Characterization of the oxytocin system regulating affiliative behavior in female prairie voles

Abstract

Oxytocin regulates partner preference formation and alloparental behavior in the socially monogamous prairie vole (Microtus ochrogaster) by activating oxytocin receptors in the nucleus accumbens of females. Mating facilitates partner preference formation, and oxytocin-immunoreactive fibers in the nucleus accumbens have been described in prairie voles. However, there has been no direct evidence of oxytocin release in the nucleus accumbens during sociosexual interactions, and the origin of the oxytocin fibers is unknown. Here we show for the first time that extracellular concentrations of oxytocin are increased in the nucleus accumbens of female prairie vole during unrestricted interactions with a male. We further show that the distribution of oxytocin-immunoreactive fibers in the nucleus accumbens is conserved in voles, mice and rats, despite remarkable species differences in oxytocin receptor binding in the region. Using a combination of site-specific and peripheral infusions of the retrograde tracer Fluorogold, we demonstrate that the nucleus accumbens oxytocin-immunoreactive fibers likely originate from paraventricular and supraoptic hypothalamic neurons. This distribution of retrogradely labeled neurons is consistent with the hypothesis that striatal oxytocin fibers arise from collaterals of magnocellular neurons of the neurohypophysial system. If correct, this may serve to coordinate peripheral and central release of oxytocin with appropriate behavioral responses associated with reproduction, including pair bonding after mating, and maternal responsiveness following parturition and during lactation.

Figure 1

In vivo microdialysis to detect extracellular oxytocin (OT) as a function of social exposure and mating. Four 30-min samples were collected and analyzed for each phase. The graph illustrates the percentage of animals yielding microdialysates in each phase with detectable OT concentrations. Under basal conditions (B) OT concentrations were below the level of detectability (<0.05 pg/sample) in all samples. Detectable OT was observed significantly more frequently during the free exposure (FE) phase compared to during the restricted exposure (RE) phase when the male was housed in a wire cage (* = Fisher’s exact test, P = 0.05). In addition, detectable OT was observed significantly more frequently during the FE phase in females that mated (M) compared to during the RE (** = Fisher’s exact test, P = 0.039). In the group of females that failed to mate (NM) during the free exposure phase, the percentage with detectable OT during that phase was not significantly different from the restricted exposure phase (Fisher’s exact test, P > 0.05). There was no significant difference in the number of females that mated vs unmated during the FE phase (Fisher’s exact test, P > 0.05).

Figure 2

Species comparison of OT immunoreactive fibers in coronal sections of the nucleus accumbens (NAcc). Prairie voles (A), meadow voles (B), mice (C) and rats (D) all displayed a comparable pattern of distribution and relative density of OT-immunoreactive fibers in the NAcc. Scale bar = 500 µm (valid for A–D). ac = anterior commisure, PVN = paraventricular nucleus.

Figure 3

Electron micrographs of OT-immunoreactive elements on the nucleus accumbens. (A and B) show strongly labeled profiles that contains darkly stained dense core vesicles (DCV). These elements did not form clear synaptic contact in the planes of sections examined. (C and D) illustrate examples of OT-immunoreactive terminal boutons (Te) forming asymmetric synapses with a spine (sp) and a dendritic shaft (Den). An unlabeled terminal forming an asymmetric axo-spinous synapse is shown in D. Scale bar in A valid for B–D.

Figure 4

Light micrograph of oxytocin-immunoreactive fibers in the paraventricular nucleus of the hypothalamus (PVN) and nucleus accumbens (NAcc) of the prairie vole from a horizontal section. Black line delineates the boundary of the striatum as determined by a marker for KChIP2. Note the diffuse pattern of immunoreactive fibers coursing toward the striatum. A few fibers deviate from the neurohypophysial pathway of the PVN and project toward the striatum. Scale bar = 100 µm. ac = anterior commisure, f = fornix.

Figure 5

Retrograde labeling from the nucleus accumbens (NAcc) of female prairie voles. A) Representative injection site of Fluorogold (FG) in the NAcc. B) FG+ cells in the medial dorsal (MD) and paratenial (PT) thalamic nuclei, one of the most heavily stained regions after these NAcc injections. C–D) Arrows indicate double labeled neurons for FG (C) and OT (D) in the paraventricular nucleus of the hypothalamus. E–F) The arrows indicates a double labeled neurons for FG (E) and OT (F) in the supraoptic nucleus of the hypothalamus. Scale bars: A = 200 µm; B = 100 µm; F = 100 µm (valid for C–E). LV = lateral ventricle, ac = anterior commisure, 3V = third ventricle.

Figure 6

Organization of the neurohypophysial system of the paraventricular nucleus of the hypothalamus (PVN). Prairie voles were injected intraperitoneally with FG and brain sections were processed for FG and OT immunoreactivity. OT immunoreactivity in the anterior (A) middle (B) and posterior (C) regions of the PVN as revealed with the immunoperoxidase method. D–F) Micrographs of immunofluorescent (Texas Red-labeled) OT-containing neurons in sections of the hypothalamus representative of those shown in A–C. G–I) FG-labeled cells labeled with Alexa Fluor 350 in the same sections shown as in D-F. J-I) Overlay of OT and FG labeling in D-I. Notice that the majority of cells in the anterior and middle levels of the PVN are labeled for both OT and FG, suggesting that they are magnocellular neurohypophysial neurons. Notice the two populations of cells in F, a small dorsal and a larger ventral group. The dorsal cluster does not have FG immunoreactivity suggesting that these are the parvocellular OT cells that project to the hindbrain and spinal cord. Scale bar in C = 200 µm (valid for A–B); bar in L = 100 µm (valid for D–K). 3V = third ventricle.