Persistent increase in hypothalamic arginine vasopressin gene expression during protracted withdrawal from chronic escalating-dose cocaine in rodents

Abstract

Arginine vasopressin (AVP) from the paraventricular nucleus (PVN) of hypothalamus has important roles in regulation of the hypothalamic-pituitary-adrenal (HPA) axis and stress-related behaviors during chronic stress. It is unknown, however, whether AVP in the PVN is involved in the modulation of HPA activity after chronic cocaine exposure. Here, we examined the gene expression alterations of AVP in the hypothalamus, and V1b receptor and pro-opiomelanocortin (POMC) in the anterior pituitary, as well as HPA hormonal changes, in Fischer rats after chronic cocaine and withdrawal, using two different chronic (14-day) 'binge' pattern administration regimens: steady-dose cocaine (SDC, 45 mg/kg/day) and escalating-dose cocaine (EDC, 45 up to 90 mg/kg/day). There was a significant (7-fold) plasma adrenocorticotropic hormone (ACTH) elevation after chronic EDC (but not SDC), coupled with increased V1b and POMC mRNA levels in the anterior pituitary. From acute (1-day) to protracted (14-day) withdrawal from chronic EDC (but not from SDC), we found persistent elevations of both plasma ACTH and corticosterone levels and AVP mRNA levels in the PVN. Selective V1b antagonist SSR149415 (5 mg/kg) attenuated acute withdrawal-induced HPA activation after EDC. To study potential roles of endogenous opioids in modulating the AVP gene, we administered naloxone (1 mg/kg); we found that opioid receptor antagonism increased AVP mRNA levels in cocaine-naive rats, but not in cocaine-withdrawn rats, suggesting less tonic opioid inhibition of PVN AVP neurons after chronic EDC. To assess the effects of cocaine withdrawal on sub-populations of PVN AVP neurons, we utilized AVP-enhanced green fluorescent protein (EGFP) promoter transgenic mice and found that acute withdrawal following chronic EDC increased the number of AVP-EGFP neurons in the parvocellular PVN (pPVN). These results suggest that during protracted withdrawal, enhanced pPVN AVP gene expression is associated with persistent elevations of basal HPA activity; a hyposensitivity of PVN AVP gene expression to naloxone is indicative of reduced opioidergic tone. Our studies indicate that the AVP and its V1b receptor system may be a potential therapeutic target for treating anxiety and depressive symptoms associated with cocaine addiction.

Figure 1

Timelines for cocaine administration regimens with antagonist applications.

Figure 2

Effects of chronic (14-day) escalating-dose (45 up to 90 mg/kg/day) ‘binge' cocaine (EDC) or chronic (14-day) steady-dose (45 mg/kg/day) ‘binge' cocaine (SDC), acute (1-day) withdrawal and chronic (10- or 14-day) withdrawal with opioid receptor antagonist naloxone (1 mg/kg) on plasma ACTH levels (a, b) and corticosterone (CORT) levels (c, d). Data shown in graphs are treatment group mean+SEM. Significant differences are indicated: ^*p<0.05, ^**p<0.01 or ^***p<0.005 vs saline control; +p<0.05, two-way ANOVA with a significant main effect for 1-day or 14-day cocaine withdrawal (n=6–7).

Figure 3

Effects of chronic (14-day) escalating-dose (45 up to 90 mg/kg/day) ‘binge' cocaine (EDC) followed by acute (1-day) withdrawal with V1b receptor antagonist SSR149415 (5 mg/kg) on plasma ACTH levels (a) and corticosterone (CORT) levels (b). Data shown in graphs are treatment group mean+SEM. Significant differences are indicated: ^*p<0.05 or ^**p<0.01 vs saline control; +p⩽0.05 vs 1-day withdrawal (n=6–8).

Figure 4

Effects of chronic (14-day) escalating-dose (45 up to 90 mg/kg/day) ‘binge' cocaine (EDC) or chronic (14-day) steady-dose (45 mg/kg/day) ‘binge' cocaine (SDC), acute (1-day) withdrawal and chronic (10- or 14-day) withdrawal with opioid receptor antagonist naloxone (1 mg/kg) on mRNA levels of AVP in the PVN (a, b) and V1b in the anterior pituitary (c, d). ^*p<0.05 or ^***p<0.001 vs saline control; ++p<0.01, two-way ANOVA with a significant main effect for 1-day or 14-day withdrawal (n=6–7).

Figure 5

Effects of chronic (14-day) escalating-dose (45 up to 90 mg/kg/day) ‘binge' cocaine (EDC) or chronic (14-day) steady-dose (45 mg/kg/day) ‘binge' cocaine (SDC), acute (1-day) withdrawal and chronic (10- or 14-day) withdrawal with opioid receptor antagonist naloxone (1 mg/kg) on POMC mRNA levels in the anterior pituitary (a, b) and neuro-intermediate lobe/posterior lobe of the pituitary (c, d). ^*p<0.05 or ^**p<0.01 vs saline control; #p<0.05 vs EDC+1-day withdrawal (n=6–7).

Figure 6

Colocalization of AVP-immunoreactive (ir) and EGFP in AVP-EGFP-expressing neurons of paraventricular nucleus (PVN, Bregma −0.82 mm) (a) and medial preoptic area (MPOA, Bregma −0.22 mm) (b) of AVP-EGFP(+) promoter transgenic mice: AVP-EGFP neurons expressing EGFP (green), AVP-ir neurons labeled with AVP antibody (red), and AVP-EGFP neurons co-expressing AVP-ir and EGFP (yellow) in the PVN and MPOA. The scale bar represents 50 μm. PaDC, paraventricular hypothalamic nucleus, dorsal cap; PaLM, paraventricular hypothalamic nucleus, lateral magnocellular part; PaMM, paraventricular hypothalamic nucleus, medial magnocellular part; PaMP, paraventricular hypothalamic nucleus, medial parvocellular part.

Figure 7

Effects of acute (1-day) withdrawal from chronic (14-day) escalating-dose (45 up to 90 mg/kg/day) ‘binge' cocaine on the number of AVP-EGFP-expressing neurons in the paraventricular nucleus (PVN) of AVP-EGFP(+) promoter transgenic mice. Sections of the PVN from saline control (upper) and acute cocaine withdrawal (lower) treated AVP-EGFP(+) mice. The scale bar represents 50 μm. AVP-EGFP expression in the PVN was quantified by measuring the number of EGFP-expressing cells (Table 2).

Figure 8

Effects of acute (1-day) withdrawal from chronic (14-day) escalating-dose (45 up to 90 mg/kg/day) ‘binge' cocaine on plasma corticosterone (CORT) levels in both AVP-EGFP(+) (n=6) and AVP-EGFP(−) (n=6–7) promoter transgenic mice. ^*p<0.05 vs saline control.

Figure 9

Schematic representation of the alterations of HPA axis after chronic escalating-dose cocaine exposure in rodents. After acute cocaine, previous work has demonstrated that CRF (but not AVP) contributes to acute stimulatory effects of cocaine on HPA activity (left side). After chronic cocaine, however, there are increases in AVP promoter activity and gene expression in the parvocellular division of the paraventricular nucleus (pPVN) following acute withdrawal (right side). This is also paralleled with the increases in V1b gene expression in the anterior pituitary, and the elevations of plasma ACTH and corticosterone levels. The increase in pPVN AVP gene expression persists into protracted (14-day) cocaine withdrawal.