1. A behaviorally specific oxytocin pathway from the PVN to the VMH?
Jeffrey M. Maneval1, Sarah Ferri-Kolwicz2, Loretta M. Flanagan-Cato2, 1Carleton College; Northfield, MN 2Department of Psychology, University of Pennsylvania; Philadelphia, PA
Introduction:
Oxytocin (OT) is a nonapeptide hormone released in response to a variety of stimuli, including sexual behavior, dehydration, stress, nursing, pregnancy, and hemorrhage. It is synthesized in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus. PVN axons project to the pituitary gland, which then releases OT into the bloodstream. The ventrolateral portion of the ventral medial nucleus of the hypothalamus (vlVMH), which is responsible for sexual/lordosis behavior, does not itself produce OT but instead contains OT receptors mediated by estradiol.
Hypothesis:
A subset of OTergic neurons in the PVN project specifically to the vlVMH and therefore are designated for sexually stimulated release.
Experimental Design:
We triple labeled with fluorogold, dextran amines, and OT antibody in order to visualize the innervation of the VMH, OTergic neurons, and projections from the PVN and SON to the pituitary gland and the vlVMH.
Fluorogold- Retrograde tracer which does not cross the blood brain barrier. When injected intraperitoneally (i.p), it is taken up by pituitary gland axons. It will travel to the cell bodies from where those projections originated, which we hypothesize to be the PVN.
Dextran amines (Rhodamine)- Retrograde tracer iontophoretically infused into the VMH. It will travel back to the areas which project to the VMH, which will likely be the PVN or possibly the SON.
OT antibody- will co-stain tracer-labeled neurons to confirm OT exists in these projection pathways.
Female Sprague-Dawley rats were anesthetized, placed in stereotax. Rhodamine was applied via iontophoresis into the VMH. After four days, rats were injected (i.p) with Fluorogold. 24 hours later rats were perfused, brains soaked in fixative and cut on a freezing sliding microtome. Samples were immunolabeled for OT and analyzed with a confocal microscope.
Figure 2: The iontophoresis machine. We used this method to insert dextran amines into the VMH. A B
VMH
Paraventricular Nucleus
Ventral Medial Hypothalamus
Posterior Pituitary
Expected Results:
Although this study is currently a work-in-progress, the expected results of our experiment are shown above. First, we expect that the Fluorogold (shown above in green) will show neural connections between the PVN and the posterior pituitary gland. Next, we expect that the dextran amine (rhodamine) (shown above in red) will show projections from the PVN to the fiber plexus adjacent to the VMH. Finally, OT (shown above in blue) should appear green throughout these neural networks. Thus, we expect that our experiment will show separate OT-VMH and OT-pituitary pathways.
150 m
Rhodamine
Figure 3A- The Fiber Plexus and the VMH
Figure 3B- Estradiol receptors (green) inside the VMH and the OT (red) outside the VMH
Importance of Fiber Plexus:
Potential Implications:
According to Millhouse (1973), the fiber plexus is a dense cluster of cell bodies and dendrites which run adjacent to and may make contact and synapse with the VMH. Figure 3A is a diagram from this paper which details this inference. However, these fibers never actually innervate the VMH. In unpublished findings, Gerald Griffin supported Millhouse’s hypothesis and found that OTergic neurons do synapse with the VMH. Figure 3B shows the location of OTergic fibers in relation to estradiol receptors located in the VMH. Estradiol receptors (marked in green), which usually coexist with OT receptors, are located inside the VMH. Oxytocin is actually found almost completely outside the VMH (above stained red with OT-antibody). From this, we can assume that there are OTergic neural receptors located inside the VMH which extend out into the fiber plexus. This could be the point of reception of OT in the VMH from axons extending from the PVN.
This experiment will clarify the complex mechanism of OT release and will determine whether the release is “all or none.”
This has clinical implications as well. OT has effects on social behavior, including affiliation and trust. The VMH/PVN system is located in a very conserved portion of the brain, and has not changed much between rats and humans. Thus, it could shed some light on human clinical deficiencies in bonding, such as autism.
Acknowledgements:
PVN
vlVMH
The authors would like to thank Lyngine Calizo, Laura Felgendreger, Dr. Daniel Yee, Emily Muncy-Keller, and Gerald Griffin for their technical support. Research was supported by the Kolenkow and Reitz Undergraduate Fellowship.
Figure 1: A rat brain coronal section. Although not shown, the SON is found anterior to this section and beneath and to the right of the VMH.