Maternal Deprivation in Rats Decreases the Expression of Interneuron Markers in the Neocortex and Hippocampus

Abstract

Early life stress has profound effects on the development of the central nervous system. We exposed 9-day-old rat pups to a 24 h maternal deprivation (MD) and sacrificed them as young adults (60-day-old), with the aim to study the effects of early stress on forebrain circuitry. We estimated numbers of various immunohistochemically defined interneuron subpopulations in several neocortical regions and in the hippocampus. MD rats showed reduced numbers of parvalbumin-expressing interneurons in the CA1 region of the hippocampus and in the prefrontal cortex, compared with controls. Numbers of reelin-expressing and calretinin-expressing interneurons were also reduced in the CA1 and CA3 hippocampal areas, but unaltered in the neocortex of MD rats. The number of calbinin-expressing interneurons in the neocortex was similar in the MD rats compared with controls. We analyzed cell death in 15-day-old rats after MD and found no difference compared to control rats. Thus, our results more likely reflect the downregulation of markers than the actual loss of interneurons. To investigate synaptic activity in the hippocampus we immunostained for glutamatergic and inhibitory vesicular transporters. The number of inhibitory synapses was decreased in the CA1 and CA3 regions of the hippocampus in MD rats, with the normal number of excitatory synapses. Our results indicate complex, cell type-specific, and region-specific alterations in the inhibitory circuitry induced by maternal deprivation. Such alterations may underlie symptoms of MD at the behavioral level and possibly contribute to mechanisms by which early life stress causes neuropsychiatric disorders, such as schizophrenia.

Keywords: cerebral cortex; hippocampus; interneurons; maternal deprivation; schizophrenia; synapses.

Figure 1

Parvalbumin-expressing interneurons. (A) Representative images of immunofluorescence staining for parvalbumin in the CA1 region of the hippocampus of a control (CON, left panel) and an maternal deprivation (MD; right panel) rat at P60. Pyr indicates a pyramidal cell layer. (B) Shown are mean values + standard error of the mean (SEM) for profile densities (number of immunopositive cells per area) of parvalbumin+ neurons in the hippocampus. (C) Representative images of immunofluorescence staining for parvalbumin in the prefrontal cortex of a CON (left panel) and an MD (right panel) rat at P60. (D) Shown are mean values + standard error of the mean (SEM) for profile densities (number of immunopositive cells per area) of parvalbumin+ neurons in the neocortex (*p < 0.05, t-test, n = 6 animals per group). Scale bars: 20 μm.

Figure 2

Calbindin-expressing neurons. (A) Representative images of immunofluorescence staining for calbindin in the prefrontal cortex of a control (CON, left panel) and MD (right panel) rat at P60. (B) Shown are mean values + standard error of the mean (SEM) for profile densities (number of immunopositive cells per area) of calbindin+ neurons in the neocortex of MD and CON rats (p > 0.05, t-test, n = 6 animals per group). Scale bars: 20 μm.

Figure 3

Calretinin-expressing interneurons. (A) Representative images of immunofluorescence staining for calretinin in the CA3 region of the hippocampus a control (CON, left panel) and an MD (right panel) rat at P60. Pyr indicates a pyramidal cell layer. (B) Shown are mean values + standard error of the mean (SEM) for profile densities (number of immunopositive cells per area) of calretinin+ neurons in the hippocampus of MD and control rats (*p < 0.05, t-test, n = 6 animals per group). (C) Representative images of immunofluorescence staining for calretinin in the retrosplenial cortex of a control (CON, left panel) and MD (right panel) rat at P60. (D) Shown are mean values + standard error of the mean (SEM) for profile densities (number of immunopositive cells per area) of calretinin+ neurons in the neocortex of MD and CON rats (p > 0.05, t-test, n = 6 animals per group). Scale bars: 20 μm.

Figure 4

Reelin-expressing interneurons. (A) Representative images of immunofluorescence staining for reelin in the CA3 region of the hippocampus of a control (CON, left panel) and an MD rat at P60. (B) Shown are mean values + standard error of the mean (SEM) for profile densities (number of immunopositive cells per area) of reelin+ neurons in the hippocampus of MD and control rats (*p < 0.05, t-test, n = 6 animals per group). (C) Representative images of immunofluorescence staining for reelin in the retrosplenial cortex of a control (CON, left panel) and MD (right panel) rat at P60. (D) Shown are mean values + standard error of the mean (SEM) for profile densities (number of immunopositive cells per area) of reelin+ neurons in the neocortex of MD and CON rats (p > 0.05, t-test, n = 6 animals per group). Scale bars: 20 μm.

Figure 5

Cell death in the cortex at P15. (A,B) Representative images of the FlouroJade (A) and activated caspase 3 (B) stainings in the prefrontal cortex of P15 rats. Left panels show control (CON) and right panels MD rats. (C) Shown are mean values + standard error of the mean (SEM) for profile densities of labeled cells (p > 0.05, t-test, n = 4 animals per group). Scale bars: 20 μm.

Figure 6

Inhibitory and excitatory synapses. (A) Representative image of immunofluorescence staining for VGAT in the CA1 region of the hippocampus of a control (CON, left panel) and maternally deprived (MD, right panel) rat at P60. (B) Shown are mean values + standard error of the mean (SEM) for linear densities (number of immunopositive puncta per unit length) of VGAT+ puncta in the pyramidal layers (Pyr) of the CA1 and CA3, and granular layer of dentate gyrus (DG) of MD and control rats (*p < 0.05, t-test, n = 6 animals per group). (C) Representative image of immunofluorescence staining for VGLUT1 in the hippocampus a control (CON, left panel) and maternally deprived (MD, right panel) rat at P60. (D) Shown are mean values + standard error of the mean (SEM) for mean fluorescence intensity of VGLUT1 in the stratum oriens (Or), radiatum (Rad) and lacunosum-moleculare (LM) of the CA1 hippocampal region of MD and CON rats (p > 0.05, t-test, n = 6 animals per group). Scale bars: 50 μm.