Kv4.2 Mediates Histamine Modulation of Preoptic Neuron Activity and Body Temperature

Histamine regulates arousal, circadian rhythms, and thermoregulation. Activation of H3 histamine receptors expressed by preoptic GABAergic neurons results in a decrease of their firing rate and hyperthermia. Here we report that an increase in the A-type K+ current in preoptic GABAergic neurons in response to activation of H3 histamine receptors results in decreased firing rate and hyperthermia in mice. The Kv4.2 subunit is required for these actions in spite of the fact that Kv4.2−/− preoptic GABAergic neurons display A-type currents and firing characteristics similar to those of wild-type neurons. This electrical remodeling is achieved by robust upregulation of the expression of the Kv4.1 subunit and of a delayed rectifier current. Dynamic clamp experiments indicate that enhancement of the A-type current by a similar amount to that induced by histamine is sufficient to mimic its robust effect on firing rates. These data indicate a central role played by the Kv4.2 subunit in histamine regulation of body temperature and its interaction with pERK1/2 downstream of the H3 receptor. We also reveal that this pathway provides a mechanism for selective modulation of body temperature at the beginning of the active phase of the circadian cycle

Upregulation of the level of expression of Kv4.1 subunits in Kv4.2−/− preoptic neurons.

<p>A. Single channel confocal images showing immunoreactivity to Kv4.1 (<i>Left</i>, green) and Kv4.2 (<i>Middle</i>, red) and their superimposition (<i>Right</i>, merge) obtained from dissociated w-t (upper row) and Kv4.2−/− (lower row) preoptic neurons. B. Cumulative histograms of the Kv4.1 staining intensity in w-t (blue circles, n = 708 neurons) and Kv4.2−/− neurons (red triangles, n = 811 neurons). The distributions were significantly different (P<0.01, Kolmogorov-Smirnov test). C. qPCR analysis of Kv4.1 (left) and Kv4.3 gene (right) expression in preoptic area from w-t and Kv4.2−/− mice (n = 6 mice each). For normalization GAPDH was selected as housekeeping gene. Bars represent averaged normalized concentrations ± SD. ** represents significant differences between w-t and Kv4.2−/− groups P<0.01 (t-test).</p

Voltage-gated K⁺ currents recorded in w-t and Kv4.2−/− preoptic GABAergic neurons.

<p>A. Voltage-gated K⁺ currents recorded in the presence of TTX (1 µM) in w-t (left) and Kv4.2−/− (right) neurons. The currents were elicited by the voltage step protocol depicted in the inset. Note the large I_DR present in the Kv4.2−/− neuron. B. Separation of I_A in the presence of TEA and 4-AP. The currents were elicited by the same voltage step protocol as in A. Recordings were from the same neurons as in A (upper traces). Lower traces represent the same traces on an expanded timescale. C. I–V plot for peak I_A amplitude in w-t (•) and Kv4.2−/− neurons (□) recorded in the presence of TEA and 4-AP. D. I–V plot for the steady state component of the current at the end of the depolarizing steps (Iss) in w-t (•) and Kv4.2−/− neurons (□) recorded in standard extracellular solution. C, D. Data points represent averages ±S.D. from n = 14 w-t neurons and n = 15 Kv4.2−/− neurons. The data points at −70, +10 and +20 mV are from a subset of n = 6 w-t neurons and n = 6 Kv4.2−/− neurons. E. I_A currents recorded in the presence of TEA and 4-AP in response to the voltage step protocol depicted in the inset. A depolarizing step to 0 mV was preceded by a 200 ms conditioning step to voltages ranging from −130 to −30 mV. F. Voltage-dependence of inactivation of I_A recorded in w-t (•, n = 8) and Kv4.2−/− neurons (□, n = 8). Data presented as averages ± S.D.</p

Dynamic clamp experiments reveal the role of I_A in the pacemaking activity of preoptic neurons.

<p>A. Comparison between the recorded I_A (black) and those generated by the corresponding model (red). The currents were elicited by step depolarizations from −110 mV holding potential to the indicated test potentials. B. Spontaneous firing activity in a preoptic neuron in control (left upper trace) and when the I_A current is increased by 10% (middle upper trace) or 20% (right upper trace). The lower traces represent the I_A injected by the dynamic clamp. Note the prolonged ramp-like potentials that precede a spike and the presence of subthreshold membrane potential oscillations (*) during dynamic clamp. C. Voltage-dependence of activation (•) and inactivation (▴) of I_A recorded from w-t neurons (data replotted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029134#pone-0029134-g003" target="_blank">Fig. 3 C and F</a>). The lines represent voltage-dependence of activation (dashed line) and inactivation (dotted line) of I_A generated by our model.</p

Activation of H3 subtype histamine receptors reduces the firing rate of preoptic w-t GABAergic neurons but has no effect on the firing rate of Kv4.2−/− neurons.

<p>A. Single channel confocal images showing immunoreactivity to Kv4.2 (<i>Left</i>, green) and Kv4.3 (<i>Middle</i>, red) and their superimposition (<i>Right</i>, merge) obtained from dissociated w-t preoptic neurons. B. Immunoreactivity to H3R (<i>Left</i>, green) and GAD1 (<i>Middle</i>, red) and their superimposition (<i>Right</i>, merge) obtained from dissociated Kv4.2−/− preoptic neurons. Note the immunoreactivity to both H3R and GAD1 of a Kv4.2−/− neuron. C, D. Expanded fragments of a recording showing “pacemaker” firing of the neuron in control (upper trace), during R-α-methylhistamine (1 µM) application (middle), and during washout (lower) from a w-t (C) and a Kv4.2−/− (D) dissociated neuron. E. Average firing rate (for every 10 s) recorded before, during, and after application of R-α-methylhistamine (1 µM). The filled circles (•) and squares (□) correspond to the experiment presented in B and C, respectively.</p

Intra-MnPO injection of PaTx1 shifts the CBT circadian profile in w-t mice.

<p>A. CBT (upper panel) and MA (lower panel) responses to intra-MnPO injection of PaTx1 (800 nM, 0.2 µL) (black) and aCSF (0.2 µL) (red) injections in Kv4.2−/− mice B. CBT (upper panel) and MA (lower panel) responses to intra-MnPO injection of PaTx1 (800 nM, 0.2 µL) (black) and aCSF (0.2 µL) (red) injections in w-t mice. The toxin induced a ∼1 h delay in the CBT rise associated with the active phase (two-way repeated measures ANOVA followed by t-tests for each time point, * P<0.05) and did not affect motor activity (two-way repeated measures ANOVA followed by t-tests for each time point, P>0.3). A–B. The line graphs represent averages±S.D. through the 24 h recording period. Experiments were carried out in parallel in 2 groups of 6 and Kv4.2−/− and w-t mice, respectively.</p

Circadian CBT and MA profiles and responses to H3 antagonist infusions.

<p>A. Intra-MnPO injection of aCSF (0.2 µL) had no effect on CBT (upper panel) or MA (lower panel) in w-t and Kv4.2−/− mice. The circadian CBT profile of Kv4.2−/− mice was shifted by 1 h (two-way repeated measures ANOVA followed by t-tests for each time point, * P<0.05). Data were pooled and averaged from 6 w-t (black) and 6 Kv4.2−/− (red) neurons. Data are plotted at 12 min intervals to emphasize the shift in circadian rhythm. The MA of Kv4.2−/− mice was not significantly different (two-way repeated measures ANOVA followed by t-tests for each time point, P>0.2). B. Intra-MnPO injection of H3 antagonist thioperamide (1 µM, 0.2 µl) delayed the CBT rise by 1 h in w-t (red) mice and had no effect in Kv4.2−/− mice (black). After thioperamide infusion the CBT profiles of w-t and Kv4.2−/− mice were not different (two-way repeated measures ANOVA followed by t-tests for each time point, P>0.1).</p

Properties of preoptic GABAergic w-t and Kv4.2−/− neurons.

<p>Values are presented as mean ± S.D., * and ** indicate P<0.05 and P<0.01, respectively (unpaired t-tests), (n)-number of cells.</p