Temperature modulates PVN pre-sympathetic neurones via transient receptor potential ion channels

Abstract

The paraventricular nucleus (PVN) of the hypothalamus plays a vital role in maintaining homeostasis and modulates cardiovascular function via autonomic pre-sympathetic neurones. We have previously shown that coupling between transient receptor potential cation channel subfamily V Member 4 (Trpv4) and small-conductance calcium-activated potassium channels (SK) in the PVN facilitate osmosensing, but since TRP channels are also thermosensitive, in this report we investigated the temperature sensitivity of these neurones. Methods: TRP channel mRNA was quantified from mouse PVN with RT-PCR and thermosensitivity of Trpv4-like PVN neuronal ion channels characterised with cell-attached patch-clamp electrophysiology. Following recovery of temperature-sensitive single-channel kinetic schema, we constructed a predictive stochastic mathematical model of these neurones and validated this with electrophysiological recordings of action current frequency. Results: 7 thermosensitive TRP channel genes were found in PVN punches. Trpv4 was the most abundant of these and was identified at the single channel level on PVN neurones. We investigated the thermosensitivity of these Trpv4-like channels; open probability (Po) markedly decreased when temperature was decreased, mediated by a decrease in mean open dwell times. Our neuronal model predicted that PVN spontaneous action current frequency (ACf) would increase as temperature is decreased and in our electrophysiological experiments, we found that ACf from PVN neurones was significantly higher at lower temperatures. The broad-spectrum channel blocker gadolinium (100 µM), was used to block the warm-activated, Ca²⁺-permeable Trpv4 channels. In the presence of gadolinium (100 µM), the temperature effect was largely retained. Using econazole (10 µM), a blocker of Trpm2, we found there were significant increases in overall ACf and the temperature effect was inhibited. Conclusion: Trpv4, the abundantly transcribed thermosensitive TRP channel gene in the PVN appears to contribute to intrinsic thermosensitive properties of PVN neurones. At physiological temperatures (37°C), we observed relatively low ACf primarily due to the activity of Trpm2 channels, whereas at room temperature, where most of the previous characterisation of PVN neuronal activity has been performed, ACf is much higher, and appears to be predominately due to reduced Trpv4 activity. This work gives insight into the fundamental mechanisms by which the body decodes temperature signals and maintains homeostasis.

Keywords: PVH; PVN; TRPV4; computational model; hypothalamus; ion channel; patch clamp; thermoregulation.

FIGURE 1

Thermosensitive TRP channel gene expression in PVN punches from young mice. Matlab heatmap standardized (by column) difference ((delta)) in cycle threshold (ΔCt) levels for seven known TRP channel mRNA in punches of the PVN (from 6 animals, 3 young adult and 3 old adults). Red genes are relatively highly expressed and the green are low expression. Mean ΔCt was lowest (highest mRNA abundance) for Trpv4 (see values in the text). A full dataset of 84 ion channel mRNA ΔCt levels measured in younger (6–8 months) and older (26 months) mice are given together with heatmaps in the Supporting Material.

FIGURE 2

Single channel properties of Trpv4-like channels from PVN neurones (A). Representative single-channel current fluctuations through Trpv4-like channels from mouse PVN neurones. Holding potentials are indicated on the trace. The open and closed channel levels are indicated by O and C, respectively. This trace is representative of 10 experiments where Trpv4-like channels were observed and these currents were absent in the presence of the Trpv4 inhibitor GSK2193874. (B). The amplitude histogram is shown for ion channels shown A, at Vm −70 mV. (C). Current-voltage relationship for Trpv4-like channels. Mean ± SEM is shown (n = 9).

FIGURE 3

The gating of PVN Trpv4-like channels is temperature sensitive. (A) Representative single-channel current fluctuations through Trpv4-like channels from mouse PVN neurones at 37°C, 32°C and 22°C. The open and closed channel levels are indicated by O and C, respectively. (B). Current-voltage relationship for Trpv4-like channels at 37°C (red circles) and 22°C (blue circles). (C). The Po of Trpv4-like channels at 37°C, 32°C and 22°C is shown. (D). Mean open and closed times for Trpv4-like channels. Mean ± SEM is shown (n = 7 at 22°C, n = 7 at 32°C and n = 9 at 37°C).

FIGURE 4

Kinetics of Trpv4-like channels from PVN neurones. Kinetic analysis of Trpv4-like channel dwell-times from PVN neurones recorded in cell attached-patch mode at 37°C (A), 32°C (B) and 22°C (C). Closed (left) and open (right) dwell-times were fitted with 3 exponentials (solid lines). Data are transformed with log-binning (x-axis) and square root of frequency (y-axis) so that exponential time constants are visible as peaks (Sigworth and Sine, 1987). Mean values are given in Table 1 and a kinetic schema in Figure 5.

FIGURE 5

In silico model of PVN neurones. (A) The simple scheme adapted from (Feetham et al., 2015b) whereby influx of Ca²⁺ increases K_Ca channel activity which hyperpolarizes the cell and increases the inward flux of Ca²⁺, by increasing the driving force for Ca²⁺ entry. (B). A computer model was adapted from (Feetham et al., 2015b) in NEURON, which includes thermosensitive TRP channels and allows an accumulation of Ca²⁺ into the cell, which is linked to a K_Ca channel. Within the model, we can change temperature and simulate action currents, shown in (C). (D). Increase in action potential frequency when temperature is decreased (n = 5 simulation runs, mean and SD of the 5 runs shown in (D).

FIGURE 6

Temperature decreases action current frequency of PVN neurones. (A). Representative spontaneous firing of action currents from PVN neurones are shown at physiological temperature (37°C) and at lower temperatures of 32°C (B), 27°C (C) and 22°C (D). (E). Representative frequency histogram showing action current response of a PVN neurone to decreasing temperature. (F). The mean temperature responses are shown for PVN neurones. Data is presented as mean ± SEM (n = 6, ***p < 0.001, Friedman Test).

FIGURE 7

Pharmacological inhibition of various TRP channels on temperature sensitivity of PVN neurones. Representative spontaneous firing of action currents from PVN neurones are shown at physiological temperature (37°C) and at lower temperatures of 32°C, 27°C and 22°C in the presence of bath applied (A) gadolinium or (B) econazole. In (C), the mean temperature responses are shown for PVN neurones in the presence of the non-specific inhibitor gadolinium, 100 µM (brown) or the Trpm2 blocker econazole, 10 µM (green). Data is presented as mean ± SEM (n = 6 for control, n = 4 for gadolinium, n = 5 for econazole, *p < 0.05, **p < 0.01, Friedman Test).

FIGURE 8

Thermosensitive TRP channel genes. Established distribution of transient receptor potential (TRP) channels in PVN tissue as a function of their temperature threshold. TRP channels may be activated by increases in temperature (orange) or by lowering the temperature (blue) (Klein et al., 2015). Image modified under BY4.0 Creative Commons Licence from (Lamas et al., 2019).