Neurogenesis of oxytocin-containing neurons in the paraventricular nucleus (PVN) of the female pig in 3 reproductive states: puberty gilts, adult gilts and lactating sows.

Evidence suggests that neurogenesis occurs in the adult hypothalamus, including centers containing oxytocin and vasopressin producing neurons. The present study was undertaken to look at one of these centers, the paraventricular nucleus (PVN), to describe its morphology, confirm the presence of neurogenesis and examine the effect of reproductive status on the incidence of neurogenesis. Serial sections of the paraffin-embedded hypothalamus were made from five puberty gilts, four adult gilts and four lactating sows. Specific sections were Nissl-stained for PVN morphology, while others were stained with an oxytocin (OT) primary antibody, which binds to the cytoplasm of oxytocin-containing neurons, and proliferating cell nuclear antigen (PCNA) primary antibody, which binds to PCNA, a protein expressed in the nucleus during cell division. Cells labeled with both OT and PCNA were considered to be oxytocin-containing neurons that had recently divided, signifying the recent synthesis of a mature neuron. The general morphology of the PVN was similar in all pigs, and three subnuclei were identified and named based on cytoarchitecture. Neurogenesis was consistently observed in OT-containing neurons of all pigs studied. However, a significantly greater number of double-labeled (OT + PCNA) cells occurred in the PVN of lactating sows and adult gilts, when compared to puberty gilts. These observations confirm the process of neurogenesis in the hypothalamus of the adult female pig and suggest that the up-regulation of OT-containing neurons is correlated to age and possibly driven by sexual maturation, but not necessarily lactation.

The use of proliferating cell nuclear antigen immunohistochemistry with a unique functional marker to detect postnatal neurogenesis in paraffin-embedded sections of the mature pig brain.

Until recently, evidence supporting postnatal neurogenesis was controversial. Much of the debate has centered on the identification of the dividing cells as neurons versus glia. Because neurogenesis has become a well-documented phenomenon, there is a need for reliable protocols to identify recently divided neurons in a wide range of situations. To facilitate the investigation of postnatal neurogenesis of magnocellular neurons in the pig hypothalamus, a sequential immunohistochemical staining technique was developed for use on serial sections of paraffin-embedded tissue. Proliferating neurons were labeled using mouse-derived monoclonal antibodies to detect proliferating cell nuclear antigen (PCNA) and vasopressin (VP). PCNA, a nuclear protein essential for cell division, identifies recently divided cells in the brains of healthy animals. VP is a unique functional marker for a mature neuron. The presence of a cell with VP positive cytoplasm and a PCNA positive nucleus demonstrates the presence of a VP-producing neuron that has recently divided. This protocol allowed us to safely and accurately label recently proliferated neurons in the mature pig hypothalamus and can be used on archived tissue. This data can be used for further morphometric analysis, as serial sectioning allows for three-dimensional reconstruction of hypothalamic nuclei.

Postnatal neurogenesis in the vasopressin and oxytocin-containing nucleus of the pig hypothalamus.

The vasopressin and oxytocin-containing nucleus (VON) of the pig hypothalamus demonstrates dramatic postnatal growth in nucleus size, both volume and neuron number, during puberty, and continues to increase in size in the adult sexually mature female pig throughout its reproductive prime. This study was designed to show that postnatal neurogenesis is responsible for the VON growth that occurs between adolescence and maturity. Recently divided neurosecretory cells of the hypothalamus were identified in adolescent and mature non-lactating female pigs using a sequential immunohistochemistry double-labeling technique with monoclonal mouse antibodies to detect vasopressin and proliferating cell nuclear antigen (PCNA), a protein associated with the S phase of the cell cycle. A computer-assisted image-analysis system was used to assess nucleus volume and neuron counts. The VON of the mature dry sows was significantly larger in volume and number of vasopressin neurons than the VON of the adolescent pigs. Double-labeled cells were noted in the VON of both adolescent and mature dry sows, but the number and proportion of double-labeled cells was significantly higher in adolescent pigs. Our results indicate the presence of neurons containing PCNA in the VON of the pig hypothalamus. This suggests that mitosis of neurogenic precursors plays a role in the growth of the nucleus.