Oxytocin Removes Estrous Female vs. Male Preference of Virgin Male Rats: Mediation of the Supraoptic Nucleus Via Olfactory Bulbs

Abstract

Social functions of oxytocin (OT) have been explored extensively; however, relationship between the effect of intranasally applied OT (nasal OT) on the social preference (SP) and intracerebral actions of endogenous OT remains unclear. To resolve this question, we first observed effects of nasal OT on the SP of virgin young adult male rats toward unfamiliar virgin estrous female (EF) vs. virgin male rats. The results showed that the test male rats exhibited significantly more times and longer duration accessing the female than the male, which were acutely eliminated by nasal OT. Then, we examined the approaches mediating nasal OT effects on the activity of potential brain targets in Western blots and found that nasal OT activated the olfactory bulbs (OBs) and the supraoptic nucleus (SON), but not the piriform cortex, amygdala and hippocampus as shown by significant changes in the expression of c-Fos and/or phosphorylated extracellular signal-regulated protein kinase (pERK) 1/2. Moreover, microinjection of TTX into the OBs blocked nasal OT-evoked increases in pERK1/2 levels as well as the molecular association between ERK1/2 and OT-neurophysin in the SON. Electrolytic lesions of the lateral olfactory tract did not significantly change the basal levels of pERK 1/2 in the SON; however, upon nasal OT, pERK 1/2 levels in the SON reduced significantly. Lastly, microinjection of L-aminoadipic acid (gliotoxin) into the SON to reduce OT levels reduced the duration of the test male's accessing the EF and blocked the nasal OT-evoked increase in the duration of test male's accessing the male while significantly increasing pERK1/2 levels in the amygdala. These findings reveal for the first time that nasal OT acutely eliminates virgin males' SP to EFs via the OB-SON route and that OT neurons could mediate the social effects of nasal OT by suppressing social phobia generated in the amygdala.

Keywords: intranasal drug delivery; olfactory bulbs; oxytocin; social preference; supraoptic nucleus.

Figure 1

Intranasally-applied oxytocin (OT) removes test virgin males’ social preference (SP) to novel virgin estrous female (EF) vs. virgin male rats. (A) The flowchart of this study. (B,C) show the frequency (times/10 min) and access index (total duration) of the male rats’ accessing females vs. males in response to intranasal application of saline (open bar) or 0.1 nM OT (IAO, solid bar), respectively. The insets are a photo of the 8-arm radial maze (a) and a microscopic image of the vaginal smear of an EF rat (b); numbers in the bars are that of rats; *P < 0.05 and **P < 0.01 compared to female group; ^‡P < 0.05 and ^‡‡P < 0.01 compared to control group.

Figure 2

Effects of nasal OT on phosphorylated extracellular signal regulated protein kinase (pERK) 1/2 and c-Fos expressions in different brain regions. (A) Exemplary protein bands showing the expressions of c-Fos (top panel), pERK 1/2 (middle panel) and total (t)ERK 1/2 (bottom panel), respectively from the piriform cortex (PC), amygdale (Amy), hippocampus (Hip), supraoptic nucleus (SON) and main olfactory bulb (OB). (B) Bar graph summarizing the effect of nasal OT on the expressions of pERK 1/2 and c-Fos at different brain areas; **P < 0.01 compared to the control group. Other annotations refer to Figure 1.

Figure 3

Effects of blocking neural conduction in the OBs and through the lateral olfactory tract (LOT) on nasal OT-elicited pERK 1/2 expressions in the SON. (A) Microscopic images of nasal OT-evoked pERK 1/2 expression in the SON showing (from left to the right) OT-neurophysin (NP), pERK 1/2 and their merge, respectively. (B) Exemplary protein bands (Ba) showing result of co-immunoprecipitation of ERK 1/2 (middle) with OT-NP (bottom) following nasal OT without and with prior intra-OB application of TTX and their summary (Bb). IgG-HC, the heavy chain of immunoglobulin G. (C) Effects of pretreatment of the main OBs with vehicle (Veh., artificial cerebrospinal fluid, aCSF) or TTX (2 μM, 0.5 μl) on nasal OT-evoked pERK 1/2 expressions. Exemplary Western blot bands (Ca) and average levels (Cb, % relative to 10 min of 0.45% saline) of pERK 1/2 in the SON 10 min after nasal OT in rats. (D) Effects of electrolytic lesion of the LOT (LOT-L) on nasal OT-evoked pERK 1/2 expressions. (Da) Exemplary Western blot bands. (Db) Bar graph summarizing the effect of nasal OT without and with LOT lesions based on six rats in each group. **P < 0.01 compared to the control group; ^#P < 0.05; ^##P < 0.01 compared to IAO or the LOT lesion group. Other annotations refer to Figures 1, 2.

Figure 4

Effects of l-aminoadipic acid (L-AAA) on the expression of glial fibrillary acidic protein (GFAP) and OT-NP in the SON. (A) Central loci (upper panels, marked with colored dots) of intra-SON microinjection of aCSF (Aa, 2 μl) and L-AAA (Ab, 2 mM, 2 μl) containing 0.4% Trypan blue and their effects on GFAP and OT-NP in microscopic immunostaining images (lower panels), respectively. The images are present in bright field (left) and in merged images of nuclei (in blue), GFAP (in green) and OT-NP (in red). (B) Microscopic images showing immunostaining of nuclei (in blue), GFAP (in green), OT-NP (in red) and their merges without (top panels, vehicle, veh) and with (bottom panels) L-AAA treatment. (C) Western blotting bands showing GFAP (Ca) and OT-NP (Cb) expression in the SON without and with intranuclear L-AAA injection. Other annotations refer to Figure 3.

Figure 5

Effects of disturbance of supraoptic integrity on nasal OT-changed SP. (A) The flowchart of this study. (B) Mechanical disturbing the SON with aCSF on nasal OT-evoked switch of SP. (Ba,Bb) The times (a) and access index (b) of the male rats’ accessing the female vs. the male after nasal application of saline (open bar) or 0.1 nM OT (solid bar) at 4 days after intra-SON application of aCSF, respectively. (C) Influence of disruption of the SON by intranuclear application of L-AAA on nasal OT-evoked switch of SP. (Ca,Cb) are the same as (Ba,Bb). *P < 0.05 and **P < 0.01 compared to female group; ^‡P < 0.05 and ^‡‡P < 0.01 compared to control group. Other annotations refer to Figures 1, 4.

Figure 6

Effects of disrupting the SON on nasal OT modulation of the activity of amygdala. (A,B) Western blot bands (A) and levels (B) of pERK 1/2 vs. actin10 min after nasal application of saline (CTR) or OT (0.1 nM, right two lanes) with pretreatment of the SON with aCSF (left two lanes) or L-AAA (the right lane), respectively. *P < 0.05 compared to the control group; ^#P < 0.05 compared to IAO group. Other annotations refer to Figure 4.

Figure 7

Hypothetical OB-SON-social brain pathway mediating nasal OT-evoked SP change of male rats. Abbreviations: CSF, cerebrospinal fluid; MFC, medial frontal cortex; POA, preoptic area; Pit, posterior pituitary; PVN, paraventricular nucleus. Other annotations refer to Figures 1, 2.