Simulated GABA synaptic input and L-type calcium channels form functional microdomains in hypothalamic gonadotropin-releasing hormone neurons

Abstract

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons integrate the multiple internal and external cues that regulate sexual reproduction. In contrast to other neurons that exhibit extensive dendritic arbors, GnRH neurons usually have a single dendrite with relatively little branching. This largely precludes the integration strategy in which a single dendritic branch serves as a unit of integration. In the present study, we identify a gradient in L-type calcium channels in dendrites of mouse GnRH neurons and its interaction with GABAergic and glutamatergic inputs. Higher levels of L-type calcium channels are in somata/proximal dendrites (i.e., 0-26 μm) and distal dendrites (∼130 μm dendrite length), but intervening midlengths of dendrite (∼27-130 μm) have reduced L-type calcium channels. Using uncaging of GABA, there is a decreasing GABAergic influence along the dendrite and the impact of GABA(A) receptors is dependent on activation of L-type calcium channels. This results in amplification of proximal GABAergic signals and attenuation of distal dendritic signals. Most interestingly, the intervening dendritic regions create a filter through which only relatively high-amplitude, low-frequency GABAergic signaling to dendrites elicits action potentials. The findings of the present study suggest that GnRH dendrites adopt an integration strategy whereby segments of single nonbranching GnRH dendrites create functional microdomains and thus serve as units of integration.

Figure 1.

Dynamic clamping distinguishes GnRH neurons that respond to simulated GABAergic excitation with action potentials (“responder”) from GnRH neurons that do not generate GABA-driven action potentials (nonresponder). Representative activity of a responder GnRH neuron to simulated GABAergic excitation using synapses with unitary conductances of 500 pS (A) and 1250 pS (B). Representative firing of a nonresponder GnRH neuron to GABAergic excitation with synapses using unitary conductances of 500 pS and reversal potentials of simulated inputs of either −36.5 mV (C) or 0 mV (D). C and D show the response of the same GnRH neuron to the same GABAergic input profiles in which only the reversal potential of the simulated inputs was changed during recording. The arrows in C and D indicate when application of the simulated conductance profile ceased and the membrane potential returned to its resting state some 10–20 mV lower than when simulated GABAergic excitation was applied. Shown are average firing rates of all GnRH neurons (E). Shown are average firing rates of only GnRH neurons that responded to GABAergic excitation with at least one action potential (F). Numbers of neurons are indicated in parentheses. The asterisks indicate significance at p ≤ 0.05 within groups of a given conductance level. Error bars indicate SEM.

Figure 2.

Blocking L-type calcium currents eliminates most action potentials induced by simulated GABAergic excitation in responder GnRH neurons. A, Traces show firing patterns in two different GnRH neurons before (no block) and after (block) bath application of nimodipine (10 μm). The asterisks indicate action potentials that were eliminated following bath application of nimodipine (block). The same profile of simulated GABAergic conductances was used in the control and nimodipine-treated conditions in individual GnRH neurons to allow for a direct comparison within the same neuron. Different profiles of GABAergic excitation (but with the same overall input frequencies) were applied between GnRH neurons to control for any effects of a particular conductance profile. B, Spike-triggered averaging on conductance indicates that responder GnRH neurons require higher conductance magnitudes of GABAergic inputs to generate action potentials when L-type calcium channels are pharmacologically blocked.

Figure 3.

Cell-attached recordings from GnRH neurons indicate that L-type calcium channels contribute to high-frequency discharges. In the presence of functional L-type calcium channels, GnRH neurons in adult male mice exhibit short bursts of action potentials (A). Following addition of nimodipine (10 μm) to the bath perfusate, the pattern of discharge shifts to an irregular pattern with a reduced frequency of action potentials (B). Recorded neurons express L-type calcium channels in somata and proximal dendrites (C).

Figure 4.

Dendritic gradients in the distribution of L-type calcium channels in GnRH neurons. Approximately one-half of GnRH neurons express L-type calcium channels in somata and proximal dendrites (at arrowheads in A; the asterisk indicates a GnRH somata without immunoreactivity for L-type calcium channels). A GnRH neuron with only modest staining for L-type calcium channels in somata (B). A GnRH neuron with L-type calcium channels in the proximal dendrite (at arrowheads in C). Representative GnRH neurons with L-type calcium channels in the distal dendrites (at arrowheads in D and E). F, Distribution of L-type calcium channels in GnRH somata and dendrites.

Figure 5.

Uncaging GABA along the length of GnRH dendrites results in decreasing EPSC amplitude. Large somatic EPSCs occur in GnRH somata in response to uncaging GABA (A). At 100 μm of dendrite length (B) and 200 μm of dendrite length (C), responses to uncaging GABA are reduced. Significantly smaller EPSCs occur in GnRH dendrites in response to the same uncaging stimulus used on somata. D, The asterisks indicate significance at p ≤ 0.05 within groups between neuronal regions. Error bars indicate SEM.

Figure 6.

Blocking L-type calcium channels reduces the response to uncaging GABA. Each trace shows a repeated response to GABA uncaging (top traces) and GABA uncaging in the absence of functional L-type calcium channels (bottom traces). Robust action potentials occur when GABA is uncaged on GnRH somata (A; top trace). Action potentials are reduced when L-type calcium channels are blocked (A; bottom trace). Action potentials are elicited by uncaging GABA at 100 μm of dendrite length (B; top traces) and reduced when L-type calcium channels are blocked (B; bottom traces). Action potentials are not generated when GABA is uncaged on GnRH dendrites at 100 μm of dendrite length (C; top trace). Significantly lower action potential frequencies occur in response to uncaging GABA on GnRH dendrites compared with GnRH somata (D; black bars). Blocking L-type calcium channels reduces response to uncaging GABA on both GnRH somata and dendrites (D; gray bars). The asterisks indicate significance at p ≤ 0.05 within groups between neuronal regions (black bars) and within regions between treatments (gray bars). Error bars indicate SEM.

Figure 7.

The response of GnRH neurons to simulated AMPA-type inputs (A, black traces) is not altered by addition of nimodipine (10 μm) to the bath perfusate (A, gray traces). In contrast, the response to uncaging of glutamate on GnRH somata and along GnRH dendrites (B, C, black bars; n = 8) was completely eliminated in the presence of nimodipine (10 μm) in the bath perfusate (B, gray bars; n = 4) but was only reduced in a second population of GnRH neurons (C, gray bars; n = 4). The asterisks indicate significance at p ≤ 0.05 within groups at the same location. Error bars indicate SEM.

Figure 8.

GnRH neurons relocated after GABA uncaging experiments and analyzed for L-type calcium channel immunoreactivity. Biocytin-filled GnRH neurons (A, B). Distribution of L-type calcium channels in GnRH neurons used during GABA uncaging experiments (C, D).