For microinjections into the ventral/dorsal subiculum or the PMCo, a stainless-steel chronic guide cannula (22 gauge) aimed at the ventral subiculum, the dorsal subiculum or the PMCo was stereotaxically implanted in the skull of male rats under chloral hydrate anaesthesia. Animals were given 2 days to recover from surgery; each rat was used only once. Oxytocin dissolved in saline or vehicle alone was injected into the dorsal or ventral subiculum or the PMCo in a volume of 0.3 lL over a period of 2 min via an internal cannula, which extended 1.5 mm and 5.3 mm, for the dorsal and ventral subiculum, respectively, and 7.2 mm for the PMCo, below the tip of the guide cannula, and was connected by polyethylene tubing to a 10-lL Hamilton syringe driven by a Stoelting microsyringe pump.
When d(CH2)5Tyr(Me)2-Orn8-vasotocin was used, the oxytocin antagonist was dissolved in saline and injected into the ventral subiculum or the PMCo in a volume of 0.3 lL over a period of 2 min, 15 min before oxytocin. After the injections, the tip of the cannula was left in the injection site for 30 s to allow spread of the injected solution. The injection of 0.3 lL of distilled water coloured with Neutral Red into the ventral subiculum or into the PMCo allowed us to rule out leakage of the injected solutions into the lateral ventricle under the experimental conditions described above.
Microinjections into the ventral subiculum and microdialysis in the nucleus accumbens
described and aimed unilaterally at the ipsilateral nucleus accumbens, were implanted stereotaxically in the skulls of male rats during the same stereotaxic surgery under chloral hydrate anaesthesia. Rats were used 2 days after stereotaxic surgery.
For intracerebral microdialysis, the microdialysis probe aimed at the shell of the nucleus accumbens was perfused with Ringer’s solution containing 147 mm NaCl, 3 mm KCl and 1.2 mm CaCl2, at a constant flow rate of 2.5 lL/min, by using a Stoelting microsyringe pump. After a 2-h equilibration period, the dialysate was collected
every 15 min in aliquots of 37.5 lL in polyethylene tubes kept at 10–15 degrees C for the determination of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations, as described below.
After the collection of three dialysate aliquots, oxytocin dissolved in saline or saline alone was injected into the ventral subiculum or the PMCo in a volume of 0.3 lL over a period of 2 min, as described above. Rats were observed for 75 min, during which time five additional dialysate fractions of 37.5 lL each were collected every 15 min, and penile erection episodes were counted. In those experiments in which d(CH2)5Tyr(Me)2-Orn8-vasotocin was used, the compound was dissolved in saline and microinjected into the ventral subiculum or the PMCo in a volume of 0.3 lL 15 min before oxytocin.
Rats in which only chronic guide cannulas were implanted were placed individually in Plexiglas cages. After a 30-min habituation period, oxytocin or vehicle alone was injected into the ventral subiculum or the PMCo in a volume of 0.3 lL over a period of 2 min through the microinjection cannula connected by polyethylene tubing to a 10-lL Hamilton syringe driven by a Stoelting microsyringe pump.
When d(CH2)5Tyr(Me)2-Orn8-vasotocin was used, the compound was injected into the ventral subiculum or the PMCo 15 min before oxytocin.
When cis-flupentixol and (+)MK-801 were used, the compounds were injected into the nucleus accumbens or the VTA 15 min before oxytocin. When microdialysis was
performed, the microdialysis probe was connected via polyethylene tubing to a 2500-lL Hamilton syringe driven by a Stoelting microsyringe pump at one end and to the polyethylene collecting loop at the other end.
After a 2-h period of equilibration of the dialysis probe with Ringer’s solution, three dialysate aliquots of 37.5 lL were collected for the determination of basal levels of dopamine and DOPAC, oxytocin or vehicle was injected into the ventral subiculum or the PMCo, and ?ve additional dialysate aliquots were collected.
When d(CH2)5Tyr(Me)2-Orn8-vasotocin was used, this was injected into the ventral subiculum or the PMCo 15 min before oxytocin.
In all of the above experimental conditions, after treatments rats were observed for the entire duration of the experiment in order to count penile erection episodes and, in those experiments during which microdialysis was performed, to replace filled loops with empty ones every 15 min. Penile erections were scored when the penis emerged from the penile sheath, which was usually accompanied by penile grooming and hip flexions.
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