Influence of chronic amphetamine treatment and acute withdrawal on serotonin synthesis and clearance mechanisms in the rat ventral hippocampus

Abstract

Amphetamine withdrawal in both humans and rats is associated with increased anxiety states, which are thought to contribute to drug relapse. Serotonin in the ventral hippocampus mediates affective behaviors, and reduced serotonin levels in this region are observed in rat models of high anxiety, including during withdrawal from chronic amphetamine. This goal of this study was to understand the mechanisms by which reduced ventral hippocampus serotonergic neurotransmission occurs during amphetamine withdrawal. Serotonin synthesis (assessed by accumulation of serotonin precursor as a measure of the capacity of in vivo tryptophan hydroxylase activity), expression of serotonergic transporters, and in vivo serotonergic clearance using in vivo microdialysis were assessed in the ventral hippocampus in adult male Sprague Dawley rats at 24 h withdrawal from chronic amphetamine. Overall, results showed that diminished extracellular serotonin at 24 h withdrawal from chronic amphetamine was not accompanied by a change in capacity for serotonin synthesis (in vivo tryptophan hydroxylase activity), or serotonin transporter expression or function in the ventral hippocampus, but instead was associated with increased expression and function of organic cation transporters (low-affinity, high-capacity serotonin transporters). These findings suggest that 24 h withdrawal from chronic amphetamine reduces the availability of extracellular serotonin in the ventral hippocampus by increasing organic cation transporter-mediated serotonin clearance, which may represent a future pharmacological target for reversing anxiety states during drug withdrawal.

Figure 1

Representative coronal diagrams of microdialysis probe membrane placements in saline (SAL) and amphetamine (AMP) pretreatment groups. Black bars represent the membrane of one or more probes. Figure adapted from Paxinos & Watson (1996, bregma −5.20 mm).

Figure 2

Effects of KCl-induced depolarization on extracellular 5-HT concentrations in the ventral hippocampus of saline (SAL) and amphetamine (AMP) pretreated rats. KCl was added to the perfusate at a concentration of 100 mM during a sample period (20 min) marked by the horizontal black bar. Data are mean ±SEM. ^#Significantly different from pre-infusion levels (P < 0.05). *Significant differences between saline and amphetamine pretreatment groups (P < 0.05).

Figure 3

Tissue concentrations of 5-hydroxytryptophan (5-HTP) in the (A) ventral hippocampus and (B) median raphe nucleus within saline (SAL) or amphetamine (AMP) pretreated rats 30 minutes after rats were treated with either vehicle or the amino acid decarboxylase (AADC) inhibitor NSD-1015 (100 mg/kg, i.p.). Data represent mean +/− SEM. Bars with different superscript letters are significantly different from one another (P < 0.001).

Figure 4

(A) Serotonin transporter (SERT) and (B) organic cation transporter 3 (OCT3) protein levels in ventral hippocampal tissue of saline (SAL) and amphetamine (AMP) pretreated rats. Levels of SERT and OCT3 protein were determined by western immunoblot with antibodies recognizing a SERT band at 75 kDa and an OCT3 band at 53 kDa (Insert: examples from a single animal). Means ± S.E.M. are shown for all groups. * Significant difference between saline and amphetamine pretreatment groups (P < 0.05).

Figure 5

Effect of local paroxetine perfusions on extracellular 5-HT concentrations in the ventral hippocampus in saline (SAL) and amphetamine (AMP) pretreated rats, expressed as percentage of baseline. Horizontal black bar = perfusion of (A) vehicle, (B) 0.1 μM paroxetine (C) 0.5 μM paroxetine, and (D) 3μM paroxetine. Data represent mean ± SEM. ^#sSignificantly different from pre-perfusion levels in saline pretreated rats, ^#aSignificantly different from pre-perfusion levels in amphetamine pretreated rats, ^#Significantly different from pre-perfusion levels in both saline and amphetamine pretreated rats, (P < 0.05).

Figure 6

Effect of local D-22 perfusion on extracellular 5-HT levels in the ventral hippocampus in saline (SAL) and amphetamine (AMP) pretreated rats, expressed as percentage of baseline. Horizontal black bar = perfusion of (A) vehicle, (B) 30 μM D-22 and (C) 100μM D-22. Data represent mean ± SEM. ^#Significantly different from pre-perfusion levels in both saline and amphetamine pretreated rats, (P < 0.05). *Significantly different from amphetamine pretreated rats, (P < 0.05).