Periaqueductal gray/dorsal raphe dopamine neurons contribute to sex differences in pain-related behaviors

Abstract

Sex differences in pain severity, response, and pathological susceptibility are widely reported, but the neural mechanisms that contribute to these outcomes remain poorly understood. Here we show that dopamine (DA) neurons in the ventrolateral periaqueductal gray/dorsal raphe (vlPAG/DR) differentially regulate pain-related behaviors in male and female mice through projections to the bed nucleus of the stria terminalis (BNST). We find that activation of vlPAG/DR^DA+ neurons or vlPAG/DR^DA+ terminals in the BNST reduces nociceptive sensitivity during naive and inflammatory pain states in male mice, whereas activation of this pathway in female mice leads to increased locomotion in the presence of salient stimuli. We additionally use slice physiology and genetic editing approaches to demonstrate that vlPAG/DR^DA+ projections to the BNST drive sex-specific responses to pain through DA signaling, providing evidence of a novel ascending circuit for pain relief in males and contextual locomotor response in females.

Keywords: SABV; bed nucleus of the stria terminalis; dopamine; dorsal raphe; pain; periaqueductal gray.

Figure 1.. Activation of vlPAG/DR^DA+ Produces Sex-Specific Reductions in Pain Sensitivity

(A) Chemogenetic approach for vlPAG/DR^DA+ activation. (Top) Diagram of virus infusion. (Bottom) Representative image of hM3Dq (orange), TH (green), and colocalization (yellow) in vlPAG/DR. Scale bar, 100 μm. (B) Timeline with schematic of pain sensitivity testing and CFA treatment. (C-D) Thermal nociceptive sensitivity of (C) male (n = 7) and (D) female (n = 7) TH-Cre mice following saline or CNO injection. (E-F) Mechanical nociceptive sensitivity of (E) male (n = 7) and (F) female (n = 7) TH-Cre mice following saline or CNO injection. (G-H) Post-CFA thermal nociceptive sensitivity in (G) male (n = 7) and (H) female (n = 7) TH-Cre mice following saline or CNO injection. (I-J) Post-CFA mechanical nociceptive sensitivity in (I) male (n = 7) and (J) female (n = 7) TH-Cre mice following saline or CNO injection. Data are shown as mean ±SEM. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Figure 2.. vlPAG/DR^DA+-BNST Drives Anti-Nociceptive Behaviors in Male, but not Female, Mice

(A) Optogenetic approach for vlPAG/DR^DA+ terminal activation in the BNST. (Left) Diagram of virus infusion and optical fiber implantation. (Right) AAV-DIO-ChR2 (green) expression in vlPAG/DR and BNST. Scale bar, 200 μm for vlPAG/DR; 100 μm for BNST. (B) Schematic of photostimulation during thermal and mechanical nociceptive sensitivity testing. Epochs of laser exposure (i.e. trials [T1–8] for Hargreaves, days for Von Frey) are indicated for each assay. (C-D) Thermal nociceptive sensitivity averaged by laser status in (C) male (n = 6–7) and (D) female (n = 8–10) TH-Cre mice. (E-F) Mechanical nociceptive sensitivity averaged by laser status in (E) male (n = 7–8) and (F) female (n = 8–9) TH-Cre mice. (G-H) Post-CFA thermal nociceptive sensitivity averaged by laser status in (G) male (n = 6–7) and (H) female (n = 4–6) TH-Cre mice. (I-J) Post-CFA mechanical nociceptive sensitivity averaged by laser status in (I) male (n = 6–7) and (J) female (n = 4–6) TH-Cre mice. Data are shown as mean ±SEM. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Figure 3.. vlPAG/DR^DA+-BNST Drives Pain-Related Locomotor Behaviors in Female, but not Male, Mice

(A) Schematic of photostimulation during visceral nociceptive sensitivity testing. (B-C) Cleared nesting zones with ddH2O/Acetic Acid treatment following optogenetic activation of vlPAG/DR^DA+-BNST in (B) male (n = 5) and (C) female (n = 5–6) TH-Cre mice. (D-E) Writhing by Light ON and OFF sessions in (D) male (n = 5) and (E) female (n = 5–6) subjects. (F-G) Writhing averaged by laser status in (F) male (n = 5) and (G) female (n = 5–6) subjects. (H-I) Distance traveled by Light ON and OFF sessions in (H) male (n = 5) and (I) female (n = 5–6) subjects. (J-K) Distance traveled by laser status in (J) male (n = 5) and (K) female (n = 5–6) subjects. Data are shown as mean ±SEM. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Figure 4.. vlPAG/DR^DA+-BNST Increases Context-Dependent Locomotor Behaviors in Female, but not Male, Mice

(A-C) Real-time place preference/aversion with optogenetic activation of vlPAG/DR^DA+-BNST. (A) Schematic of RTPP/RTPA with designated sides for 20 Hz light stimulation (purple) and no stimulation (grey). Comparisons by sex are shown for (B) duration in stimulus zone (n = 5–6) and (C) distance traveled (n = 5–6). (D-H) Sociability test with a same sex conspecific mouse and novel object. (D) Schematic of sociability test. (E-F) Ratio of mouse and object exploration by laser status and (G-H) distance traveled in (E, G) male (n = 7–8) and (F, H) female (n = 5–6) TH-Cre mice. (I-M) Sociability test with an opposite sex conspecific mouse and novel object. (I) Schematic of sociability test. (J-K) Ratio of mouse and object exploration by laser status and (L-M) distance traveled in (J, L) male (n = 7–8) and (K, M) female (n = 8) TH-Cre mice. (N-R) Sociability test with a male conspecific mouse and novel object following social isolation. (N) Schematic of post-isolation sociability test. (O-P) Ratio of mouse and object exploration by laser status and (Q-R) distance traveled for (O, Q) male (n = 4–5) and (P, R) female (n = 5–6) TH-Cre mice. Data are shown as mean ±SEM. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Figure 5.. Sex Differences in vlPAG/DR^DA+-BNST Transmission and Connectivity

(A) Experimental schematic illustrating whole cell patch clamp in ACSF. Recordings were performed in the BNST following optogenetic activation of vlPAG/DR^DA+ terminals. (B) Representative o(E/I)PSC traces from BNST neurons following activation of vlPAG/DR^DA+ terminals with a single 1 ms pulse of 473 nm light (blue). Onset latency (green) and amplitude (pink) are indicated to highlight the distinct properties of excitatory and inhibitory transmission between vlPAG/DR^DA+ and BNST. (C) E/I ratio comparison in male (n = 16 cells) and female (n = 5 cells) TH-Cre mice. (D) Onset latency comparison for oEPSCs in male (n = 20 cells) and female (n = 14 cells) subjects. (E) Amplitude comparison for oEPSCs in male (n = 20 cells) and female (n = 14 cells) subjects. (F) Paired-pulse ratio of oEPSCs in male (n = 19–20 cells at each ISI) and female (n = 12–14 cells at each ISI) subjects. (G) Percentage of responsive and non-responsive cells for oEPSCs in male (n = 52 cells) and female (n = 53 cells) subjects. (H) Onset latency comparison for oIPSCs in male (n = 18 cells) and female (n = 5 cells) subjects. (I) Amplitude comparison for oIPSCs in male (n = 18 cells) and female (n = 5 cells) subjects. (J) Paired-pulse ratio of oIPSCs in male (n = 12–15 cells at each ISI) and female (n = 3–4 cells at each ISI) subjects. (K) Percentage of responsive and non-responsive cells for oIPSCs in male (n = 52 cells) and female (n = 53 cells) subjects. Data are shown as mean ±SEM. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Figure 6.. Sex Differences in vlPAG/DR^DA+-BNST Dopaminergic Transmission

(A) Experimental schematic illustrating fast-scan cyclic voltammetry in ACSF. Recordings were performed in the BNST following optogenetic activation of vlPAG/DR^DA+ terminals. (B-C) Peak DA current following photostimulation of vlPAG/DR^DA+ terminals in the BNST in male (n = 6–7 slices in ACSF; n = 6 slices in ACSF + sulpiride) and female (n = 8 slices in ACSF; n = 6 slices in ACSF + sulpiride) TH-Cre mice. Recordings with (B) varying light pulses were performed in ACSF, then (C) repeated with varying frequencies in ACSF + 2 μM sulpiride. Drug effects were quantified using the percentage change in peak DA current following D2R antagonism. (D) Experimental schematic illustrating whole cell patch clamp in ACSF + 3 mM kynurenic acid + 25 μM picrotoxin. Recordings were performed in the BNST following optogenetic activation of vlPAG/DR^DA+ terminals. (E) Representative traces of BNST neurons responding to optically-evoked DA transmission from vlPAG/DR^DA+ terminals. (Left) 20 Hz (5 ms width, 20 pulses) stimulation in ACSF + kynurenic acid (KA) / picrotoxin (P) produces depolarization, hyperpolarization, or a mix of the two responses. (Middle-Right) These responses are blocked by the addition of SCH-23390 or sulpiride to ACSF + KA/P. Scale bars, x = time (4 sec), y = membrane potential (2 mV). (F-I) Change in membrane potential of BNST neurons following optically-evoked DA transmission from vlPAG/DR. Comparison of (F) depolarization and (G) hyperpolarization size between male (depolarization: n = 26 cells; hyperpolarization: n = 13 cells) and female (depolarization: 23 cells; hyperpolarization: 14 cells) TH-Cre mice. DA receptor antagonism of (H) depolarization (n = 14 cells) and (I) hyperpolarization (n = 6 cells) is shown with individual cells from male (blue) and female (pink) subjects. (J) Percentage of BNST neurons showing depolarization, hyperpolarization, and no response following light-evoked DA transmission from vlPAG/DR^DA+ terminals in male (n = 53 cells) and female (n = 56 cells) subjects. Data are shown as mean ±SEM. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Figure 7.. vlPAG/DR^DA+ Anti-Nociception and Locomotion is Dopamine-Dependent

(A) Chemogenetic approach for vlPAG/DR^DA+ activation with local genetic deletion of TH. (Top-Left) Diagram of virus infusion. (Top-Right) Detailed schematic of TH CRISPR provided by Zweifel lab. (Bottom-Left) Validation of TH deletion in vlPAG/DR for male (n = 7) and female (n = 7) TH-Cre mice. (Bottom-Right) hM3Dq expression (orange), TH immunoreactivity (green), and colocalization (yellow) in vlPAG/DR. Scale bar, 100 μm. (B) Schematic illustrating the measurement of thermal and mechanical nociceptive sensitivity and locomotion following treatment with saline or CNO. (C-D) Thermal nociceptive sensitivity of (C) male (n = 7) and (D) female (n = 7) TH-Cre mice following saline or CNO injection. (E-F) Mechanical nociceptive sensitivity of (E) male (n = 7) and (F) female (n = 7) TH-Cre mice following CNO or saline injection. (G-H) Locomotor activity of (G) male (n = 7) and (H) female (n = 7) TH-Cre mice following a saline injection. (I-J) Locomotor activity of (I) male (n = 7) and (J) female (n = 7) TH-Cre mice following a CNO injection. (K) Pre-Saline Habituation (I). Averaged locomotor activity prior to saline treatment in male (n = 7) and female (n = 7) TH-Cre mice. (L) Saline (I). Averaged locomotor activity after saline treatment in male (n = 7) and female (n = 7) TH-Cre mice. (M) Pre-Saline Habituation (II). Averaged locomotor activity prior to CNO treatment in male (n = 7) and female (n = 7) TH-Cre mice. (N) CNO (II). Averaged locomotor activity after CNO treatment in male (n = 7) and female (n = 7) TH-Cre mice. Data are shown as mean ±SEM. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.

Figure 8.. Morphine Anti-Nociception and Locomotion is Not Dependent on vlPAG/DR Dopamine

(A) Pharmacological approach for opiate-induced vlPAG/DR^DA+ activation with local genetic deletion of TH. Schematic illustrating measurement of thermal and mechanical nociceptive sensitivity and locomotion following systemic treatment with saline or morphine. (B-C) Thermal nociceptive sensitivity of (B) male (n = 7) and (C) female (n = 7) TH-Cre mice following saline or morphine injection. (D-E) Mechanical nociceptive sensitivity of (D) male (n = 7) and (E) female (n = 7) TH-Cre mice following saline or morphine injection. (F-G) Locomotor activity of (F) male (n = 7) and (G) female (n = 7) TH-Cre mice following a saline injection. (H-I) Locomotor activity of (H) male (n = 7) and (I) female (n = 7) TH-Cre mice following a morphine injection. (J) Pre-Saline Habituation (III). Averaged locomotor activity prior to saline treatment in male (n = 7) and female (n = 7) TH-Cre mice. (K) Saline (III). Averaged locomotor activity after saline treatment in male (n = 7) and female (n = 7) TH-Cre mice. female (n = 7) TH-Cre mice. (L) Pre-Morphine Habituation (IV). Averaged locomotor activity prior to morphine treatment in male (n = 7) and female (n = 7) TH-Cre mice. (M) Morphine (IV). Averaged locomotor activity after morphine treatment in male (n = 7) and female (n = 7) TH-Cre mice. Data are shown as mean ±SEM. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001.