Quantal release at a neuronal nicotinic synapse from rat adrenal gland.

Abstract

The neuronal nicotinic synapse in tissue slices of the adrenal medulla was studied with whole-cell patch-clamp. Excitatory postsynaptic currents (EPSCs) were evoked by local field stimulation or occurred spontaneously especially when external [K+l was increased. EPSCs were carried by channels sharing biophysical and pharmacological properties of neuronal-type nicotinic receptors (nAChRs). A single-channel conductance (y) of 43-45 pS was found from nonstationary variance analysis of EPSCs. Spontaneous EPSCs were tetrodotoxin-insensitive and Ca2+-dependent and occurred in burst-like clusters. Quantal analysis of spontaneous EPSCs gave a quantal size of 20 pA and amplitude histograms were well described by binomial models with low values of quantal content, consistent with a small number of spontaneously active release sites. However, rare large amplitude EPSCs suggest that the total number of sites is higher and that extrajunctional receptors are involved. Our estimates of quantal content and size at the chromaffin cell neuronal nicotinic synapse may be useful in characterizing central neuronal-type nicotinic receptor-mediated cholinergic synaptic transmission.The neuronal nicotinic receptor (nAChR) family displays a large diversity of a and ,B subunit composition and, correspondingly, differences in pharmacological profile, singlechannel conductance, and distribution in the central nervous system and autonomic ganglia (1, 2). Activation of central somatic nAChRs either depolarizes neurons from prefrontal cortex (3), medial habenula (4), and locus coeruleus (5) or hyperpolarizes neurons from dorsolateral septal nucleus (6). As well, nAChRs appear to be involved in presynaptic control of transmitter release (7,8). However, apart from the early discovery of nicotinic excitatory synaptic potentials in cat Renshaw cells (9), postsynaptic nAChRs have not been shown to be directly involved in central synaptic transmission. Thus, neuronal nicotinic synapses have been mostly studied in vertebrate autonomic ganglia (10-13) and recently in the guineapig adrenal gland (14, 15). Acetylcholine (ACh) release has been shown to be quantal at ganglionic synapses (16), but detailed quantal analysis was not done due to the poor resolution associated with intracellular recording. Detection of the postsynaptic action of a single quantum is a prerequisite to quantal analysis and determination of quantal parameters (17), thereby allowing study of pre-and postsynaptic factors involved in synaptic transmission (18,19).Cholinergic synapses arising from splanchnic nerve terminals are present on chromaffin cells (20). These cells are small, rounded, electrically compact and well suited for patch-clamp studies. ACh-induced current in chromaffin cells is due to the activation of a3 subunit containing nAChRs (21,22), which are also expressed in the hippocampus, medial habenula, lateral geniculate, and granular layer of cerebellum (2, 23). The (32 and perhaps also the 134 subunit appear to be coexpressed in chromaffin cells, given the se...

Functional Characterization of α9-Containing Cholinergic Nicotinic Receptors in the Rat Adrenal Medulla: Implication in Stress-Induced Functional Plasticity

“…The respective contribution of ␣9 and/or ␣9/␣10, ␣3, and ␣7 nAChR channels to sEPSCs still remains to be quantified. Based on the blocking effects of ␣-RgIA and hexamethonium, ␣3 nAChRs appear to preferentially mediate sEPSCs in control rats, as previously reported (Barbara and Takeda, 1996), whereas ␣9-containing nAChRs predominantly mediate sEPSCs in cold-stressed rats. The contribution of ␣7 nAChRs needs further investigation, keeping in mind that ␣7 and ␣10 subunits can be spatially associated (Lips et al, 2006).…”

“…Regarding cholinergic neurotransmission at rat splanchnic nerve-chromaffin cell synapses, we provide evidence that, in addition to ␣3 and ␣7 nAChRs (Barbara and Takeda, 1996;Martin et al, 2003), nAChRs built by assembly of ␣9 and/or ␣9/␣10 subunits are activated during synaptic release of ACh. Addition- Figure 6.…”

“…In some cells, a quantal analysis of sEPSCs was performed. Note that sEPSCs are independent of presynaptic action potentials at the splanchnic nerve-chromaffin cell synapse (Barbara and Takeda, 1996;Kajiwara et al, 1997). sEPSCs were analyzed by using Clampfit 9.0 (Molecular Devices).…”

“…Multiple ␣ and ␤ subunits have been identified in rat chromaffin cells (Di Angelantonio et al, 2003;Sala et al, 2008). Although the ␣3 subunit constitutes a major component of nAChRs activated in response to synaptically released acetylcholine (Criado et al, 1992;Nooney and Feltz, 1995;Barbara and Takeda, 1996), ␣7-built receptors are also involved in acetylcholine-mediated responses (Ló pez et al, 1998;Martin et al, 2003Martin et al, , 2005. The expression of ␣9 and ␣10 subunits in rat chromaffin cells (L. A. Olivos-Oré, L. Solís, C. Montiel, and A. R. Artalejo, unpublished observations) extends the tissular distribution of ␣9/␣10 nAChRs to a neuroendocrine tissue and raises the question of their role.…”

Ca2+ Imaging in Perfused Adrenal Medullae

“…Measuring the amount of catecholamines secreted, however, would sacrifice the time resolution in response. In contrast to the measurement of secretion, the record-ing of electrical activity in a slice preparation has a high time-resolution [7][8][9]. Thin, about 300 µm, slices of the adrenal gland are made with a vibratome and immersed in the balanced salt solution, which are continuously gassed with a certain mixture of O 2 and CO 2 .…”