Pregnancy-related changes in connections from the cervix to forebrain and hypothalamus in mice

Abstract

The transneuronal tracer pseudorabies virus was used to test the hypothesis that connections from the cervix to the forebrain and hypothalamus are maintained with pregnancy. The virus was injected into the cervix of nonpregnant or pregnant mice, and, after 5 days, virus-labeled cells and fibers were found in specific forebrain regions and, most prominently, in portions of the hypothalamic paraventricular nucleus. With pregnancy, fewer neurons and fibers were evident in most brain regions compared to that in nonpregnant mice. In particular, little or no virus was found in the medial and ventral parvocellular subdivisions, anteroventral periventricular nucleus, or motor cortex in pregnant mice. By contrast, labeling of virus was sustained in the dorsal hypothalamus and suprachiasmatic nucleus in all groups. Based upon image analysis of digitized photomicrographs, the area with label in the rostral and medial parvocellular paraventricular nucleus and magnocellular subdivisions was significantly reduced in mice whose cervix was injected with virus during pregnancy than in nonpregnant mice. The findings indicate that connections from the cervix to brain regions that are involved in sensory input and integrative autonomic functions are reduced during pregnancy. The findings raise the possibility that remaining pathways from the cervix to the forebrain and hypothalamus may be important for control of pituitary neuroendocrine secretion, as well as for effector functions in the cervix as pregnancy nears term.

Figure 1

Photomicrographs of cells and fibers in forebrain and hypothalamus after the cervix of mouse #8 was injected with pseudorabies virus on day 15 of pregnancy. Top panels are anteroventral periventricular area (AvPe) and periventricular region (Pe), medial preoptic area (MPA), and retrochiasmatic area (RCh). Bottom panels are lateral hypothalamus (LH) and sensorimotor cortex (M1). Other neuroanatomical structures are optic chiasm (ox), third ventricle (3V), corpus callosum (cc), and lateral ventricle (LV). Insets illustrate fine fibers that emanate from soma and diversity of somas that were labeled with virus. Scale bar is 50 µm for all panels and 25 µm for all insets.

Figure 2

Photomicrographs of cells and fibers in forebrain and hypothalamus after the cervix of a nonpregnant mouse (top panels) or day 15 pregnant mouse (bottom panels) was injected with pseudorabies virus. Left panels are from the anterior hypothalamus (AH). Right panels are of the suprachiasmatic nuclei (SCh). Other neuroanatomical structures are the third ventricle (3V) and optic tract (opt). Insets highlight morphology of cells and fibers labeled with virus. Scale bar is 50 µm for all panels and 25 µm for all insets.

Figure 3

Photomicrographs of pseudorabies virus-labeled cells and fibers in the hypothalamic paraventricular nucleus 5 days after injection of virus into the cervix of nonpregnant mice, and mice on day 15 or 18 of pregnancy (top, middle, or bottom panels respectively). Based on the Paxinos & Franklin (2004) mouse brain atlas, designations correspond to rostral (Figure 36), middle (Figures 38 and 39), and caudal (Figures 40 and 41) portions of the paraventricular nucleus. Insets highlight virus label in individual and clusters of cells, fine fibers, as well as single or multiple projections. Subdivisions of the paraventricular nucleus are parvocellular, i.e. ventral (PaV), anterior (PaAP), and posterior (PaPo), or magnocellular, i.e. lateral (PaLM), medial (PaMM), and posterior (PaMP). Scale bar is 50 µm for all panels and 50 µm for all insets.

Figure 4

Digitized images of pseudorabies virus label in sections of paraventricular nucleus in Fig. 2 using Image Pro Plus 6 software. Subdivision designations were based upon the mouse atlas by Paxinos & Franklin (2004). Pixels with pseudorabies virus label were counted to calculate the area of the paraventricular nucleus with cells and fibers that were infected 5 days after injection of virus into the cervix. Small structures and isolated thin fibers were often below the limit of detection. Scale bar is 50 µm.

Figure 5

Area of PRV-labeled cells and fibers in subdivisions of paraventricular nucleus from nonpregnant mice (NP, n = 3) and mice injected with pseudorabies virus into the cervix on day 15 or 18 post-breeding (Preg, n = 6). Data are the mean sum of pixels (±s.e.m.) from Image Pro Plus 6-analyzed digitized images of photomicrographs (details in Materials and Methods). *Indicates P < 0.05 versus NP group.