Development × environment interactions control tph2 mRNA expression

Abstract

Adverse early life experience is thought to increase an individual's susceptibility to mental health disorders, including anxiety and affective disorders, later in life. Our previous studies have shown that post-weaning social isolation of female rats during a critical period of development sensitizes an anxiety-related serotonergic dorsal raphe nucleus (DR) system in adulthood. Therefore, we investigated how post-weaning social isolation, in combination with a challenge with the anxiogenic drug, N-methyl-beta-carboline-3-carboxamide (FG-7142; a partial inverse agonist at the benzodiazepine allosteric site on the GABAA receptor), affects home cage behavior and serotonergic gene expression in the DR of female rats using in situ hybridization histochemistry. Juvenile female rats were reared in isolation or groups of three for a 3-week period from weaning (postnatal day (PD) 21 to mid-adolescence (PD42)), after which all rats were group-reared for an additional 16 days until adulthood. Among vehicle-treated rats, isolation-reared rats had decreased rodent tryptophan hydroxylase 2 (tph2) mRNA expression in ventral and ventrolateral subdivisions of the DR, a pattern observed previously in a rat model of panic disorder. Isolation-reared rats, but not group-reared rats, responded to FG-7142 with increased duration of vigilance and arousal behaviors. In addition, FG-7142 decreased tph2 expression, measured 4h following treatment, in multiple subregions of the DR of group-reared rats but had no effect in isolation-reared rats. No treatment effects were observed on 5-HT1A receptor or serotonin transporter gene expression. These data suggest that adolescent social isolation alters tph2 expression in specific subregions of the DR and alters the effects of stress-related stimuli on behavior and serotonergic systems.

Figure 1

Diagram illustrating the experimental timeline and sequence of procedures. Abbreviations: HC, home cage; PD, postnatal day; veh, vehicle.

Figure 2

Neuroanatomic atlas of rat tph2 mRNA expression in the midbrain dorsal raphe nucleus (DR) from a female rat in the present study. Displayed (from left to right) are 5 representative coronal sections (12 μm) taken from one rat that were used to measure tph2 mRNA expression in all subdivisions of the DR from rostral (−7.580 mm bregma, designated level +7) to caudal (−8.672 mm bregma, designated level −6). Photomicrographs are autoradiographic images revealing the localization of hybridized ³⁵S-labeled cRNA probe complementary to tph2 mRNA. Dashed lines indicate borders between subdivisions of the DR. Abbreviations: DRC, dorsal raphe nucleus, caudal part; DRD, dorsal raphe nucleus, dorsal part; DRI, dorsal raphe nucleus, interfascicular part; DRV, dorsal raphe nucleus, ventral part; DRVL, dorsal raphe nucleus, ventrolateral part; VLPAG, ventrolateral periaqueductal gray. Scale bar: 1 mm.

Figure 3

Graphs illustrating the duration and frequency of selected home cage behaviors 30 to 40 min following injection of vehicle or FG-7142 (a partial inverse agonist at the benzodiazepine allosteric site on the γ-aminobutyric acid (GABA)_A receptor, 7.5 mg/kg, i.p.). Graphs represent A) the duration of spontaneous non-ambulatory motor activity (SNAMA), B) the duration of locomotion, C) the duration of feeding-related behavior, and D) the frequency of rearing. Data are presented as means (+ S.E.M.). *p < 0.05 versus vehicle-treated controls within the same housing condition; ^#p < 0.05 versus group-reared rats within the same drug condition; Fisher's protected least significant difference (LSD) test. Sample size, n = 9 for all treatment groups. Abbreviations: SNAMA, spontaneous non-ambulatory motor activity.

Figure 4

Graphs illustrating the effects of adolescent social isolation or group-rearing followed by subsequent administration of either FG-7142 (7.5 mg/kg) or vehicle on the expression of tph2 mRNA in the dorsal raphe nucleus (DR). Shown is the mean (+ S.E.M.) tph2 mRNA expression in the whole DR (A) and in each of the 5 subdivisions of the DR studied (B-F). (G) Representative autoradiograms of tph2 mRNA expression at −8.168 mm bregma (designated level 0) in each treatment group. *p<0.05 versus vehicle-treated controls within the same housing condition; ^#p < 0.05 versus group-reared rats within the same drug condition; Fisher's protected least significant difference (LSD) test. Sample size: n = 9, group-reared/vehicle-treated; n = 8, group-reared/FG-7142-treated; n = 9, isolation-reared/vehicle-treated; n = 7, isolation-reared/FG-7142-treated. Abbreviations: DRC, dorsal raphe nucleus, caudal part; DRD, dorsal raphe nucleus, dorsal part; DRI, dorsal raphe nucleus, interfascicular part; DRV, dorsal raphe nucleus, ventral part; DRVL, dorsal raphe nucleus, ventrolateral part; VLPAG, ventrolateral periaqueductal gray. Scale bar: 1 mm.

Figure 5

Graph illustrating the positive correlation between mean tph2 mRNA expression in the combined DRD/DRC and the total social interaction (SI) time. Only data from vehicle-treated rats is shown. Open circles, group-reared/vehicle-treated; filled circles isolation-reared/vehicle-treated.

Figure 6

Graph illustrating a positive correlation between mean htr1a mRNA expression and mean tph2 mRNA expression across all subdivisions of the dorsal raphe nucleus. Data are mean gray value × area for each gene. Open circles, group-reared/vehicle-treated; open triangles, group-reared/FG-7142-treated; filled circles, isolation-reared/vehicle-treated; filled triangles, isolation-reared/FG-7142-treated.