Exploring the effects of adolescent social isolation stress on the serotonin system and ethanol-motivated behaviors

Abstract

Rationale: Alcohol is one of the most frequently used drugs of abuse and has a major impact on human health worldwide. People assigned female at birth and those with adverse childhood experiences are stress-vulnerable and more likely to report drinking as a means of "self-medication." Prior studies in our laboratory showed that adolescent social isolation stress (SIS) increases vulnerability to ethanol (EtOH) intake and consumption despite negative consequences in female rats.

Objectives: Here, we explored modulation of the dorsal raphe nucleus (DRN)-serotonin (5-HT) system, a sexually dimorphic neurotransmitter system involved in stress-reward interactions, to determine its contribution to EtOH-motivated behaviors in rats that have undergone SIS.

Results: We employed electrophysiological and functional neuroanatomy strategies to show that both SIS and EtOH exposure induce persistent hypofunction of the DRN 5-HT system, particularly in females. Chemogenetic activation of DRN 5-HT neurons attenuated reward value for both EtOH and sucrose and elevated punished responding for EtOH in a stress-dependent manner.

Conclusions: Our results highlight an inverse relationship between EtOH consumption and the 5-HT system, the sex- and stress-dependent nature of this relationship, and a connection between DRN 5-HT signaling and acute responding to rewards and punishment. These data support the DRN 5-HT system as a potential target to treat aberrant alcohol consumption and drinking despite negative consequences in stress-vulnerable populations.

Keywords: Chemogenetics; DREADDs; Early life stress; Electrophysiology; Ethanol operant conditioning; Punishment-resistant drinking; Rats; Serotonin; Sex differences; Tph2-iCre.

Fig. 1

Excitability of DRN 5-HT neurons in male and female EtOH-naïve rats. a Experimental timeline. b Post hoc immunohistochemical identification of Tph2 recorded neurons, biocytin-filled recorded neuron (green) and Tph2 + neurons (red) is shown. SIS females showed hypoexcitability of DRN 5-HT neurons in adulthood relative to other treatment groups (c), vs. male SIS at currents of 100, 120 and 160pA (d) and vs. female GH controls at currents of 60-160pA (e). Asterisks indicate significant differences by post hoc Sidak tests (*p < 0.05, **p < 0.01, ***p < 0.001). Total n = 28 cells. GH males n = 8 (solid gray circles), SIS males n = 8 (open black circles), GH females n = 6 (solid gray squares), SIS females n = 6 (open black squares)

Fig. 2

Excitability and c-Fos expression of DRN 5-HT neurons in female EtOH-exposed rats. a Experimental timeline. b When split by stress, no significant differences were observed between treatment groups. c When median-split by EtOH intake over the last three self-administration sessions, higher drinking rats showed significant hypoexcitability of DRN 5-HT neurons compared to lower drinking rats. d However, the correlation between individual EtOH intake and excitability of DRN 5-HT neurons did not reach statistical significance. Asterisks indicate significant differences by post hoc Sidak test (*p < 0.05). Total n = 10 cells. a; GH females n = 5 (solid gray squares), SIS females n = 5 (open black squares). b; lower drinking n = 6 (solid black upside-down triangles), higher drinking n = 4 (open gray triangles). e and f Representative photomicrographs of immunofluorescence staining in the DRN following punished SA. Images show staining of Tph2 (red), c-Fos (green), and DAPI (blue). e Merged image at 20x magnification outlining the locations of the different DRN subdivisions. f Higher magnification images of the framed area in panel e. The images show (from top left to bottom right) staining of c-Fos, Tph2, and a merged image, respectively. Arrowheads in the merged image indicate cells that are single-labeled for c-Fos (green) or Tph2 (red), as well as cells with dual expression (white). Scale bars indicate 500 μm and 75 μm for images in panels e and f, respectively. Aq = cerebral aqueduct, DRN = dorsal raphe nucleus, DRD = dorsal DRN, DRV = ventral DRN, DRVL = ventrolateral DRN and mlf = medial longitudinal fasciculus

Fig. 3

The effect of Gq-DREADD activation of DRN 5-HT neurons on reinforcers earned during EtOH and sucrose self-administration in GH female rats. a Experimental timeline. b EtOH dose-effect curve showing the average reinforcers earned at escalating concentrations of EtOH, with peak responding at 30–40%. CNO activation of Gq DREADDs significantly reduced reinforcers earned for 20% EtOH (c), 50% EtOH (d) and 20% sucrose (e). Pound signs indicate significant differences by post hoc Tukey tests (#p < 0.05). Asterisks indicate significant differences by two-tailed paired t-tests (*p < 0.05, ***p < 0.001). Total GH females n = 5 (b) and n = 4 (c-e)

Fig. 4

The effect of Gq-DREADD activation of DRN 5-HT neurons on footshock-punished EtOH SA in female rats. CNO activation of Gq DREADDs produced stress-specific effects on punished EtOH SA. a Experimental timeline. b CNO elevated average punishment resistance scores in SIS but not GH rats. c Punishment resistance scores over three days of punished SA revealed that CNO elevated punishment resistance in SIS rats and reduced punishment resistance in GH rats compared to their respective vehicle controls. Asterisks indicate significant differences by post hoc Sidak tests (*p < 0.05, **p < 0.01). Total rats n = 18, GH vehicle n = 3, GH CNO n = 4, SIS vehicle n = 7, SIS CNO n = 4