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Comparative Study
. 1997 Jan 15;17(2):586-96.
doi: 10.1523/JNEUROSCI.17-02-00586.1997.

Heterogeneity of Nicotinic Receptor Class and Subunit mRNA Expression Among Individual Parasympathetic Neurons From Rat Intracardiac Ganglia

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Free PMC article
Comparative Study

Heterogeneity of Nicotinic Receptor Class and Subunit mRNA Expression Among Individual Parasympathetic Neurons From Rat Intracardiac Ganglia

K Poth et al. J Neurosci. .
Free PMC article

Abstract

Neurons have the potential to form thousands of distinct neuronal nicotinic receptors from the eight alpha and three beta subunits that currently are known. In an effort to determine how much of this potential complexity is realized among individual neurons, we examined the nicotinic pharmacological and biophysical properties and receptor subunit mRNA expression patterns in individual neurons cultured from rat epicardial ganglia. Analysis of the whole-cell pharmacology of these neurons showed a diversity of responses to the agonists acetylcholine, nicotine, cytisine, and 1,1-dimethyl-4-phenylpiperazinium, suggesting that a heterogeneous population of nicotinic receptor classes, or subtypes, is expressed by individual neurons. Single-channel analysis demonstrated three distinct conductances (18, 24, and 31 pS), with patches from different neurons containing different combinations of these channel classes. We used single-cell RT-PCR to examine nicotinic acetylcholine receptor (nAChR) subunit mRNA expression by individual neurons. Although mRNAs encoding all eight neuronal nAChR subunits for which we probed (alpha 2-alpha 5, alpha 7, beta 2-beta 4) were present in multicellular cultures, we found that individual epicardial neurons express distinct subsets of these nAChR subunit mRNAs. These results suggest that individual epicardial neurons express distinct arrays of nAChR subunits and that these subunits may assemble into functional receptors with distinct and variable subunit composition. This variable receptor subunit expression provides an explanation for the diversity of pharmacological and single-channel responses we have observed in individual neurons.

Figures

Fig. 1.
Fig. 1.
Whole-cell currents in rat parasympathetic neurons evoked by nicotinic agonists. A, Whole-cell current responses evoked by 10 msec focal applications of 100 μmconcentrations of ACh, cytisine, nicotine, and DMPP in two different cells. Rank order of potency was determined by the peak current amplitude evoked by each agonist. One cell responded with a rank order of agonist potency of cytisine ≥ ACh > nicotine > DMPP (top current records), whereas the second cell responded in the order ACh > DMPP > cytisine > nicotine (bottom current records). Holding potential, −90 mV. B, Rank order of potency to nicotinic agonists in 11 parasympathetic intracardiac neurons. Current amplitude evoked by cytisine (filled bars), nicotine (hatched bars), and DMPP (shaded bars) is plotted relative to ACh. Current records shown in A correspond to cell 1 (top records) and cell 11 (bottom records). Cells are plotted in the order of decreasing response to cytisine.
Fig. 2.
Fig. 2.
Interpatch variability of single-channel conductance levels activated by ACh. A, Unitary currents evoked by ACh in three separate outside-out membrane patches held at −90 mV. Patches exhibited either the small and medium conductance (i, 9 of 24 patches), the medium and large conductance (ii, 6 of 24 patches), or all three conductance levels (iii, 9 of 24 patches). Conductance levels are designated by the dashed lines, and the closed states of the channels are indicated. Currents were filtered at 3 kHz.B, Amplitude histograms of the experimental records from which the data shown in A were taken. The current amplitude distributions are fit by Gaussian curves with mean ± SD (relative area) of (i) −1.2 ± 0.1 pA (37 ± 3%) and −2.0 ± 0.3 pA (63 ± 4%), (ii) −2.2 ± 0.2 pA (13 ± 4%) and −3.1 ± 0.3 pA (87 ± 4%), and (iii) −1.6 ± 0.2 pA (36 ± 3%), −2.2 ± 0.1 pA (14 ± 3%), and −2.7 ± 0.2 pA (50 ± 4%).
Fig. 3.
Fig. 3.
Single-channel currents evoked by exogenous ACh in rat cultured parasympathetic neurons. A, Unitary ACh-activated currents obtained in an excised outside-out membrane patch at the membrane potentials indicated. The closed states of the channels (c) are indicated. Three distinct levels are evident in this patch (indicated by the dashed lines in the bottom trace), corresponding to slope conductances of 27.4, 21.8, and 16.6 pS. B, Current–voltage (i–V) relations for single-channel currents activated by ACh. Each point represents the mean ± SEM from 16 excised outside-out patches obtained from different cells, with 65–628 openings per patch at each membrane potential. The data are fit by linear regression and give slope conductances of 18 pS (triangles), 24 pS (squares), and 31 pS (circles). Current records were filtered at 3 kHz and digitized at 67 μsec/point (15 kHz).
Fig. 4.
Fig. 4.
RT-PCR identification of mRNA encoding α2, α3, α4, α5, α7, β2, β3, and β4 in rat brain RNA.A, Second-round PCR products. Lane 1, 100 bp standards; lanes 2–6 are the PCR products amplified with the α2/3/4, β2/4, α5, β3, and α7 primers, respectively. In this experiment, α5 and β3 were amplified separately.Lane 7 is the negative control. B, Restriction digestion of PCR products. Lanes 1, 7, 100 bp standards; lanes 2–6 contain digested second-round PCR products for α2/3/4, β2/4, α5, β3, and α7 primers, respectively. Fragments identifying each subunit are labeled to theleft of each lane. For enzymes used and exact fragment sizes, see Table 2. For clarity in presentation, we have removed the undigested sample routinely run next to each digested product.
Fig. 5.
Fig. 5.
Neuronal nicotinic receptor subunit mRNAs encoding α2, α3, α4, α5, α7, β2, β3, and β4 are expressed by cultures of intracardiac parasympathetic neurons. A, Second-round PCR products. Lane 1, 100 bp standards;lanes 2–5 are the PCR products obtained by using the α2/3/4, β2/4, α5/β3, and α7 primers, respectively. In this experiment, α5 and β3 products were amplified in the same reaction (lane 4). Lanes 6 and7 are blank. Lane 8 is the negative control. B, Restriction digestion of PCR products.Lanes 1, 7, 100 bp standards; lanes 2–6contain the digested fragments for the α2/3/4, β2/4, α5, β3, and α7 products, respectively. Note that the α5 and β3 products, although amplified in the same reaction, are digested separately because of different buffer requirements. Fragments identifying each subunit are labeled to the left of each lane. For enzymes used and exact fragment sizes, see Table 1. For clarity in presentation, we have removed the undigested sample routinely run next to each digested product.
Fig. 6.
Fig. 6.
Individual intracardiac parasympathetic neurons express diverse arrays of nAChR subunit mRNAs. Second-round PCR (i) and restriction digestion (ii) results for neurons A–E are shown. A, Neuron expressing α2, α3, and α4 (lanes 2i and 2ii), α7 (lanes 5i and 4ii), and β2 (lanes 3i and 3ii). B, Neuron expressing α3 (lanes 2i and2ii), α5 (lanes 4i and4ii), and β4 (lanes 3i and3ii). C, Neuron expressing α3 (lanes 2i and 2ii), α5 (lanes 4i and 4ii), α7 (lanes 6i and5ii), and β2 (lanes 3i and3ii). D, Neuron expressing α3 and α4 (lanes 2i and 2ii), β3 (lanes 5i and 4ii), and β4 (lanes 3iand 3ii). E, Neuron expressing α3 (lanes 2i and 2ii), α5 (lanes 4i and 4ii), and β2 (lanes 3iand 3ii). Neurons C and D were harvested from the same culture dish. Negative controls are run in lane 6 for neuron A and lane 7 for neurons B–E. 100 bp size standards are run in lane 1 of each second-round PCR gel (Ai–Ei); lanes 1 and 5 inAii, Bii, Dii, and Eii; and lanes 1 and 5 in Cii. For clarity in presentation, we have removed the lanes containing undigested samples routinely run next to each digested product.

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