Synaptic potentials mediated by alpha 7 nicotinic acetylcholine receptors in supraoptic nucleus

Abstract

Brain slice preparations preserving projections from nearby forebrain cholinergic neurons to the supraoptic nucleus (SON) were used to study synaptic potentials mediated by nicotinic acetylcholine receptors (nAChRs) in the hypothalamus. Paired-pulse electrical stimulation in an area anterior to the SON that was rich in cholinergic cells confirmed the monosynaptic nature of the connections to putative oxytocin and vasopressin SON neurons. With ionotropic glutamate and GABA(A) transmission blocked, this stimulation evoked fast, atropine-insensitive EPSPs that were sensitive to nAChR antagonists. Evoked EPSPs were blocked by methyllycaconitine and alpha-bungarotoxin, antagonists that are selective for nAChRs containing the alpha7 subunit, but not by dihydro-beta-erythroidine at concentrations known to antagonize alpha4beta2 nAChRs. Although anatomical evidence exists for postsynaptic alpha4beta2 nAChRs in the SON, these results indicate that postsynaptic alpha7 nAChRs are primarily responsible for the cholinergically mediated EPSPs. Repetitive stimulation suggested partial desensitization of the receptors. With ionotropic glutamate transmission blocked, inhibition of AChE increased spontaneous EPSP frequency and amplitude, suggesting spontaneous ACh release. ACh, nicotine, and choline (a selective alpha7 nAChR agonist) were effective in evoking action potentials and repetitive firing with synaptic transmission blocked by low Ca2+, high Mg2+ medium. These agonists were also effective in evoking the type of phasic bursts characteristic of vasopressin neurons, long thought to be completely dependent on activation of NMDA receptors (NMDARs). Because phasic bursting is Ca2+-dependent, the functional equivalence of alpha7 nAChR and NMDAR activation in this regard is likely attributable to their large Ca2+ fluxing capacities. This is the first demonstration that synaptically released ACh results in fast, alpha7 nAChR-mediated EPSPs in hypothalamic neurons.

Fig. 1.

A, Micrograph of a transilluminated hypothalamic slice in a static recording chamber, showing the plane of the horizontal slice preparation used. CTX, Cortex;HDB, horizontal limb of the diagonal band of Broca;OC, optic chiasm; OT, optic tract;REC, recording electrode (tip indicated byarrow); STIM, stimulating electrode; SON, supraoptic nucleus pars anterior;3V, third ventricle. B, Diagram of horizontal section on which cholinergic neurons near the SON are plotted. (Modified from Mason et al., 1983.) Arrowsindicate the area stimulated in this study.

Fig. 2.

Recordings (averages of five traces) of two cells maintained in medium containing (in μm): 6 CNQX, 50dl-APV, and 20 BIC. Left, Synaptic responses to single- and paired-pulse stimulation of a cholinergic cell area.Right, Synaptic responses to repetitive stimulation of the same area. Cells subjected to repetitive stimulation were hyperpolarized by 2–5 mV.

Fig. 3.

Continuous recording showing synaptic responses to single and high-frequency repetitive stimulation of a cholinergic cell area in medium containing (in μm): 6 CNQX, 50dl-APV, and 20 BIC. Indicated EPSPs are shown at higher sweep speed (insets below each trace). Subsequent single stimuli evoked attenuated EPSPs, which then recovered to amplitudes at or above those seen before stimulation. Cells were hyperpolarized by 5 mV from the resting state to enhance EPSP amplitude.

Fig. 4.

Synaptic responses to cholinergic cell stimulation (averages of five traces). Medium contained 6 μm CNQX and 50 μmdl-APV. Responses were blocked by 10 μm but not 1 μm dihydro-β-erythroidine, which is an α4β2 nAChR-selective antagonist at concentrations up to 1 μm. Arrows indicate stimulus artifacts.

Fig. 5.

Reversible blockade of cholinergically mediated synaptic responses in SON neurons with initial spontaneous firing patterns that were either phasic (A) or continuous (B) by the α7 nAChR-selective antagonist MLA. MLA added to the medium in A1 orB1 reversibly abolished the synaptic responses in both cell types. Arrows indicate stimulus artifacts.

Fig. 6.

Blockade of synaptic responses in two different cells with α7-selective antagonists. A1, Cholinergic synaptic responses at resting and hyperpolarized potentials remained in the presence of 6 μm CNQX, 50 μmdl-APV, and 20 μm BIC. A2, A3, Recordings at hyperpolarized potentials show that these responses were completely and reversibly blocked by 50 nm MLA.B1, Synaptic responses that remained when glutamate transmission was blocked were reversibly eliminated by medium containing 9 mm Mg²⁺ and 0.05 mm Ca²⁺ (B2, B3). Synaptic responses were partially (B4) and then completely (B5) antagonized by 20 and 100 nmα-bungarotoxin. All records are the averages of five traces, with five cells in each treatment. Arrowsindicate stimulus artifacts.

Fig. 7.

Effects of AChE inhibition by galanthamine.A, Spontaneous EPSPs were observable with ionotropic glutamate and GABA_A receptors blocked. B, Addition of galanthamine resulted in a doubling of EPSP frequency and depolarization. C, Washout with the medium used inA reduced EPSP frequency (n = 3).D, At higher concentrations (different cell), galanthamine induced larger depolarizations and prolonged excitability (n = 3).

Fig. 8.

Enhancement of cholinergic EPSPs by AChE inhibition. A, Addition of galanthamine to the medium resulted in larger amplitudes and faster rising phases of evoked EPSPs.B, Similar enhancements were seen with 1 mmcholine, which also may inhibit AChE (see Results). That choline EPSPs are α7-mediated is suggested by their blockade with MLA. Averages of five traces are shown; n = 3 in each treatment. *Calibration pulse in A, 10 mV, 5 msec.Arrows indicate stimulus artifacts.

Fig. 9.

Effects of ACh on SON neurons showing two spontaneous firing patterns. A, ACh application increased the firing rates of continuously firing cells with slight membrane depolarization (n = 5). B, Firing of phasic cells was increased by ACh application, which usually also led to membrane potential oscillations (n = 5). C, The postsynaptic site of ACh action is suggested by its efficacy with synaptic transmission blocked (n = 3) .

Fig. 10.

Effect of nicotine on phasically firing neurons. Although ineffective at 5 μm (A), nicotine prolonged phasic bursts at 10 μm(B) and 20 μm(C). D, Blocking synaptic transmission typically eliminates phasic bursts in the SON, but nicotine application, presumably acting postsynaptically during this blockade, was able to evoke such bursts (n = 5).

Fig. 11.

Effects of choline on phasically and continuously firing neurons. A, Choline applied by bath evoked bursts in neurons for which spontaneous phasic activity was silenced by glutamate receptor antagonists. B, MLA blocked these choline-induced responses, and washout reinstated them (C). D, Continuous firing persists with glutamate receptors antagonized, and choline application was effective in enhancing firing rates. E, F, MLA reversibly blocked the choline-induced effect.