Glutamatergic Neurokinin 3 Receptor Neurons in the Median Preoptic Nucleus Modulate Heat-Defense Pathways in Female Mice

Abstract

We have proposed that arcuate neurons coexpressing kisspeptin, neurokinin B, and dynorphin (KNDy neurons) contribute to hot flushes via projections to neurokinin 3 receptor (NK3R)-expressing neurons in the median preoptic nucleus (MnPO). To characterize the thermoregulatory role of MnPO NK3R neurons in female mice, we ablated these neurons using injections of saporin toxin conjugated to a selective NK3R agonist. Loss of MnPO NK3R neurons increased the core temperature (TCORE) during the light phase, with the frequency distributions indicating a regulated shift in the balance point. The increase in TCORE in the ablated mice occurred despite changes in the ambient temperature and regardless of estrogen status. We next determined whether an acute increase in ambient temperature or higher TCORE would induce Fos in preoptic enhanced green fluorescent protein (EGFP)-immunoreactive neurons in Tacr3-EGFP mice. Fos activation was increased in the MnPO but no induction of Fos was found in NK3R (EGFP-immunoreactive) neurons. Thus, MnPO NK3R neurons are not activated by warm thermosensors in the skin or viscera and are not warm-sensitive neurons. Finally, RNAscope was used to determine whether Tacr3 (NK3R) mRNA was coexpressed with vesicular glutamate transporter 2 or vesicular γ-aminobutyric acid (GABA) transporter mRNA, markers of glutamatergic and GABAergic neurotransmission, respectively. In the MnPO, 94% of NK3R neurons were glutamatergic, but in the adjacent medial preoptic area, 97% of NK3R neurons were GABAergic. Thus, NK3R neurons in the MnPO are glutamatergic and play a role in reducing TCORE but are not activated by warm thermal stimuli (internal or external). These findings suggest that KNDy neurons modulate thermosensory pathways for heat defense indirectly via a subpopulation of glutamatergic MnPO neurons that express NK3R.

Figure 1.

Focal injections of NK₃-SAP ablated NK₃R neurons in the MnPO and adjacent MPA in Tacr3-EGFP mice. Representative (A, B) computer-assisted maps and (C, D) photomicrographs of EGFP-ir neurons in (Left) blank-SAP and (Right) NK₃-SAP–injected mice. (A) Scale bar, 250 µm for (A) and (B). (C) Scale bar, 100 µm for (C) and (D). 3V, third ventricle; ac, anterior commissure; HDB, horizontal limb of the diagonal band; oc, optic chiasm.

Figure 2.

Ablation of NK₃R neurons in the MnPO increased the average T_CORE during the light phase in both (Left) OVX and (Right) OVX plus E₂ treated mice. (A, B) Average T_CORE lines were generated using a moving average of five data points. (C, D) T_CORE data were binned into 3-hour blocks (beginning at 7:00 am) for statistical comparisons. (A–D) Black bars on the x-axes denote the dark phase. (E–H) Frequency distributions of T_CORE during 6-hour intervals of the (E, F) light phase (10:00 am to 4:00 pm) and (G, H) dark phase (10:00 pm to 4:00 am). n = 8 to 9 mice per group. *P < 0.01, blank-SAP vs NK₃-SAP; ⁺P < 0.01, OVX vs OVX + E₂, within blank-SAP; ^#P < 0.01, OVX vs OVX + E₂, within NK₃-SAP.

Figure 3.

Ablation of NK₃R neurons in the MnPO did not alter circadian rhythms of (A, B) T_SKIN or (C, D) activity in (Left) OVX or (Right) OVX + E₂ mice. The data were binned into 3-hour blocks (beginning at 7:00 am) for statistical comparisons. Black bars on the x-axes denote the dark phase. n = 7 to 9 mice per group. ⁺P < 0.01, OVX vs OVX + E₂, within blank-SAP; ^#P < 0.05, OVX vs OVX + E₂, within NK₃-SAP.

Figure 4.

Ablation of preoptic NK₃R neurons resulted in a higher T_CORE at multiple T_AMBIENT. (A, B) Average T_CORE in (Left) OVX or (Right) OVX + E₂ mice at various T_AMBIENT. n = 7 to 11 mice per group. *P = 0.01, blank-SAP vs NK₃-SAP; ⁺P < 0.05, OVX vs OVX + E₂ (applies to both NK₃-SAP and blank-SAP mice); ^#P < 0.01, elevated at T_AMBIENT of 35°C compared with all other T_AMBIENT.

Figure 5.

Graphs showing the (A, B, D) average T_CORE and (C, E) T_AMBIENT in experiments (A–C) 2 and (D, E) 3. At time 0, the thermostat setting of the environmental chamber remained at 25°C or was increased to 35°C (experiment 2) or 36°C (experiment 3). Experiment 2, n = 8 to 9 mice per group; experiment 3, n = 4 mice per group. *P < 0.01, T_CORE in mice exposed to the higher T_AMBIENT compared with the low T_AMBIENT of 25°C.

Figure 6.

An acute increase in T_AMBIENT activated Fos in the MnPO but not in preoptic NK₃R neurons. (A, B) Maps of NK₃R (EGFP-ir neurons, green squares), Fos-ir neurons (black circles), and dual labeled neurons (red stars) in a representative OVX mouse exposed to T_AMBIENT of (A) 25°C and (B) 35°C. (C, D) Photomicrographs showing the increase in Fos-ir neurons in the MnPO (black; nuclear stain) at a T_AMBIENT of 35°C. EGFP-ir neurons not expressing Fos can also be seen (brown; cytoplasmic stain). (A) Scale bar, 250 µm for (A) and (B). (C) Scale bar, 100 µm for (C) and (D). 3V, third ventricle; ac, anterior commissure.

Figure 7.

An increase in the T_AMBIENT and T_CORE increased Fos in the MnPO and MPA but not in preoptic NK₃R neurons. (A, B) Maps of NK₃R (EGFP-ir neurons, green squares), Fos-ir neurons (black circles), and dual-labeled neurons (red stars) in mice exposed to T_AMBIENT of (A) 25°C and (B) 36°C. Photomicrographs of the MnPO from mice at T_AMBIENT of (C) 25°C and (D) 36°C showing the increase in Fos-ir neurons (black, nuclear stain) in mice with the elevated T_CORE. EGFP-ir neurons not expressing Fos can also be seen (brown; cytoplasmic stain). (E–G) Photomicrographs of the MnPO from mice exposed to 36°C showing no Fos (black nuclear staining) expression in EGFP (brown cytoplasmic staining) neurons. (A) Scale bar, 250 µm for (A) and (B). (C) Scale bar, 100 µm for (C) and (D). (E) Scale bar, 25 µm for (E)–(G). 3V, third ventricle; ac, anterior commissure; HDB, horizontal limb of the diagonal band.

Figure 8.

Dual-label fluorescent in situ hybridization reveals predominantly glutamatergic NK₃R neurons in the MnPO and GABAergic NK₃R neurons in the MPA. (A, B) Maps of single-labeled NK₃R neurons (green squares), dual-labeled NK₃R/VGLUT2 neurons (red circles), and dual-labeled NK₃R/VGAT neurons (blue circles). (C, D) Graphs showing the percentage of neurons expressing NK₃R mRNA that were single-labeled (green bars), dual-labeled with VGLUT2 mRNA (red bars), or dual-labeled with VGAT mRNA (blue bars). Photomicrographs of dual-label fluorescent in situ hybridization in the (Top) MnPO and (Bottom) MPA using probes complementary to (E, F, I, J) NK₃R and VGLUT2 mRNA or (G, H, K, L) NK₃R and VGAT mRNA. The white arrows in (E) and (F) show a cell that is dual-labeled for NK₃R and VGLUT2 mRNA in the MnPO. The white arrows in (K) and (L) show a cell that is dual-labeled for NK₃R and VGAT mRNA in the MPA. Single-labeled NK₃R neurons are marked with white arrowheads, single-labeled VGLUT2 or VGAT neurons are marked with yellow arrowheads. (A) Scale bar, 250 µm for (A) and (B). (I) Scale bar, 25 µm for (E)–(L).

Figure 9.

Schematic diagram of the relationship between KNDy neurons (blue) and NK₃R-expressing neurons in the MnPO (green) and the central thermosensory heat defense pathway (red) in the mouse. We found that ablation of NK₃R neurons in the MnPO increases the T_CORE without causing thermoregulatory failure. In addition, NK₃R neurons in the MnPO are glutamatergic but do not express Fos when mice are exposed to an acute increase in T_AMBIENT or higher T_CORE. We hypothesize that KNDy neurons activate the central thermosensory pathway for heat defense indirectly via a subpopulation of glutamatergic NK₃R-expressing neurons in the MnPO.