Early-life prefrontal cortex inhibition and early-life stress lead to long-lasting behavioral, transcriptional, and physiological impairments

Abstract

Early-life stress has been linked to multiple neurodevelopmental and neuropsychiatric deficits. Our previous studies have linked maternal presence/absence from the nest in developing rat pups to changes in prefrontal cortex (PFC) activity. Furthermore, we have shown that these changes are modulated by serotonergic signaling. Here we test whether changes in PFC activity during early life affect the developing cortex leading to behavioral alterations in the adult. We show that inhibiting the PFC of mouse pups leads to cognitive deficits in the adult comparable to those seen following maternal separation. Moreover, we show that activating the PFC during maternal separation can prevent these behavioral deficits. To test how maternal separation affects the transcriptional profile of the PFC we performed single-nucleus RNA-sequencing. Maternal separation led to differential gene expression almost exclusively in inhibitory neurons. Among others, we found changes in GABAergic and serotonergic pathways in these interneurons. Interestingly, both maternal separation and early-life PFC inhibition led to changes in physiological responses in prefrontal activity to GABAergic and serotonergic antagonists that were similar to the responses of more immature brains. Prefrontal activation during maternal separation prevented these changes. These data point to a crucial role of PFC activity during early life in behavioral expression in adulthood.

Figure 1.

DREADDs expression and response to CNO in early-life. a-c) Time course of AAV-hSyn-hM4D response to CNO. a) Mice were injected with AAV8-hSyn-hM4D-mCherry or a control virus (AAV8-hSyn-mCherry) into the PFC at P1. b) PFC slices were collected at several points after infusion (P2, P4, P7 and P14). The response to CNO was measured by VSD imaging and normalized to pre-CNO baseline. c) Illustration of the anatomical reference of Layer2/3, marked in blue, for analysis for voltage dye imaging. Gray images: baseline fluorescence. Colored images: Variation in fluorescence in response to CNO. d) mCherry expression in the PFC was observed at P2, 24 hours after surgery. DAPI (blue) and mCherry (red) are shown. Scale bar 50 μm in 20x figure. e-h) Electrophysiological response of HM3D-positive neurons, compared to mCherry controls, to CNO bath application at P2 (24h post viral infusion). e) HM3D (n=6) and mCherry (n=6) cells in response to a 300 pA depolarizing steps, representative current-clamp traces (right) from PFC positive cells for HM3D before and after CNO application. After CNO application, HM3D neurons fire more action potentials indicating increased intrinsic excitability compared to before CNO application and to mCherry-positive cells. f) The resting membrane potential Vm (mV) of mCherry and HM3D-positive cells at baseline and after exposure to CNO showed no statistical difference. F) Spike threshold (mV) of mCherry and HM3D-positive cells at baseline and after exposure to CNO during the recording indicates that HM3D neurons were more excitable when exposed to CNO. h) The majority of HM3D neurons (n=11) responded to CNO with increased spiking, while mCherry neurons (n=7) showed no changes in spiking when exposed to CNO. Pre-CNO activity was analyzed for 20 seconds, while post-CNO was analyzed for 60 seconds. *p<0.05, **p<0.01, ***p<0.001. Data presented as mean ± SEM.

Figure 2.

Maternal separation leads to cognitive deficits. a) Experimental design; created with https://biorender.com. b) Maternally separated animals displayed no statistical difference from littermate controls on distance travelled during habituation. c) Maternally separated animals displayed no statistical difference in exploration of two equal objects in the sample phase. d) Maternal separated animals performed significantly worse than standard reared controls in the object recognition task. e) Standard reared control mice took longer to reach the delayed non-match to sample test (DNMS) criteria than maternally separated males. f) Maternal separated animals performed significantly worse than standard reared controls in the DNMS. In MS animals’ performance declined as the delay increased. NOR: nCTR=9M+13F; nMS=8M+9F. *p<0.05, **p<0.01, ***p<0.001. Data presented as mean ± SEM.

Figure 3.

Early-life PFC inhibition leads to cognitive deficits. a) Experimental design; created with https://biorender.com. b) Right: Density plot showing the viral spread in all animals. Red colored areas indicate a higher number of animals with expression in that area. Left: Representative image of viral spread. DAPI (blue) and mCherry (red). c) Distance travelled during habituation was similar in all groups. d) Time exploring the objects was similar in all groups during the sample phase. e) HM4D/CNO mice performed significantly worse than standard reared control animals in the object recognition test. f) Days to criteria in the DNMS was similar in all groups tested. g) HM4D/CNO animals performed significantly worse than controls in the DNMS. In HM4D/CNO animals’ performance declined as the delay increased. nmCherry/SAL = 8M+8F; nHM4D/SAL = 7M+7F; nmCherry/CNO = 12M+10F; nHM4D/CNO = 7M+7F. *p<0.05, **p<0.01, ***p<0.001. Data presented as mean ± SEM.

Figure 4.

Early-life PFC excitation blocks cognitive deficits induced by maternal separation. a) Experimental design; created with https://biorender.com. b) Right: Density plot showing the viral spread in all animals. Red colored areas indicate a higher number of animals with expression in that area. Left: Representative image of viral spread. DAPI (blue) and mCherry (red). c) CTR/HM3D and MS/HM3D mice, presented significantly higher distance traveled in the habituation phase. d) Time exploring the objects was similar in all groups during the sample phase. e) Maternal separated animals (MS/mCherry) performed significantly worse than standard reared animals in the object recognition test. Mice in which we excited the PFC during MS performed at the same level as standard reared animals. f) Days to criteria in the DNMS was similar in all groups tested. g) Maternal separated animals (MS/mCherry) performed significantly worse than standard reared animals in the DNMS. In MS/mCherry animals’ performance declined as the delay increased. nCTR/mCherry=8M+16F;nMS/mCherry=13M+12F; nCTR/HM3D=9M+19F; nMS/HM3D=11M+8F. *p<0.05, **p<0.01, ***p<0.001. Data presented as mean ± SEM.

Figure 5.

Transcriptome-based cell classification and differential comparison by age and rearing in the mouse PFC. a) Multiplex single-nucleus RNAseq procedure; created with https://biorender.com. b-c) UMAP showing the clustering of cells by broad cell type (b) and age and rearing status (c). d) Number of significantly up or downregulated genes for each cell type in different comparison groups (adjusted p value<0.05). nMS/P70 = 3; nControl/P70 = 4; nMS/P13 = 4; nControl/P13 = 4.

Figure 6.

Maternal separation affected pathways related to development, glutamatergic/GABAergic and serotonergic function, in interneurons. a) Pathway analysis of DGE in interneurons in development (P13 vs P70; left) and in maternal separated versus standard-reared animals (right). IPA z-score indicates down- (green shading) and up- (pink shading) regulation of pathways b-d) Schematic of gene expression changes in interneurons. Blue text on image represents downregulated genes, red text on image represents upregulated genes. b) Glutamatergic receptor signaling changes in interneurons in adult MS versus control (top) and control-P13 versus control-adults (bottom). c) GABAergic receptor signaling changes in interneurons in adult MS versus control (top) and control-P13 versus control-adults (bottom). d) Serotonergic pathway changes in interneurons in adult MS versus control. Created with https://biorender.com. nMS/P70 = 3; nControl/P70 = 4; nMS/P13 = 4; nControl/P13 = 4.

Figure 7.

PFC responses to GABAergic and serotonergic modulation. a, c) Experimental designs; created with https://biorender.com. b) Bicuculline application increased excitation in slices from control mice but not from MS mice. 5HT application excited MS and inhibited slices from control animals. Ketanserin and WAY application excited slices from control animals. d) Bicuculline application increased excitation in slices from adult animals. 5HT.application excited P13 and inhibited slices from adult animals. Ketanserin and Way application excited slices from adult animals. nCTR=10–9, nMS=5; nPAdult=10, nP13=11. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data presented as mean ± SEM.

Figure 8.

PFC responses to GABAergic and serotonergic modulation. a, c) Experimental designs; created with https://biorender.com. b) Bicuculline application increased excitation in slices from control animals but not from CNO/HM4D animals. 5HT application excited slices from CNO/HM4D animals. ketanserin and Way application excited slices from control animals but not from CNO/HM4D animals. d) Bicuculline application increased excitation in slices from all animals except MS/mCherry. 5HT application excited slices from MS/mCherry animals. Ketanserin and Way application excited slices from all animals except MS/mCherry. nSAL/mCherry=13, nSAL/HM4D=14, nCNO/mCherry=16, nCNO/HM4D=13; CTR/mCherry=8, MS/mCherry=8, CTR/HM3D=11, MS/HM3D=9. ***p<0.001. Data presented as mean ± SEM.