Early postnatal gene expression in the developing neocortex of prairie voles (Microtus ochrogaster) is related to parental rearing style

Abstract

The earliest and most prevalent sensory experience includes tactile, thermal, and olfactory stimulation delivered to the young via contact with the mother, and in some mammals, the father. Prairie voles (Microtus ochrogaster), like humans, are biparental and serve as a model for understanding the impact of parent/offspring interactions on the developing brain. Prairie voles also exhibit natural variation in the level of tactile stimulation delivered by the parents to the offspring, and this has been well documented and quantified. Previous studies revealed that adult prairie vole offspring who received either high (HC) or low (LC) tactile contact from their parents have differences in the size of cortical fields and the connections of somatosensory cortex. In the current investigation, we examined gene expression, intraneocortical connectivity, and cortical thickness in newborn voles to appreciate when differences in HC and LC offspring begin to emerge. We observed differences in developmentally regulated genes, as well as variation in prelimbic and anterior cingulate cortical thickness at postnatal Day 1 (P1) in HC and LC voles. Results from this study suggest that parenting styles, such as those involving high or low physical contact, impact the developing neocortex via very early sensory experience as well as differences in epigenetic modifications that may emerge in HC and LC voles.

Keywords: RRID: NCBITaxon_79684; RRID: SCR_002677; RRID: SCR_003070; cortical connections; gene expression; neocortical development; parental care.

Figure 1.. Breeding and experimental timeline.

Breeding pairs of voles were scored for amounts of parental care they provided to their offspring during an initial offspring generation. Once sorted and categorized by the parental care amounts, top quartile and bottom quartile pairs were assigned as either high contact (HC) or low contact (LC) pairs, respectively. These same pairs generated a second litter which were subsequently referred to as HC or LC offspring. One day following parturition, pups were sacrificed, brain tissue was removed, and 3 experimental approaches were applied to offspring from each group as outlined in rightmost yellow boxes.

Figure 2.. Dye placement and thalamic verification.

Representative whole hemisphere views of brains with dye placements in putative ACC (a) and S1 (c). b, Coronal section of P1 vole hemisphere demonstrating retrograde labeling in the mediodorsal nucleus of the thalamus (MD) stemming from ACC dye placement as seen in a. d, Coronal section of P1 vole hemisphere demonstrating retrograde labeling in the ventral posterior nucleus of the thalamus (VP) stemming from S1 dye placement as seen in (c). Image oriented dorsal (D) up, rostral (R) right in (a); lateral (L) up, rostral (R) right in (c); dorsal (D) up, medial (M) right in (b,d). Scale bars = 2 mm (a,c) and 500 μm (b,d).

Figure 3.. Somatosensory and anterior cingulate intraneocortical connections (INC) in postnatal day 1 (P1) pups.

Vibratome-cut 100 μM coronal sections from P1 hemispheres are arranged in a rostral (top) to caudal (bottom) series following crystal placement of DiI (red) or DiA (green) in putative anterior cingulate cortex (ACC; a2, b2, stars) and somatosensory (S1; a4, b4, stars) cortex of high contact (HC; a1–6), and low-contact (LC; b1–6) offspring brains. Sections were counterstained with DAPI (blue). Analysis of ACC and S1 connections revealed no difference between LC and HC offspring. Sections are oriented dorsal (D) up and lateral (L) to the right. Scale bar = 500 μm.

Figure 4.. Flattened reconstructions of LC and HC brains at P1.

Drawn, ‘flattened’ neocortex images, reconstructed from coronal sections of three different HC (a1–3) and LC (b1–3) brains illustrating the retrograde connections of ACC (red) and S1 (green) at postnatal day 1. A standard cortical outline with dorsal, ventral, rostral, and caudal limits was applied to all cases for the transformation of serial coronal sections into reconstructed flattened cortical image. Black lines: Cortical outline; arrow/red patches: DiI anterior cingulate dye placement locations (DPLs); arrow/green patches: DiA somatosensory DPLs; green/red dots: Retrogradely labeled cell bodies. Reconstructions are oriented medial (M) up and caudal (C) to the right. Scale bar =1,000 μm.

Figure 5.. Quantitative analysis of dye labeling.

a, Putative anterior cingulate cortex (ACC) dye placement location (DPL) spread as a function of total cortical length. No differences are present among groups. b, ACC projection zones as a function of total cortical length. No differences are present among groups. c, Putative primary somatosensory cortex (S1) dye placement location (DPL) spread as a function of total cortical length. No differences are present among groups d, S1 projection zones as a function of total cortical length. No significant differences are present between HC and LC voles. Bars represent group means ± SEM.

Figure 6.. Analysis of neocortical expression of RZRβ and Id2.

High magnification P1 coronal sections of high-contact (a1–4) and low-contact (b1–4) offspring in situ hybridized to RZRβ, as well as sections of high-contact (c1–4), low-contact (d1–4) offspring in situ hybridized to Inhibitor of DNA binding 2 (Id2). White arrowheads (a2–3, b2–3) indicate strong RZRβ expression in the developing barrel cortex in S1, similar to expression patterns in mouse development. Black arrowheads in c3–4 and d3–4 indicate the border of superficial Id2 expression, which is shifted laterally in LC pups compared to HC pups. Images oriented dorsal (D) up, lateral (L) right. Scale bar = 1,000 μm.

Figure 7.. Semi-quantitative analysis of Id2 transcript density within somatosensory cortex ROI.

a, b, Representative coronal sections of HC (a) and LC (b) P1 vole hemispheres hybridized to Id2. Note the lateral shift in layer II/III Id2 expression in LC voles (arrow, b) compared to HC voles (arrow, a). c, Line drawing of the anatomical level (using Paxinos et al., 2007 as a guide) in which a static electronically-drawn region of interest (ROI) was placed on sections of binary-converted ISH experiments to quantify levels of mRNA expression. d, LC brains display an increase in Id2 transcript densities compared to HC in the ROI defined in (c) (***p=0.008, Student’s t). Bars represent group means ± SEM. ISH images and line drawing oriented dorsal (D) up, lateral (L) right. Scale bar = 1,000 μm.

Figure 8.. Analysis of RZRβ and Id2 expression at the sensory-motor border.

P1 coronal sections of HC (a1–3) and LC (b1–3) offspring hybridized to RZRβ (a1, b1) and Id2 (a2, b2) at the level of the somatosensory-motor cortex. Merging the expression patterns together (a3, b3) reveals the primarily complementary patterning of RZRβ and Id2 expression at this level, as well as a thin overlapping region in both LC and HC offspring. Purple, Id2 expression; yellow, RZRβ expression. Images oriented dorsal (D) up, lateral (L) right. Scale bar = 1,000 μm.

Figure 9.. Cortical thickness at P1.

Coronal 40 μm sections in HC (top row, a1–e1) and LC P1 voles (middle row, a2–e2). Arrows indicate measurements of cortical thickness. No differences were observed in putative frontal cortex (a3), somatosensory (d3), and visual cortices (e3). However, a significant reduction was detected in putative prelimbic (b3; *p < 0.05) and anterior cingulate cortex (c3; *p < 0.05) in the LC group compared to HC voles. Bars represent group means ± SEM. Images oriented dorsal (D) up, lateral (L) left. Scale bar in (a2) applies to (a1–2). Scale bar in (e2) applies to all images in (b–e). Both scale bars = 500 μm.