Nicotinic receptors promote susceptibility to social stress in female mice linked with neuroadaptations within VTA dopamine neurons

Abstract

There are about twice as many women as men who experience depression during their lifetime. Although life circumstances and especially exposure to stressful situations constitute a major risk factor to develop depression, the underlying mechanisms have yet to be unraveled. We employed the chronic social defeat procedure to elicit depressive-like symptoms in females and ketamine to validate the model. We performed ex-vivo patch clamp recordings to assess cellular adaptations and used pharmacological agents to dissect these deregulations. Chronic social defeat exposure triggers a hyperactivity of VTA putative dopamine (DA) neurons in females susceptible to stress but not resilient ones. This hyperactivity was fully reversed by a single administration of ketamine. In virally-identified brain circuits of both susceptible and resilient females, we found a hypercholinergic tone to the VTA arising from the laterodorsal tegmentum. Application of puffs of nicotine revealed a decreased sensitivity of DA neurons in resilient mice when compared to naive or susceptible ones. The in vivo acute administration of the positive allosteric modulator for α7 nicotinic acetylcholine receptors (nAChRs) not only increased susceptibility to stress by enhancing activity of VTA DA neurons, but also triggered a switch in phenotype from resilient to susceptible. Our data unravel dysregulations of VTA DA neurons activity exclusively in females exhibiting depressive-like symptoms and identify VTA nAChRs as key molecular substrates that exacerbate susceptibility to stress.

Fig. 1. Chronic social defeat promotes depressive-like behaviors in female mice.

a Schematic of the social defeat procedure. b Experimental timeline. c Susceptible mice displayed social avoidance, while resilient females showed similar social interaction ratio than naive mice. d Sucrose preference was decreased in susceptible female mice. e Defeated females exhibited increased anxiety responses in the O-Maze test. (n/group: Naive = 9; Resilient = 8; Susceptible = 8) f Naive and susceptible females were injected with saline or ketamine (i.p, 20 mg/kg) after the SI test on day 11, and the social exploration was re-tested 24 h following drug injection. Ketamine reversed the social avoidance in susceptible females, without changes in the performance of naive mice. (n/group: Naive-Saline = 6; Naive-Ketamine = 4; Susceptible-Saline = 4; Susceptible-Ketamine = 7). All data are expressed as the mean ± SEM.

Fig. 2. Chronic social defeat alters the firing rates and excitatory synaptic transmission of VTA DA neurons in females.

a Schematic experimental timeline. b Percentage of cells with or without spontaneous firing for each condition (N/n: Naive = 13/4; Resilient = 13/3; Susceptible = 13/4). c VTA DA displayed increased excitability in susceptible mice. (N/n: Naive = 13/4; Resilient = 13/3; Susceptible = 13/4). d Representative voltage traces are shown. e Ketamine administration reverses the VTA DA cells hyperexcitability in susceptible mice. f Representative traces of recordings. g Graph plot and frequency distribution histogram depict significant increases in frequency of sEPSC in VTA DA neurons of susceptible mice. (N/n: Saline = 11/2; Ketamine = 13/2). h Graph plot and frequency distribution histogram show increased amplitude of sEPSC of VTA DA neurons in both susceptible and resilient groups. (N/n: Naive = 13/4; Resilient = 13/3; Susceptible = 13/4). i Representative traces of sEPSC. (N/n: Naive = 13/4; Resilient = 13/3; Susceptible = 13/4). Data are expressed as the mean ± SEM except for (g, h) where data are expressed as the median and interquartile range. N/n Number of cells/mice.

Fig. 3. Chronic social defeat alters cholinergic signals in the VTA in females.

a Schematic experimental timeline to assess the chronic social defeat effect on excitability of LDTg cholinergic cells projecting to the VTA. Three weeks before chronic social defeat, ChAT-Cre mice were injected with retro-AAV-CAG-FLEX-tdTomato bilaterally in the VTA. Confocal image shows tdTomato-tagged LDTg^→VTA cholinergic neurons. b No change in membrane potential and membrane resistance of LDTg^→VTA cholinergic neurons following chronic social defeat. c Cholinergic LDTg^→VTA neurons exhibited increased excitability profiles in both susceptible and resilient female mice. Representative voltage traces are shown (N/n: Naive = 14/2; Resilient = 11/2; Susceptible = 17/2).

Fig. 4. Chronic social defeat alters nicotine responses of VTA DA neurons in females.

a Schematic experimental timeline to evaluate the response to nicotine of VTA DA cells in naive and defeated females. b Decreased nicotine-elicited currents from VTA DA neurons were found in resilient females. Representative recordings are shown. c Decreased decay constant of nicotine-elicited currents in resilient mice when compared to naive and susceptible (N/n Naive = 12/5; Resilient = 16/4; Susceptible = 19/9). Representative traces are shown.Representative traces are shown.

Fig. 5. The modulation of α7-nAChRs triggers susceptibility to social stress in females.

a Schematic of cellular actions of PNU-120596. b Bath application of PNU-120596 produces a significant increase in nicotine-elicited currents when compared to the bath application of vehicle alone. (N/n: vehicle = 10/3; PNU = 9/3). c Schematic of the subthreshold defeat procedure. d To evaluate the role of α7-nAchRs in the vulnerability to social stress, females received PNU-120596 (PNU) or vehicle injection paired with subthreshold social defeat and tested 1 day later in the SI test. PNU-injected females displayed significant social avoidance. (n/group: Naive-Vehicle = 8; Naive-PNU = 8; SubSD-Vehicle = 10, SubSD-PNU = 10). e VTA DA neurons displayed increased excitability only in female mice, which were submitted to subthreshold defeat stress paired with PNU-120596 injection. (N/n: Naive-Vehicle = 22/4; Naive-PNU = 21/4; SubSD-Vehicle = 19/4, SubSD-PNU = 25/4). f Experimental design to evaluate whether α7-nAchRs modulation can switch a resilient phenotype to a susceptible one. The graph depicts the social exploration of resilient mice during Test 1 (1 day following chronic social defeat) and Test 2 (1 day after T1 and 15 min following PNU administration). The positive modulation of α7-nAchRs promotes susceptibility to social stress in resilient females (n/group: Resilient-Vehicle = 5; Resilient-PNU = 7). Data are expressed as the mean ± SEM.