Opposing actions of co-released GABA and neurotensin on the activity of preoptic neurons and on body temperature

Abstract

Neurotensin (Nts) is a neuropeptide acting as a neuromodulator in the brain. Pharmacological studies have identified Nts as a potent hypothermic agent. The medial preoptic area, a region that plays an important role in the control of thermoregulation, contains a high density of neurotensinergic neurons and Nts receptors. The conditions in which neurotensinergic neurons play a role in thermoregulation are not known. In this study optogenetic stimulation of preoptic Nts neurons induced a small hyperthermia. In vitro, optogenetic stimulation of preoptic Nts neurons resulted in synaptic release of GABA and net inhibition of the preoptic pituitary adenylate cyclase-activating polypeptide (PACAP) neurons firing activity. GABA-A receptor antagonist or genetic deletion of VGAT in Nts neurons unmasked also an excitatory effect that was blocked by a Nts receptor 1 antagonist. Stimulation of preoptic Nts neurons lacking VGAT resulted in excitation of PACAP neurons and hypothermia. Mice lacking VGAT expression in Nts neurons presented changes in the fever response and in the responses to heat or cold exposure as well as an altered circadian rhythm of body temperature. Chemogenetic activation of all Nts neurons in the brain induced a 4-5 °C hypothermia, which could be blocked by Nts receptor antagonists in the preoptic area. Chemogenetic activation of preoptic neurotensinergic projections resulted in robust excitation of preoptic PACAP neurons. Taken together our data demonstrate that endogenously released Nts can induce potent hypothermia and that excitation of preoptic PACAP neurons is the cellular mechanism that triggers this response.

Keywords: GABA; body temperature; hyperthermia; hypothermia; neurotensin; neurotensinergic neuron; preoptic area.

Figure 1.. Electrophysiological characteristics of MPO^Nts;hChR2 neurons

A. Representative example of spontaneous firing activity of MPO^Nts;hChR2 neurons recorded in whole-cell (up) or cell-attached configuration (down). B. Membrane potential responses to hyperpolarizing current steps of −40 and −20 pA reveal the presence of a low threshold spike (LTS) upon depolarization to the resting membrane potential. Depolarizing current injections of 20 and 40 pA (right) elicit burst firing activity. The neuron fires 2–3 action potentials during each burst. C. Nts (up) and VGAT (down) transcripts are present in MPO^Nts;hChR2 neurons. Representative results from 10 MPO^Nts;hChR2 neurons. The expected sizes of the PCR product are 149 and 137 base pairs, respectively. Negative (−) control was amplified from a harvested cell without reverse-transcription, and positive control (+) was amplified using 1 ng of hypothalamic mRNA. Nts transcripts were detected in 7 out of 10 neurons while VGAT was detected in 8 neurons. 6 neurons expressed both transcripts.

Figure 2.. Effects of optogenetic stimulation of MPO^Nts;hChR2 neurons on the firing activity of nearby MPO neurons

A. Optogenetic stimulation of a MPO^Nts;hChR2 neuron decreases the spontaneous firing rate of a nearby MPO neuron (upper trace) from 2.2 Hz to 0.9 Hz and increases the frequency of IPSC from 0.5 Hz to 21.8 Hz (see expanded trace). Gabazine (5 μM) (middle trace) increased the spontaneous firing activity of the neuron and abolished the activation of IPSCs by optogenetic stimulation. In the presence of Gabazine optogenetic stimulation increased the firing activity of the neuron from 4.1 Hz to 9.6 Hz. This increase in firing activity was blocked by pe-incubation with the NtsR1 antagonist SR48692 (100 nM) (lower trace). B. Bar charts summarizing the effects of optogenetic stimulation of MPO^Nts;hChR2 neurons on the spontaneous firing rates of nearby MPO neurons in control and in the presence of Gabazine (5 μM) and/or the NtsR1 antagonist SR48692 (100 nM). Bars represent means ± S.D. of the normalized firing rate relative to the control. The control value for the firing rate was calculated as the average value during the 5 min period preceding the optogenetic stimulation. Data pooled from n=9 neurons in each condition. There was a statistically significant difference between groups as determined by one-way ANOVA (F(5,40)= 77.71, p=1.08×10⁻⁸) followed by Tukey’s test between conditions; ** indicates statistical significance of P<0.01. The P-values of the Tukey’s statistical comparisons among groups are presented in Supplementary Table 2. C. PACAP transcripts are present in MPO neurons inhibited by optogenetic stimulation of nearby MPO^Nts;hChR2 neurons. Representative results from 8 recorded MPO neurons. The expected size of the PCR product is 103 base pairs. Negative (−) control was amplified from a harvested cell without reverse-transcription, and positive control (+) was amplified using 1 ng of hypothalamic mRNA. PACAP transcripts were detected in 6 out of 8 neurons.

Figure 3.. Optogenetic stimulation of MPO^Nts;hChR2 neurons increases the frequency of IPSCs and activates an inward current in nearby MPO neurons

A. Optogenetic stimulation of a MPO^Nts;hChR2 neuron activates IPSCs in a nearby MPO neuron. Recordings were performed in the presence of CNQX (20 μM), AP-5 (50 μM) and the NtsR1 antagonist SR48692 (100 nM). The sIPSCs were abolished by Gabazine (5 μM) (lower trace). The neuron was held at −50 mV. B. Optogenetic stimulation of the whole field of view containing several MPO^Nts;hChR2 neurons for 20 s activates IPSCs in a nearby MPO neuron (upper trace). Longer optogenetic stimulation (80 s) of the same neurons activated both IPSCs and an inward current (middle traces). The inward current was abolished by the NtsR1 antagonist SR48692 (100 nM) (lower trace). The neuron was held at −50 mV. C,D. Bar charts summarizing the increase in the frequency of IPSCs (C) and the amplitude of the inward current (D) recorded in MPO neurons in response to optogenetic stimulation of several MPO^Nts;hChR2 neurons. C. The IPSCs frequency increased from1.75±0.97 Hz to 17.8±7.47 Hz in response to photostimulation (one-way ANOVA (F(1,18)=42.5, p=4×10⁻⁴). The control value for the firing rate was calculated as the average value during the 5 min period preceding the optogenetic stimulation. D. The average inward current activated by optogenetic stimulation decreased from 9.26±2.39 pA to 2.31±0.83 pA in the presence of the NtsR1 antagonist SR48692 (100 nM) (one-way ANOVA (F(1,18)=75.35, p=7.5×10⁻⁸). Bars represent means ± S.D. Data pooled from n=10 neurons in each condition.

Figure 4.. Optogenetic activation of MPO^Nts;hChR2 neurons induces hyperthermia while optogenetic activation of MPO^{Nts;VGAT−/−;hChR2} neurons induces hypothermia

A. DIC (left) and fluorescence (right) images of an acute slice from MPO^Nts;hChR2 mouse indicating hChR2-eYFP expression in the MPO. B. Optogenetic stimulation of MPO^Nts;hChR2 neurons (●) in vivo for 1 hour (blue light) induced a hyperthermia of 1.22±0.35 °C relative to control (Δ). The response was statistically different to the response to photostimulation of control MPO^Nts;eYFP mice (Δ) (one-way repeated measures ANOVA, F(1,111)=20.9, p=1.2×10⁻⁵, followed by Man-Whitney U tests for each time point, ** P<0.01). C. Optogenetic stimulation of MPO^{Nts;VGAT−/−;hChR2} neurons (●) in vivo for 1 hour (blue light) induced a hypothermia of 1.44±0.29 °C relative to control (Δ). The response was statistically different to the response to photostimulation of control MPO^{Nts;VGAT−/−;eYFP} mice ( Δ) (one-way repeated measures ANOVA, F(1,112)=8.27, p=4.8×10⁻³, followed by Man-Whitney U tests for each time point, ** P<0.01). B,C. The points represent averages±S.D. through the 7h recording period. Experiments were carried out in parallel in groups of 6 mice.

Figure 5.. Optogenetic stimulation of MPO^{Nts-VGAT−/−;hChR2} neurons increases the firing activity of nearby MPO neurons

A. Optogenetic stimulation of MPO^{Nts;VGAT−/−;hChR2} neurons increases the spontaneous firing rate of a nearby MPO neuron (upper trace) from 1.6 Hz to 3.9 Hz. The photostimulation-induced increase in firing activity was blocked by pe-incubation with the NtsR1 antagonist SR48692 (100 nM) (lower trace). B. Bar charts summarizing the effects of optogenetic stimulation of MPO^{Nts;VGAT−/−;hChR2} neurons on the spontaneous firing rates of nearby MPO neurons in control and in the presence of the NtsR1 antagonist SR48692 (100 nM). Bars represent means ± S.D. of the normalized firing rate relative to the control. Data pooled from n=12 neurons in each condition. There was a statistically significant difference between groups as determined by one-way ANOVA (F(2,22)= 42.45, p=2.80×10⁻⁸) followed by Tukey’s test between conditions; ** indicates statistical significance of P<0.01, * indicates P<0.05. The P-values of the Tukey’s statistical comparisons among groups are presented in Supplementary Table 3. C. Optogenetic stimulation of the whole field of view containing several MPO^Nts;hChR2 neurons for 20 s activates IPSCs in a nearby MPO neuron (upper trace). Longer optogenetic stimulation (80 s) of the same neurons activated both IPSCs and an inward current (middle traces). The inward current was abolished by the NTSR1 antagonist SR48692 (100 nM) (lower trace). The neuron was held at −50 mV. D. Bar chart summarizing the amplitude of the inward current recorded in MPO neurons in response to optogenetic stimulation in control and during incubation with the NtsR1 antagonist SR48692 (100 nM). The average inward current activated by optogenetic stimulation decreased from 12.86±2.33 pA to 2.18±1.49 pA in the presence of MPO^{Nts;VGAT−/−;hChR2} neurons the NtsR1 antagonist SR48692 (100 nM) (one-way ANOVA (F(1,22)=193.73, P= 2.49×10⁻⁸). Bars represent means ± S.D. Data pooled from n=12 neurons.

Figure 6.. Altered thermoregulatory profile of Nts^VGAT−/− mice

A. CBT responses to i.p. injection (arrow) of LPS (0.03 mg/kg) in Nts^VGAT+/+ mice (Δ, control) and Nts^VGAT−/− mice (●). LPS induced fever responses with differential profiles (one-way repeated measures ANOVA, F(1,94)=9.31, P=2.948×10⁻³, followed by Man-Whitney U tests for each time point, ** P<0.01, * P<0.05). B. CBT responses during a heat test in an incubator at 37 °C. The CBT increased faster in Nts^VGAT−/− mice (●) than in Nts^VGAT+/+ mice (Δ, control) (repeated measures ANOVA, F(1,16)= 33.49, p= 2.78×10⁻⁵, followed by Man-Whitney U tests for each time point, * P<0.05). C. Circadian CBT profiles in Nts^VGAT−/− mice (●) than in Nts^VGAT+/+ mice (Δ, control). Nts^VGAT−/− mice display a longer active phase relative to controls (repeated measures ANOVA, F(1,359)= 173.35, P=1.10×10⁻⁹, followed by Man-Whitney U tests for each time point, * P<0.05). Data for each mouse represents the average of 10 different 24 hour periods. D. CBT responses during a cold test in an incubator at 4°C. Nts^VGAT−/− mice (●), in contrast with Nts^VGAT+/+ mice (Δ, control), displayed a significant hyperthermia at the beginning as well as following the end of the cold exposure (repeated measures ANOVA, F(1,92)=220.89, p= 4.3×10⁻¹⁴, followed by Man-Whitney U tests for each time point, * P<0.05, ** P<0.01). A-D. The points represent averages±S.D. (n = 6 male mice).

Figure 7.. Chemogenetic activation of neurotensinergic neurons and projections induces hypothermia and potently excites MPO neurons.

A. I.p. injection (arrow) of CNO (20 mM, 3 μl) in Nts^hM3D(Gq) mice (Δ) and in Nts-cre mice (control, ●). CNO induced a hypothermia of 4.8±0.6 °C (repeated measures ANOVA, F(1,242)=21.72, p=2.8×10⁻⁶, followed by followed by Man-Whitney U tests for each time point, ** P<0.01). B. Role of NtsR1 and NtsR2 expressed in the MPO in the CNO-induced activation of Nts^hM3D(Gq) neurons. Nts^hM3D(Gq) mice received a bilateral infusion of aCSF (gray), NtsR1 antagonist SR48692 (300 nM, 100nl, blue), NtsR2 antagonist NTRC 824 (200 nM, 100nl, black) and NtsR1 antagonist (300 nM, 100nl) + NtsR2 antagonist (200 nM, 100nl) (red) 1.5h prior to an i.p. injection of CNO (20 mM, 3 μl). The antagonists significantly reduced the hypothermia (repeated measures ANOVA, F(3,360)=71.33, p=8.82×10⁻¹⁶). A,B. The points represent averages±S.D. Experiments were carried out in parallel in groups of 6 C. Chemogenetic activation of neurotensinergic neurons and neurotensinergic projections in the MPO by bath application of CNO (3 μM) in slices from Nts^hM3D(Gq) mice depolarizes and increases the firing rate of a MPO neuron. The firing rate increased from 0.15 Hz to 1.65 Hz. D. Bar chart summarizing the effect of chemogenetic activation of neurotensinergic neurons and projections in the MPO on the firing rates of MPO neurons. There was a statistically significant difference between groups as determined by one-way ANOVA (F(1,16)=8.99, P=8.52×10⁻³; ** indicates statistical significance of P<0.01). Bars represent means ± S.D. of the normalized firing rate relative to the control. Data pooled from n=10 neurons. E. Chemogenetic activation of neurotensinergic neurons and neurotensinergic projections in the MPO by bath application of CNO (3 μM) in slices from Nts^hM3D(Gq) mice increases the amplitudes and frequencies of both IPSCs and EPSCs and activates an inward current (upper trace). The NtsR1 antagonist SR48692 (100 nM) abolished the inward current activated by CNO (middle trace). The NtsR2 antagonist NTRC 824 (100 nM) did not change the inward current activated by CNO but significantly decreased the amplitude of sEPSCs (lower trace). The neuron was held at −50 mV. F,G,H,I. Bar charts summarizing the increase in sEPSCs frequency (F) and amplitude (G) and IPSCs frequency (H) and amplitude (I). Bars represent means ± S.D. of the normalized frequency relative to the control. Data pooled from n=6 neurons. The changes were statistically significant for sEPSCs frequency (one-way ANOVA (F(3,19)=6.94, P=3.33×10⁻³) and amplitude (one-way ANOVA (F(3,19)=9.20, P=9.03×10⁻⁴) as well as for sIPSCs frequency (one-way ANOVA (F(3,19)=12.61, P=1.71×10⁻⁴) and amplitude (one-way ANOVA (F(3,19)=4.53, P=1.76×10⁻³). The P values for the inter-group comparisons are listed in Supplementary Tables 4–7.

Figure 8.. Schematic representation of neurotensinergic neurons in a thermoregulatory preoptic pathway

Preoptic thermoregulatory PACAP neurons, assumed to be glutamatergic, project to inhibitory interneurons in other brain regions that project to neurons controlling thermogenesis and vasoconstriction. PACAP neurons’ inhibition results in increased thermogenesis and decreased vasoconstriction resulting in increased CBT. Conversely, excitation of the PACAP thermoregulatory neurons results in hypothermia. Preoptic Nts neurons are GABAergic and project to preoptic thermoregulatory PACAP neurons and modulate their activity. Preoptic astrocytes express NtsR2 receptors and their activation modulates the release of glutamate from nearby synaptic terminals.

Figure 9.. Transduction of MPO^Nts neurons with ChR2-eYFP by injecting AAV-EF1a-double floxed-hChR2(H134R)-eYFP in Nts-cre mice.

A. Brightfield image of a MPO slice. The white rectangle represents the region imaged in B. The scale bar represents 100 μm. B. Representative images of Nts transcripts visualized using RNAscope (red, left panel), eYFP (green, middle) and their superimposed images (right) in a coronal slice from a MPO^Nts;hChR2 mouse. eYFP was visible in 8 out of 10 Nts positive cells. The scale bar represents 10 μm.