Burst generation mediated by cholinergic input in terminal nerve-gonadotrophin releasing hormone neurones of the goldfish

Abstract

Peptidergic neurones play a pivotal role in the neuromodulation of widespread areas in the nervous system. Generally, it has been accepted that the peptide release from these neurones is regulated by their firing activities. The terminal nerve (TN)-gonadotrophin releasing hormone (GnRH) neurones, which are one of the well-studied peptidergic neurones in vertebrate brains, are characterised by their spontaneous regular pacemaker activities, and GnRH has been suggested to modulate the sensory responsiveness of animals. Although many peptidergic neurones are known to exhibit burst firing activities when they release the peptides, TN-GnRH neurones show spontaneous burst firing activities only infrequently. Thus, it remains to be elucidated whether the TN-GnRH neurones show burst activities and, if so, how the mode switching between the regular pacemaking and bursting modes is regulated in these neurones. In this study, we found that only a single pulse electrical stimulation of the neuropil surrounding the TN-GnRH neurones reproducibly induces transient burst activities in TN-GnRH neurones. Our combined physiological and morphological data suggest that this phenomenon occurs following slow inhibitory postsynaptic potentials mediated by cholinergic terminals surrounding the TN-GnRH neurones. We also found that the activation of muscarinic acetylcholine receptors induces persistent opening of potassium channels, resulting in a long-lasting hyperpolarisation. This long hyperpolarisation induces sustained rebound depolarisation that has been suggested to be generated by a combination of persistent voltage-gated Na(+) channels and low-voltage-activated Ca(2+) channels. These new findings suggest a novel type of cholinergic regulation of burst activities in peptidergic neurones, which should contribute to the release of neuropeptides.

Figure 2. The slow inhibitory postsynaptic potential (IPSP) is induced by the activation of muscarinic acetylcholine (Ach) receptors

A, B, attenuation of slow IPSP (current clamp; A) or inhibitory postsynaptic current (IPSC) (voltage clamp; B) by the application of gallamine (10 μm). The dashed black line, solid black line and grey line represent traces before, during and after gallamine applications. C, time course of the effect of gallamine (10 μm) on the amplitude of slow IPSC. D, E, current clamp recordings (D) or voltage clamp recordings (E) showing that the application of scopolamine abolished slow IPSP or IPSC (dashed line and continuous line represent traces before and during scopolamine applications, 20 μm and 1 μm in D and E, respectively). F, time course of the effect of scopolamine (1 μm) on the amplitude of slow IPSC. G, statistical comparison of the gallamine-induced (n= 3) and scopolamine-induced (n= 3) suppression of slow IPSC of the terminal nerve (TN)-gonadotrophin releasing hormone (GnRH) neurones in comparison with the vehicle application (n= 4). The averaged amplitude of 0.0–2.0 min before treatment was normalised to 100% and the averaged amplitude of 2.5–5.0 min after treatment was compared with this value. ***P < 0.001, Dunnett's test. H, puffer application of Ach (1 mm) on the TN-GnRH neurone immediately evoked a strong hyperpolarisation of the TN-GnRH neurone. Arrowhead indicates the time of Ach puffer application.

Figure 5. Prior bath application of Ba²⁺ reversibly diminishes acetylcholine (Ach)-induced outward currents in the terminal nerve (TN)-gonadotrophin releasing hormone (GnRH) neurones

A, representative traces showing the effect of Ba²⁺ on the Ach-induced currents (dashed line, continuous line and grey line represent traces before, during and after 500 μm Ba²⁺ application, respectively). The arrowhead indicates the time of Ach puffer application. B, time course of the effect of 500 μm Ba²⁺ application on the amplitude of Ach-induced currents. C, statistical comparison of the effect of Ba²⁺ on Ach-induced currents (n= 4) in comparison with the vehicle application (n= 5). The averaged amplitude of 3.0–5.5 min in (B) was normalised to 100% and the averaged value of 8.0–10.5 min was compared with the former. ***P < 0.001.

Figure 6. The slow rebound depolarisation consists of the combinational components of tetrodotoxin (TTX)-resistant persistent voltage-gated Na⁺ channels and low-voltage-activated Ca²⁺ channels

A, representative traces showing the effects of each pharmacological manipulation on the rebound depolarisations in the terminal nerve (TN)-gonadotrophin releasing hormone (GnRH) neurones. Aa, an application of TTX revealed that sustained rebound depolarisation follows the offset of the hyperpolarisation of the TN-GnRH neurone (grey, before 0.75 μm TTX application; black, during 0.75 μm TTX application). Dashed line denotes the baseline membrane potential before the current injection. Bath application of 1 mm Ni²⁺ (Ab) and substitution of extracellular Na⁺ with N-methyl-d-glucamine (NMDG) (Ac) did not diminish the rebound depolarisations in the presence of 0.75 μM TTX. Alternatively, sustained rebound depolarisation was diminished by bath application of 1 mm Ni²⁺ in Na⁺-free solution (Ad) or 200 μm Ni²⁺ in Na⁺-free solution (Ae) in the presence of 0.75 μM TTX (grey line, solid black line and dashed black line represent before, during and after Ni²⁺+ NMDG, respectively). The values on the left of the traces denote the membrane potentials. Scale bar, 1 s. B, Statistical comparisons of the effects of pharmacological manipulations on the rebound depolarisation. The response 0.5 min before treatment was normalised to 100%, and the value 4 min after treatment was examined. Ba, normalised change in the amplitudes of the rebound depolarisations. Bb, normalised change in the averaged value (first 5 s) of the rebound depolarisations. Bc, change in the onset of rebound depolarisations. Numbers in parentheses represent the numbers of neurones tested for the experiment. *P < 0.05, Dunnett's test.

Figure 1. Current clamp recordings show that the tonic firing activities of terminal nerve (TN)-gonadotrophin releasing hormone (GnRH) neurones are switched to burst activities by a stimulation of fibres around them (A–E) or by a hyperpolarising current injection (F–I)

A, schematic drawing of the fibre stimulation. The firing activities of the TN-GnRH neurones were recorded by whole cell recording, and the fibres around the TN-GnRH neurone cell bodies were stimulated by a bipolar electrode. B, slow inhibitory postsynaptic potential (IPSP) was elicited by a single pulse stimulation of fibres surrounding the TN-GnRH neurones. A transient change from tonic firing to burst firing was also observed. The arrowhead indicates the time of the stimulus. C, raster plot showing that the activity of a TN-GnRH neurone is altered by the stimulation. Results of 10 repetitive trials from the same neurone are shown. The arrowhead indicates the time of stimulation. Scale bar, 1 s. D, averaged peristimulus time histogram (PSTH) showing the activities of TN-GnRH neurones (n= 9). The stimulation is applied at 0 ms. The time bin is 200 ms. E, application of tetrodotoxin (TTX) abolished the slow IPSP as well as the action potentials in the TN-GnRH neurone (the dashed and continuous line represent the traces before and during 0.75 μm TTX application, respectively). The arrowhead indicates stimulus artefact. Scale bar, 20 mV and 200 ms, respectively. F, schematic drawing of the experimental condition for injecting hyperpolarising current pulses to the TN-GnRH neurone. A whole cell patch-clamp electrode was used to record TN-GnRH neuronal activity and to apply hyperpolarising current pulses (1 s). The current amplitude was adjusted to the value that hyperpolarised the neurone by more than 20 mV. G, hyperpolarising current injection also induced a transient change in the firing frequency of the TN-GnRH neurones. H, raster plots showing that the activity of a TN-GnRH neurone is altered by the current injection. The grey box indicates the period of current injection. Results of 10 repetitive trials from the same neurone are shown. I, averaged PSTH showing the activities of TN-GnRH neurones. A hyperpolarising current is applied from 0 ms to 1000 ms. The time bin is 200 ms.

Figure 3. Confocal microscopy images of a cell cluster of terminal nerve (TN)-gonadotrophin releasing hormone (GnRH) neurones double-labelled with anti-choline acetyltransferase (anti-ChAT) antiserum and anti-microtubule-associated protein (MAP)2a + 2b monoclonal antibody

A, immunoreactive fluorescent signals against MAP2a + 2b show the location of the somata and dendrites of TN-GnRH neurones in the cell cluster of goldfish olfactory bulb. B, fluorescent signals against ChAT antiserum show the location of cholinergic neuroterminals around the TN-GnRH neurones. C, merged image of MAP2a + 2b (magenta) and ChAT (green) immunocytochemistry. D–F, higher magnification images of the central regions of A–C. Dense distribution of ChAT-immunoreactive terminal buttons was observed around the dendrite and soma of TN-GnRH neurones. Scale bar: 50 μm (A–C); 20 μm (D–F).

Figure 4. Reversal potential values of the slow inhibitory postsynaptic current (IPSC) are consistent with the values for the calculated equilibrium potentials for potassium ions

A, slow IPSCs recorded at different holding potentials ranging from –120 to −40 mV (10 mV step) in 3, 6 and 9 mm[K⁺]_o. B, I–V plot of the peak value of slow IPSCs obtained from the current responses. C, average value of reversal potentials is consistent with the calculated equilibrium potentials for potassium ions in 3, 6 and 9 mm[K⁺]_o. Each small dot indicates each data point, and the dashed line denotes the equilibrium potentials of potassium ions calculated from the Nernst equation. D, acetylcholine (Ach)-induced currents recorded at different holding potentials ranging from −120 to −40 mV. The reversal potential of slow IPSC evoked by puffer application of Ach is consistent with that by electrical stimulation of the surrounding fibres. The arrowhead indicates the time of Ach puffer application. E, I–V plot of the peak value of Ach-induced currents obtained from the traces shown in (D).

Figure 7. A proposed mechanism underlying the slow inhibitory postsynaptic potential (IPSP) and the rebound burst activities in terminal nerve (TN)-gonadotrophin releasing hormone (GnRH) neurones

Stimulation of the cholinergic fibres around the TN-GnRH neurones activates the muscarinic acetylcholine receptors (mAchR) on the TN-GnRH neurones. This results in the opening of potassium channels, showing a large and long hyperpolarisation of TN-GnRH neurones. The long hyperpolarisation relieves the inactivation of persistent voltage-gated Na⁺ channels (VGSCs) and low-voltage-activated (LVA) Ca²⁺ channels. Cooperative action of persistent VGSCs and LVA Ca²⁺ channels generates the rebound depolarisations, which evoke the rebound burst discharges in the TN-GnRH neurones.