Richard J. Thielen

Serotonin 5-HT2 Receptor Stimulation of Dopamine Release in the Posterior but Not Anterior Nucleus Accumbens of the Rat

Abstract: The objective of the present study was to examine the involvement of serotonin 5‐HT2 receptors within the rat nucleus accumbens (Acc) in the regulation of dopamine (DA) release usingin vivo microdialysis. Perfusion with the 5‐HT2 agonist (+)‐1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane (DOI), at concentrations of 25‐250 μM, through microdialysis probes located in the posterior Acc increased extracellular DA levels to a maximum of 200% of baseline. DOI‐induced increases in the extracellular levels of DA were Ca2+ dependent and were inhibited by co‐perfusion with the 5‐HT2 antagonist LY‐53,857. DOI enhancement of the extracellular concentrations of DA was observed when probes were implanted in the Acc core and shell regions posterior to anteroposterior +1.2 mm from bregma, whereas a small reduction in the extracellular levels of DA was observed in the anterior Acc. There were no differences between core and shell subdivisions within either the anterior or the posterior Acc. These results suggest that activation of 5‐HT2 receptors within the posterior, but not anterior, Acc stimulates DA release, indicating rostral‐caudal differences in the interactions of 5‐HT with DA systems in the Acc.

Long-lasting alterations of the mesolimbic dopamine system after periadolescent ethanol drinking by alcohol-preferring rats.

BACKGROUND This study tested the hypothesis that ethanol consumption by alcohol-preferring (P) rats during the periadolescent period causes persistent alterations in the mesolimbic dopamine (DA) system. After ethanol drinking during periadolescence, P rats were examined for alterations in basal locomotor activity, changes in extracellular DA levels and extraction fraction in the nucleus accumbens (NAc) by using no-net-flux (NNF) microdialysis, and changes in the response of the mesolimbic DA system to ethanol. METHODS Male P rat pups were given 24-hr free-choice access to 15% (v/v) ethanol from postnatal day (PD) 30 through PD 60. On PD 70, rats were assessed for locomotor activity. On PD 70 to 80, rats were implanted with bilateral guide cannulas aimed above the NAc. After at least 5 days, microdialysis probes were inserted bilaterally; on the following day, NNF microdialysis experiments were conducted. On the day after the NNF experiment, conventional microdialysis experiments were conducted to measure extracellular levels of DA in response to intraperitoneal injection of saline or ethanol 2.5 g/kg. RESULTS Compared with the ethanol-naive group, ethanol drinking by P rats during periadolescence did not alter basal locomotor activity, nor did it alter the basal extracellular concentration of DA. There was, however, a significant increase in the extraction fraction of DA of ethanol-drinking animals relative to the controls (57.4 +/- 2.7% and 45.8 +/- 2.3%, respectively). Additionally, compared with controls, P rats with exposure to ethanol during the periadolescent period showed a prolonged increase in the extracellular levels of DA after a challenge dose of ethanol. CONCLUSIONS The results of the microdialysis experiments suggest that periadolescent ethanol drinking by P rats increases basal DA neurotransmission (as indicated by higher DA clearance while maintaining the same extracellular DA concentrations) and prolongs the response of DA neurotransmission to ethanol.

Ethanol-stimulated serotonin release in the ventral hippocampus: an absence of rapid tolerance for the alcohol-preferring P rat and insensitivity in the alcohol-nonpreferring NP rat

This study examined the acute effects of intraperitoneal administration of ethanol on the extracellular levels of serotonin (5-HT) in the ventral hippocampus (vHIP) of adult, male alcohol-preferring P and -nonpreferring NP rats. Using in vivo microdialysis coupled with HPLC and electrochemical detection, the effects of acute administration of saline or 1.0, 1.75, or 2.5 g/kg ethanol on the extracellular levels of 5-HT in the vHIP were examined. Saline and 1.0 g/kg ethanol did not alter the extracellular levels of 5-HT. However, the 1.75-g/kg dose resulted in a transient increase in 5-HT levels in the vHIP of P rats only. Administration of 2.5 g/kg ethanol increased 5-HT levels to 180% of baseline in P rats (P<.05), but was without effect on NP rats. The 2.5-g/kg dose also significantly increased the extracellular levels of 5-HT in the vHIP of P rats, which had been pretreated with the same dose of ethanol 18-24 h earlier (P<.05). Comparison of the response of ethanol pretreated P rats with animals that had been pretreated with saline 24 h earlier did not reveal any significant differences in ethanol-stimulated increases in 5-HT levels between the groups. These data suggest that ethanol may activate terminals of the median raphe 5-HT system in P rats because the vHIP receives its 5-HT inputs primarily from the median raphe nucleus (MRN). Rapid tolerance does not develop to this activation of the system in the vHIP of P rats. In addition, the data suggest that the 5-HT system in the vHIP of NP rats may be relatively insensitive to the stimulating effect of acute ethanol of 5-HT release.

Effects of ethanol on the dorsal raphe nucleus and its projections to the caudate putamen

The objective of this study was to examine the effects of intraperitoneal injection of ethanol on the activity of the dorsal raphe nucleus (DRN) serotonin (5-hydroxytryptamine [5-HT]) system and its projections to the rostral caudate putamen (CPu) and determine whether rapid tolerance to the effects of ethanol develops in this system. Adult, male, Wistar rats were used in these experiments. In experiment 1, a microdialysis procedure was used to determine (a) the effects of acute intraperitoneal administration of ethanol (1.75 and 2.5 g/kg) on the extracellular levels of 5-HT in the rostral CPu and (b) whether rapid tolerance develops to these effects. In experiment 2, firing rates of 5-HT neurons were determined in the DRN after intraperitoneal administration of 2.5 g/kg of ethanol. The results of the microdialysis experiments indicated that the 2.5-g/kg dose significantly (P < .005) increased the extracellular levels of 5-HT to 150%-160% of baseline. Compared with findings for rats pretreated with saline 24 h earlier, prior treatment 24 h earlier with 2.5 g/kg of ethanol had no effect on the extracellular levels of 5-HT produced by a challenge dose of 2.5 g/kg of ethanol. Contrary to the effects in the CPu, intraperitoneal administration of 2.5 g/kg of ethanol significantly (P<.005) decreased the firing rates of 5-HT neurons in the DRN to approximately 50% of control. Overall, the results suggest to us that there is a dissociation between the effects of acute administration of ethanol on 5-HT cell body neuronal activity and 5-HT synaptic activity. The higher extracellular levels of 5-HT in the CPu may be due to increased release of 5-HT from a direct or an indirect action of ethanol, a result of inhibiting 5-HT reuptake, or related to both of these mechanisms. In addition, the findings suggest to us that rapid tolerance did not develop to the effects of ethanol on the 5-HT system within the CPu.

Housing conditions alter GABAA receptor of alcohol-preferring and -nonpreferring rats

The effects of housing conditions on some functional properties of the GABAA benzodiazepine (BZD) receptor in the cerebral cortex were examined in the selectively bred alcohol-preferring (P) and -nonpreferring (NP) lines of rats. Compared to rats housed in pairs (P with P and NP with NP), P and NP rats housed individually had 44% (p < 0.005) and 32% (p < 0.01) lower values, respectively, for GABA-stimulated 36Cl- influx into cortical microsacs. The maximal effect (Vmax) of flunitrazepam (FNZ) to enhance GABA-stimulated 36Cl- uptake was 44% higher in individually housed P rats than pair-housed P rats (p < 0.05) and 51% higher than individually housed NP rats (p < 0.05). There was no difference between single and pair-housed NP rats for Vmax values of FNZ enhancement of GABA-stimulated 36Cl- influx. The results show housing conditions can alter some of the functional properties of the GABAA/BZD receptor in the P and NP lines of rats. The differential effect of housing conditions on FNZ enhancement of 36Cl- influx, observed between the lines, may be a result of higher levels of anxiety being produced by brief isolation in the P rat.

Regional densities of benzodiazepine sites in the CNS of alcohol-naive P and NP rats

The regional densities of benzodiazepine (BDZ) recognition sites coupled to GABAA receptors were studied in ethanol-naive alcohol-preferring (P) and -nonpreferring (NP) lines of rats by using quantitative autoradiography to measure the amount of 2 nM [3H]flunitrazepam (FNZ) binding in the absence and presence of 100 microM GABA. Lower values (p < 0.025) for [3H]FNZ binding (in the absence of GABA) were observed in the prefrontal cortex, layer 4 of the parietal cortex, and the nucleus accumbens shell of the P relative to the NP line. GABA significantly (p < 0.025) stimulated [3H]FNZ binding in all 50 central nervous system regions examined in both the P and the NP rats. The largest percent increases (190-220%) were observed in the prefrontal, cingulate, frontal, and parietal cortices; shell and core nucleus accumbens; caudate putamen; dorsal lateral, intermediate lateral, ventral lateral, and medial septal nuclei; and lateral hypothalamus. In several layers of the frontal and parietal cortices, a 25-30% greater net or percent increase (p < 0.025) in GABA-enhanced [3H]FNZ binding was observed in the P rats compared with the NP rats. In contrast, lower net or percent increases (p < 0.025) in GABA-enhanced [3H]FNZ binding were found in the entorhinal cortex, the mediodorsal thalamus, and the dorsal CA3 area and middle dentate gyrus of the posterior hippocampus of the P line relative to the NP line. The present findings suggest that there are innate regional differences between P and NP rats in the densities and/or affinities of BDZ recognition sites and in the coupling between the GABAA and BDZ binding sites.

Effects of repeated daily treatments with a 5-HT3 receptor antagonist on dopamine neurotransmission and functional activity of 5-HT3 receptors within the nucleus accumbens of Wistar rats.

A previous study indicated that pretreatment with repeated daily injections of serotonin-3 (5-HT3) receptor antagonists subsequently reduced the effectiveness of the 5-HT3 antagonists to attenuate ethanol intake under 24-h free-choice conditions; one possibility to account for this is that the functional activity of the 5-HT3 receptor may have been altered by prior treatment with the antagonists. The present experiments were conducted to examine the effects of local perfusion of the 5-HT3 agonist 1-(m-chlorophenyl)-biguanide (CPBG) on the extracellular levels of dopamine (DA) in the nucleus accumbens (ACB) and ventral tegmental area (VTA) of adult male Wistar rats that had received repeated daily injections of the 5-HT3 antagonist, MDL 72222 (MDL). In vivo microdialysis was used to test the hypothesis that alterations in 5-HT3 receptor function have occurred with repeated antagonist injections. One group was given daily injections of MDL (1 mg/kg, s.c.) for 10 consecutive days (MDL group), and the other group was administered saline for 10 days (saline group). On the day after the last treatment, rats were implanted with a unilateral guide cannula aimed at either the ACB or VTA. Two days later, the microdialysis probe was inserted into the guide cannula; on the next day, microdialysis experiments were conducted to determine the extracellular levels of DA in the ACB or VTA. Local perfusion of CPBG (17.5, 35, 70 microM) in the ACB significantly stimulated DA release in the saline- and MDL-treated animals. In terms of percent baseline, the CPBG-stimulated DA release was higher in the MDL-treated group than in the saline-treated group in both the ACB and VTA; however, on the basis of the extracellular concentration, there were no significant differences in the ACB between the two groups. Using the no-net-flux microdialysis, it was determine that the basal extracellular concentration of DA in the ACB was approximately 60% lower in the MDL group than saline group; there was no difference between the groups in the extraction fraction (clearance). Overall, the results suggest that repeated daily treatments with MDL decreased basal DA neurotransmission in the ACB and did not have a clear effect on functional activity of 5-HT3 receptors in the ACB.

In vivo time-course changes in ethanol levels sampled with subcutaneous microdialysis.

BACKGROUND The objective of this study was to determine time-course changes in in vivo ethanol (EtOH) concentrations using a novel subcutaneous (s.c.) microdialysis sampling technique. The hypothesis to be tested was that EtOH concentrations in the s.c. fluid would reflect blood EtOH concentrations. If this is the case, then s.c. microdialysis could allow a more detailed analysis of changes in in vivo levels of EtOH under different drinking paradigms. METHODS Adult male and female Wistar rats and male alcohol-preferring (P) rats were used in this study. A loop-style microdialysis probe was designed for s.c. applications. After initial in vitro characterization, probes were implanted under the skin between the shoulder blades. Animals were allowed to recover 4 to 24 hours prior to microdialysis collection (2.0 microl/min flow rate with isotonic saline). In vivo microdialysis experiments were then conducted to determine (i) the extraction fraction (or clearance) using EtOH no-net-flux (NNF) coupled with the alcohol clamp method, (ii) the dose-response and time-course effects after systemic EtOH administration and to compare with blood EtOH levels, and (iii) the time-course changes in EtOH levels during and after an EtOH drinking episode. RESULTS In vivo probe recovery (extraction fraction) obtained using the alcohol clamp method was 69 +/- 3%, and was comparable to the in vitro recovery of 73 +/- 2%. For the EtOH dose-response experiment, rats injected i.p. with 0.5, 1.0, or 2.0 g/kg EtOH showed a clear dose-response effect in the s.c. dialysate samples. Peak concentrations (70, 123, and 203 mg%, respectively) were reached by 15 minutes after injection. In an experiment comparing levels of EtOH in s.c. dialysis and arterial blood samples in rats administered 1.0 g/kg EtOH, similar time-course changes in in vivo EtOH concentrations were observed with both i.g. and i.p. EtOH administration. In P rats drinking 15% EtOH during a 1-hour scheduled access period, EtOH levels in s.c. microdialysates rose rapidly over the session and peaked at approximately 50 mg% at 60 to 80 minutes. CONCLUSIONS Overall, these experiments indicate that s.c. EtOH and blood EtOH concentrations follow a similar time course. Moreover, s.c. microdialysis can be useful as an experimental approach for determining detailed time-course changes in in vivo EtOH concentrations associated with alcohol drinking episodes.

Ethanol drinking reduces extracellular dopamine levels in the posterior ventral tegmental area of nondependent alcohol-preferring rats.

Moderate ethanol exposure produces neuroadaptive changes in the mesocorticolimbic dopamine (DA) system in nondependent rats and increases measures of DA neuronal activity in vitro and in vivo. Moreover, moderate ethanol drinking and moderate systemic exposure elevates extracellular DA levels in mesocorticolimbic projection regions. However, the neuroadaptive changes subsequent to moderate ethanol drinking on basal DA levels have not been investigated in the ventral tegmental area (VTA). In the present study, adult female alcohol-preferring (P) rats were divided into alcohol-naive, alcohol-drinking, and alcohol-deprived groups. The alcohol-drinking group had continuous access to water and ethanol (15%, vol/vol) for 8 weeks. The alcohol-deprived group had 6 weeks of access followed by 2 weeks of ethanol deprivation, 2 weeks of ethanol re-exposure, followed again by 2 weeks of deprivation. The deprived rats demonstrated a robust alcohol deprivation effect (ADE) on ethanol reinstatement. The alcohol-naïve group had continuous access to water only. In the last week of the drinking protocol, all rats were implanted with unilateral microdialysis probes aimed at the posterior VTA and no-net-flux microdialysis was conducted to quantify extracellular DA levels and DA clearance. Results yielded significantly lower basal extracellular DA concentrations in the posterior VTA of the alcohol-drinking group compared with the alcohol-naive and alcohol-deprived groups (3.8±0.3nM vs. 5.0±0.5nM [P<.02] and 4.8±0.4nM, [P<.05], respectively). Extraction fractions were significantly (P<.0002) different between the alcohol-drinking and alcohol-naive groups (72±2% vs. 46±4%, respectively) and not significantly different (P=.051) between alcohol-deprived and alcohol-naive groups (61±6% for the alcohol-deprived group). The data indicate that reductions in basal DA levels within the posterior VTA occur after moderate chronic ethanol intake in nondependent P rats. This reduction may result, in part, from increased DA uptake and may be important for the maintenance of ethanol drinking. These adaptations normalize with ethanol deprivation and may not contribute to the ADE.

GABAA receptor function in the cerebral cortex of alcohol-naive P and NP rats

Previous studies have demonstrated an innate difference in the sensitivity of ethanol-naive P and NP rats to the acute intoxicating effects of high doses of ethanol. A number of studies have suggested that the acute intoxicating effects of ethanol may be mediated in part through potentiation of GABA(A)/benzodiazepine receptor function. In the present study, the function of GABA(A)/benzodiazepine receptors was studied in ethanol-naive alcohol-preferring (P) and -nonpreferring (NP) lines of rats by measuring 36Cl- influx into cortical microsacs. GABA, in a concentration-dependent manner, increased 36Cl- influx to an equivalent extent into cortical microsacs from P and NP rats (EC50 = 9.0 +/- 1.0 and 10 +/- 1.1 microM; Emax = 30.8 +/- 1.3 and 28.1 +/- 0.9 nmol Cl-/mg protein/3 s, respectively). Pentobarbital (30 microM) enhanced GABA-stimulated 36Cl- uptake (75 and 71% increase for P and NP rats, respectively) equally well in cortical microsacs from P and NP rats. Likewise, phenobarbital potentiation of GABA-stimulated 36Cl- influx was similar in cortical microsacs from P and NP rats. Phenobarbital, at the highest concentration tested (3 mM), directly stimulated 36Cl- influx to a similar extent in P and NP rats. However, ethanol failed to alter GABA-stimulated 36Cl- uptake into cortical microsacs prepared from ethanol-naive P and NP rats. The present results suggest that the differences between P and NP rats in innate sensitivity to the high dose effects of ethanol do not appear to be due to differences in cortical GABA(A) receptor function.