Figure 4. Photostimulation of Pdyn^PBN→POA, Penk^PBN→POA, and VGLUT2^PBN→POA causes acute hypothermia by evoking thermal heat defenses.

(A) Illustration of viral injections in parabrachial nucleus (PBN) and fiber optic implantation over POA in Pdyn-Cre, Penk-Cre, or VGLUT2-Cre mice. (B) Illustration shows viral and fiber optic delivery in a Pdyn-Cre mouse along with representative expression of ChR2-eYFP (green) in PBN injection site and POA implantation site. (C) Diagram shows core body temperature measurement method and paradigm for photostimulation for 15 min and temperature recording for 65 min trials. (D–F) Body temperature vs. time graphs for 2 (yellow), 5 (orange), 10 (red), and 15 (dark red) Hz photostimulation of (D) Pdyn^PBN→POA, (E) Penk^PBN→POA, (F) VGLUT2^PBN→POA, and controls for each. Photostimulation was delivered from t = 0 to t = 15 min and led to a frequency dependent reduction in body temperature in Pdyn-Cre, Penk-Cre, and VGLUT2-Cre mice. Body temperature of control animals was stable throughout the trials. Data are presented as mean ± SEM. For experimental animals, n = 6 (D and E) and n = 8 (F). For control animals, n = 8 (D) and n = 7 (E and F). (G) Representative quantitative thermal imaging from a representative trial showing a mouse before, during, and after 10 Hz photostimulation of Pdyn^PBN→POA. Arrows show temperatures of eye, BAT, or tail. Eye and BAT temperature decreased as a result of stimulation; tail temperature increased as a result of stimulation. (H–L) Quantitative thermal imaging measurements of (H) eye, (I) tail, (J) eye minus tail, (K) BAT, and (L) BAT minus eye temperature vs. time graphs for 10 Hz photostimulation of Pdyn^PBN→POA. Photostimulation was delivered from t = 0 to t = 15 min and led to decreases in eye and BAT temperatures, an increase in tail temperature. Tail and eye temperatures equilibrated in Cre+ animals. BAT thermogenesis was suppressed with a decline in the difference between eye and BAT temperatures during stimulation. Data are presented as mean ± SEM. See Figure 4—figure supplement 1 for data from Penk-Cre animals.

Figure 4—figure supplement 1. Pdyn^PBN→POA or Penk^PBN→POA photostimulation-induced hypothermia is independent of opioid system.

(A) Illustrations of viral injections and fiber optic implantation in Pdyn-Cre and Penk-Cre mice and timeline of drug administration/photostimulation/temperature recording trials. (B) Body temperature vs. time graphs for 10 Hz photostimulation of Pdyn^PBN→POA and control with IP saline, naltrexone (red), or norBN (blue) administration. Photostimulation was delivered from t = 0 to t = 15 min and led to equivalent levels of hypothermia in Cre+ mice regardless of pharmacologic pretreatment. Body temperature of controls was consistent throughout the trial. Data are presented as mean ± SEM. n = 6–7 animals/group. (C) Body temperature vs. time graphs for 10 Hz photostimulation of Penk^PBN→POA in Penk-Cre and control mice with prior IP naloxone or saline administration. Pretreatment did not alter the responses to photostimulation in Penk-Cre mice. Data are presented as mean ± SEM. n = 5 animals in each group. (D–F) To examine effectiveness of antagonist pretreatment we examined the impact of administration naltrexone and norBNI on U50 (5 mg/kg) mediated suppression of locomotor activity. (D) Saline or antagonists were administered 30 min prior to U50 (or saline) in the combinations listed and locomotor activity tracked for 30 min after U50 injection. (E) The cumulative distance traveled and total distanced traveled show marked suppression of movement by U50 which was ameliorated by pretreatment with naltrexone or norBNI (24 hr plus 30 min prior). (F) Total distance traveled at 30 min after U50 administration significantly (p=0.002) reduced compared to saline. This decrease in locomotion was reduced significantly by pretreatment naltrexone (p=0.014) or norBNI (p=0.025). norBNI followed by U50 was not significantly different from norBNI followed by saline. n = 5 mice. (G–H) Results obtained using quantitative thermal imaging to measure tail, eye, and BAT temperatures in Penk-Cre mice during 10 Hz photostimulation as done for Pdyn-Cre mice in Figure 4. (G) Eye, (H) tail, (I) BAT, and (J) eye minus tail temperature vs. time graphs for 10 Hz photostimulation of Penk^PBN→POA. Photostimulation was delivered from t = 0 to t = 15 min and led to drops in eye and BAT temperatures. Tail temperature rose and the gradient between eye and tail temperatures declined in Cre+ animals but not control mice. Data are presented as mean ± SEM. n = 5 in each group.