Inhibitory transmission in the bed nucleus of the stria terminalis in male and female mice following morphine withdrawal

Abstract

The United States is experiencing an opioid crisis imposing enormous fiscal and societal costs and driving the staggering overdose death rate. While prescription opioid analgesics are essential for treating acute pain, cessation of use in individuals with a physical dependence induces an aversive withdrawal syndrome that promotes continued drug use to alleviate/avoid these symptoms. Additionally, repeated bouts of withdrawal often lead to an increased propensity for relapse. Understanding the neurobiology underlying withdrawal is essential for providing novel treatment options to alleviate physiological and affective components accompanying the cessation of opiate use. Here, we administered morphine and precipitated withdrawal with naloxone to investigate behavioral and cellular responses in C57BL/6J male and female mice. Following 3 days of administration, both male and female mice demonstrated sensitized withdrawal symptoms. Since the bed nucleus of the stria terminalis (BNST) plays a role in mediating withdrawal-associated behaviors, we examined plastic changes in inhibitory synaptic transmission within this structure 24 hours following the final precipitated withdrawal. In male mice, morphine withdrawal increased spontaneous GABAergic signaling compared with controls. In contrast, morphine withdrawal decreased spontaneous GABAergic signaling in female mice. Intriguingly, these opposing GABAergic effects were contingent upon activity-dependent dynamics within the ex vivo slice. Our findings suggest that male and female mice exhibit some divergent cellular responses in the BNST following morphine withdrawal, and alterations in BNST inhibitory signaling may contribute to the expression of behaviors following opioid withdrawal.

Keywords: GABA; bed nucleus of the stria terminalis; morphine; opioids; sex differences; withdrawal.

Figure 1:

Morphine withdrawal in male and female mice A) Naloxone-precipitated morphine withdrawal paradigm. B) Average withdrawal scores in male mice (SN-M n=22; MN-M n=18). C) Average withdrawal scores in female mice (SN-F n=15; MN-F n=15). D) The average number of jumps between MN-M and MN-F. E) The average number of fecal boli produced between MN-M and MN-F. F) The average number of paw tremors between MN-M and MN-F. F) The average number of paw tremors between MN-M and MN-F. G) The average number of wet dog shakes between MN-M and MN-F. H) Average weight loss in male mice (% baseline). I) Average weight loss in female mice (% baseline). Each bar represents the mean ± SEM, * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001).

Figure 2:

sIPSCs in naïve mice A) Representative trace of sIPSC in male B) Representative trace of sIPSCs in female C) Cumulative distribution plot comparing naïve male and female inter event interval D) Cumulative distribution plot comparing naïve male and female amplitude E) Bar graph of male vs. female frequency from cell averages F) Bar graph of male vs. female amplitude from cell averages

Figure 3:

mIPSCs in naïve mice A) Representative trace of sIPSC in male B) Representative trace of sIPSCs in female C) Cumulative distribution plot comparing naïve male and female inter event interval D) Cumulative distribution plot comparing naïve male and female amplitude E) Bar graph of male vs. female frequency from cell averages F) Bar graph of male vs. female amplitude from cell averages. **p<0.01, ****p<0.0001

Figure 4:

Morphine withdrawal differentially alters BNST spontaneous GABAergic transmission in male and female mice. A) Representative currents (SN-M gray, MN-M black) B) Representative currents (SN-F gray, MN-F black) C) Cumulative distribution plot comparing sIPSC inter event interval in males (**** p<0.0001, SN-M n=14; MN-M n=22). D) Cumulative distribution plot comparing sIPSC inter event interval in females (**** p<0.0001.SN-F n=17; MN-F n=19). F) Cumulative distribution plot comparing sIPSC amplitude in females from same cells as D. E) Cumulative distribution plot comparing sIPSC amplitude in males from the same cells as C. Bar graph of sIPSC frequency cell averages (## drug by sex interaction p<0.01, * P<0.05) F) Bar graph of sIPSC amplitude cell averages had main effect of sex (** p<0.01).

Figure 5:

Morphine withdrawal similarly altered mIPSCs in male and female mice. A) Representative traces of SN-M and MN-M mIPSCs. B) Representative tracels of SN-F and MN-F mIPSCs. C) Cumulative distribution plot comparing miniature-IPSC inter event interval in male mice (SN-M n=14; MN-M n=22). D) Cumulative distribution plot comparing miniature-IPSC inter event interval in female mice (SN-F n=17; MN-F n=19). E) Cumulative distribution plot comparing miniature-IPSC amplitude in male mice from the same cells as C F) Cumulative distribution plot comparing miniature-IPSC amplitude in female mice from the same cells as D. G) Bar graph of mIPSC frequency in male and female mice H) Bar graph of mIPSC amplitude in male and female mice**** (p<0.0001).

Figure 6:

Morphine withdrawal increases the paired-pulse ratio in male and female mice. A) Representative paired-pulse traces from BNST neurons in male mice (SN-M top; MN-M bottom, dashed line 0 pA, scale bar 100 pA x 25 ms). B) Representative paired-pulse traces from BNST neurons in female mice (SN-F, top; MN-F bottom, scale bar 50 pA x 10 ms). C). Bar graph of paired-pulse ratios (main effect of drug treatment, ** p<0.01; $ $ planned t-tests p<0.01, SN-M n=12; MN-M n=20, SN-F n=22; MN-F n=19). Each bar represents the mean ± SEM.