared; and, second, deletions in the rat alpha2 and beta4 nAChR subunits expressed in Xenopus oocytes were analysed. Compared to the three-dimensional structure of bovine hepatic catalase, structural coincidences were found in the motif of catalases and nAChRs. On the other hand, partial deletions of the motif in the alpha2 or beta4 subunits and injection of the mutants into oocytes was followed by a very weak expression of functional nAChRs; oocytes injected with alpha2 and beta4 subunits in which the entire motif had been deleted failed to elicit any acetylcholine currents. The results suggest that the motif may play a role in the activation of nAChRs.