Animals

Male and female Wistar rats (23-day-old) were obtained from Charles River (Raleigh, NC, USA) and maintained under standard laboratory conditions (12 h light/dark cycle, lights off at 1400 hours, 22 °C, 50% humidity, food and water ad libitum). Rats were housed in same-sex groups of four in standard rat cages (48 × 27 × 20 cm3) unless otherwise mentioned. The experiments were conducted in accordance with the guidelines of the NIH and approved by the Institutional Animal Care and Use Committee at Michigan State University and at Boston College.

Extracellular DA and NE release in the LS of juvenile male and female rats in response to LS-AVP system manipulations and social play exposure

The effect of LS-AVP administration (experiment 1), social play exposure (experiment 2), and local V1aR antagonist administration combined with social play exposure (experiment 3) on extracellular DA and NE release in the LS of 32-day-old male and female rats was determined using intracerebral microdialysis with (experiment 1 and 3) or without (experiment 2) retrodialysis. All microdialysis experiments took place in the home cage of individually housed experimental rats. At 28 days of age, experimental rats were housed individually in a standard rat cage with bedding that was not changed for the remainder of the experiments. Experimental rats were familiarized with the microdialysis setup 1 day after being housed individually. At 30 days of age, experimental rats were anesthetized with isoflurane (Butler Schein Animal Health, Dublin, OH, USA) and mounted on a stereotaxic frame (Kopf Instruments, Tujunga, CA, USA) with the tooth bar set at −4.5 mm. The u-shaped microdialysis probes (Brainlink, Groningen, The Netherlands) were unilaterally implanted into the LS according to Lukas et al. [23] at coordinates 0.4 mm caudal to bregma, −1.5 mm lateral to the midline, 5.6 mm beneath the surface of the skull at an angle of 10° to avoid damage to the sagittal sinus. The probes were filled with sterile Lactated Ringer’s solution (pH 7.4) and fixed to the skull with three stainless steel screws and dental cement. Two 5-cm-long pieces of polyethylene tubing (PE 20, Plastics One, Roanoke, VA, USA) filled with Ringer’s solution were connected to the inflow and the outflow of the probe and fixed with dental cement. After 1 day of surgery, probes were flushed with Ringer’s and rats were familiarized with the microdialysis procedure. After 2 days of surgery, microdialysis probes were perfused with Ringer’s (3.0 μl/min, pH 7.4) using a microinfusion pump. After a stabilizing period of 2 h, 6 consecutive 10-min dialysates were collected. For experiment 1–3, dialysates 1 and 2 were taken under baseline (undisturbed) conditions. For experiment 1, dialysate 3 was taken at the end of a 10-min retrodialysis period with synthetic AVP (Sigma; 1 μg/ml; this concentration will result in ~2 ng of AVP delivered locally into the LS during a 10-min retrodialysis period, based on Engelmann et al. (1992)), and dialysates 4, 5, and 6 were taken thereafter. For experiment 2, dialysate 3 was taken at the end of the 10-min social play test, and dialysates 4, 5, and 6 were taken thereafter. For the social play test, each experimental rat was exposed to an age-matched and sex-matched socially housed unfamiliar rat for a 10-min period. For experiment 3, dialysate 3 was taken at the end of a 10-min retrodialysis period with the V1aR antagonist d(CH 2 ) 5 -(Tyr(Me)2)AVP [24] (10 μg/ml; this concentration will result in ~20 ng of V1aR antagonist delivered locally into the LS during a 10-min retrodialysis period, based on Engelmann et al. (1992)), dialysate 4 was taken at the end of a second 10-min retrodialysis period with the V1aR antagonist combined with 10-min social play exposure, and dialysates 5 and 6 were taken thereafter. Microdialysates were immediately frozen on dry ice and stored at −45 °C. DA and NE concentrations were quantified in 10 μl dialysates by HPLC with tandem mass spectrometry (MS/MS) detection, using deuterated internal standards of the analytes (BrainsOnline, San Francisco), as described previously [25]. Rats were killed with CO 2 and proper probe placement in the LS (Fig. 1a) was checked on Nissl-stained 30 µm cryocut coronal brain sections. Rats with incorrect probe placement were removed from the analysis. The final number per group is indicated in the legends of Figs. 2 and 3. During microdialysis, the percentage of social play duration of male and female rats was 8.34 ± 1.53 and 7.27 ± 1.64 respectively, which is similar to our previous microdialysis study in juvenile rats [14].

Fig. 1 Microdialysis probe and microinjection placements in the lateral septum of male and female juvenile rats. Schematic representation of coronal brain sections (from Paxinos and Watson, 2007) indicating in blue the areas marking correct placement of a microdialysis probe (a) or a microinjection (b) in the lateral septum. Microdialysis probe placement was aimed at the area of the LS showing high V1aR binding density in juvenile male and female rats [40]. Cannula placement was aimed at the medial LS in order to target both hemispheres of the LS. Distance from Bregma is indicated on the right Full size image

Fig. 2 Sex-specific effects of vasopressin (AVP) and social play exposure on extracellular dopamine (DA) release in the lateral septum (LS) of juvenile rats. DA release was measured in the LS under baseline conditions and in response to LS-AVP retrodialysis (males n = 11, females n = 6; a–c), social play exposure (males n = 9, females n = 7; d–f), and LS-V1aR antagonist (V1aR-a) retrodialysis (males n = 6, females n = 7; g–i). The percent of baseline DA release increased in females, but not in males, in response to AVP retrodialysis (b) and social play exposure (e), but did not change in response to V1aR antagonist retrodialysis (h). Females showed higher DA concentrations than males after AVP administration (Post-AVP = dialysate 4; c) while no sex differences were observed under baseline conditions (Baseline = mean of dialysates 1 and 2; c, f, i), during social play exposure (Play = dialysate 3) (f), or during V1aR antagonist retrodialysis (i). Data are presented as mean+SEM; Two-way ANOVA for repeated measures (sex × dialysate) followed by LSD post-hoc testing for b, e, and h; Student’s t-test for c, f, i; *p < 0.05 vs. baseline (dialysates 1 and 2) in females, #p < 0.05 females vs. males Full size image

Fig. 3 Extracellular norepinephrine (NE) release in the lateral septum (LS) of juvenile male and female rats increased in response to vasopressin (AVP), social play exposure and V1aR antagonist (V1aR-a) treatment. NE release was measured in the LS under baseline conditions and in response to LS-AVP retrodialysis (males n = 10, females n = 10; a–c), social play exposure (males n = 8, females n = 8; d–f), and LS-V1aR-a retrodialysis (males n = 9, females n = 8; g–i). The percent of baseline NE release increased in males and females in response to AVP retrodialysis (b), during social play exposure (e), and during V1aR antagonist retrodialysis (h). No sex differences in absolute NE concentrations were observed under baseline conditions (Baseline = mean of dialysates 1 and 2; c, f, i), but females showed higher absolute NE concentrations than males during social play exposure (Play = dialysate 3; f). Data are presented as mean+SEM; Two-way ANOVA for repeated measures (sex × dialysate) followed by one-way ANOVA for repeated measures by sex (dialysate) followed by LSD post-hoc testing for b, e, and h; Student’s t-test for c, f, and i; ‡ p < 0.05, treatment effect in males, dialysate 3 vs. dialysate 2 (h) and dialysate 4 vs. dialysates 1 and 2 (b, h); *p < 0.05, treatment effect in females, dialysate 3 vs. dialysate 1 (e), dialysate 3 vs. dialysate 1 and 2 (h), dialysate 4 vs. dialysates 1 and 2 (e, h); #p < 0.05 females vs. males (f) Full size image

Effects of pharmacological manipulations of DA or NE neurotransmission in the LS on social play behavior in juvenile male and female rats

To determine the causal involvement of extracellular LS-DA and LS-NE release patterns on social play behavior, various drugs that alter DA or NE neurotransmission were administered in the LS. After 5 days of daily handling to familiarize them to the injection procedure, experimental rats (30 days of age) were anesthetized with isoflurane (Butler Schein Animal Health, Dublin, OH, USA) and mounted on a stereotaxic frame with the tooth bar set at −4.5 mm. Guide cannulae (22 gauge; Plastics One, Roanoke, VA, USA) were implanted 2 mm dorsal to the medial part of the LS according to Veenema et al. [12] at coordinates 0.4 mm caudal to bregma, −1.0 mm lateral to the midline and 3.6 mm ventral to the surface of the skull at an angle of 10° from the midsagittal plane to avoid damage to the sagittal sinus. Cannulae were fixed to the skull with three stainless steel screws and dental cement and closed with a dummy cannula (Plastics One, Roanoke, VA, USA). After surgery, rats were housed individually. After 2 days, rats received a 0.5 μl injection of either Ringer’s solution (vehicle), the non-specific DA receptor antagonist cis-(z)-flupenthixol dihydrochloride (Sigma-Aldrich F114; 15 or 30 µg/0.5 μl), the V1aR antagonist (dCH 2 ) 5 -[Tyr(Me2)]AVP (10 ng/0.5 µl), a combination of the V1aR antagonist and the DA agonist (r)-(-)-apomorphine hydrochloride (Tocris 2074; 0.2 or 2.0 µg/0.5 μl), the norepinephrine agonist DL-norepinephrine hydrochloride (Sigma-Aldrich A7256; 10 or 30 nmol/0.5 μl), the alpha adrenergic receptor antagonist phentolamine hydrochloride (Sigma-Aldrich P7547; 1.0 or 5.0 µg/0.5 μl), or the beta adrenergic receptor antagonist (s)-(−)-propranolol hydrochloride (Sigma-Aldrich P8688; 2.0 or 5.0 µg/0.5 μl). Rats were tested for social play 20 min later. Apomorphine hydrochloride and norepinephrine hydrochloride were dissolved in Ringer’s solution containing 0.1% ascorbic acid. Drug doses were chosen based on previous microinjection studies showing behavioral effects [11, 26,27,28,29,30]. Behavioral testing occurred at the beginning of the dark phase (between 1400 hours and 1500 hours) and under red light conditions. Each experimental rat was exposed to an age-matched and sex-matched socially housed unfamiliar rat for a 10-min period. Tests were videotaped for subsequent analysis of behavior by a researcher blinded to the treatment conditions using JWatcher (http://www.jwatcher.ucla.edu/). Social play duration of the experimental rats was scored according to Veenema and Neumann [31] and consisted of the time spent in playful social interactions including nape attacks, pinning and supine poses. The number of nape attacks, pinning and supine poses was also scored. After the experiments, rats were euthanized with CO 2 , and charcoal was injected as a marker to check proper cannula placement (Fig. 1b) on Nissl-stained 30 μm cryocut coronal brain sections. Rats with incorrect probe placement were removed from the analysis. The final number per group is indicated in the bars of Figs. 4 and 5.

Fig. 4 Involvement of the dopamine (DA) system in the lateral septum in the regulation of social play behavior in male and female juvenile rats. a Administration of the non-specific DA receptor antagonist cis-(z)-flupenthixol at either 15 µg/0.5 µl (CisFlup15) or 30 µg/0.5 µl (CisFlup30) reduced social play duration compared to vehicle treatment (Veh) in males, while only the higher dose (CisFlup30) reduced social play duration in females. b Administration of the V1aR antagonist (d(CH 2 ) 5 -[Tyr(Me)2]AVP at 10 ng/0.5 µl (V1aR-a) reduced social play duration compared to vehicle treatment (Veh) in females, an effect that is prevented by co-administration of the DA agonist apomorphine at 2 µg/0.5 µl (APO2) but not at 0.2 µg/0.5 µl (APO0.2). Data are presented as mean + SEM; Two-way ANOVA (sex × treatment; a) or one-way ANOVA (treatment; b) followed by Bonferroni post-hoc testing. a *p < 0.05 vs. vehicle; b *p < 0.05 vs. vehicle, V1aR-a + APO0.2, and V1aR-a + APO2.0 Full size image

Fig. 5 No effects of manipulations of the norepinephrine (NE) system in the lateral septum on social play behavior in male and female juvenile rats. a Administration of NE at either the lower dose (10 nmol/0.5 µl Ringer’s; NE10) or higher dose (30 nmol/0.5 µl Ringer’s; NE30) did not significantly alter social play duration compared to vehicle treatment (Veh) in males and females. b Administration of the alpha adrenergic receptor antagonist phentolalamine at either the lower dose (1 µg/0.5 µl Ringer’s) or higher dose (5 µg/0.5 µl Ringer’s) did not significantly alter social play duration compared to vehicle treatment (Veh) in males and females. c Administration of the beta adrenergic receptor antagonist propranolol at either the lower dose (2 µg/0.5 µl Ringer’s) or higher dose (5 µg/0.5 µl Ringer’s) did not significantly alter social play duration compared to vehicle treatment (Veh) in males and females. Data are presented as mean+SEM; Two-way ANOVA (sex × treatment) Full size image

Statistics

The percent of baseline DA release and NE release was analyzed using two-way ANOVAs for repeated measures (sex × dialysate). An LSD post hoc test was run when significant sex × dialysate interaction effects were found. When only a significant dialysate effect was found, a one-way ANOVA for repeated measures was run by sex to test for dialysate effects within each sex, followed by LSD post hoc testing. Absolute DA and NE concentrations were analyzed for sex effects using Student’s t-tests. Drug treatment effects were analyzed using two-way ANOVAs (sex × drug) or one-way ANOVA (drug) followed by Bonferroni post hoc tests. Data are presented as mean+SEM. Significance was set at p < 0.05.