Long-term effects of a double hit murine model for schizophrenia on parvalbumin expressing cells and plasticity-related molecules in the thalamic reticular nucleus and the habenula

Abstract

The exposure to aversive experiences during early-life affects brain maturation and induces changes in behavior. Additionally, when these experiences coincide with subtle neurodevelopmental alterations, they may contribute to the emergence of psychiatric disorders, such as schizophrenia. Studies in patients and animal models have identified changes in parvalbumin (PV) expressing inhibitory neurons, highlighting their significance in the etiology of this disorder. Most studies have been focused on the cortex, but PV+ neurons also provide inhibitory input to diencephalic regions, particularly to the thalamus (through cells in the thalamic reticular nucleus, TRN) and the habenula. Remarkably, alterations in both nuclei have been described in schizophrenia. Some of these changes in PV+ cells may be mediated by perineuronal nets (PNN), specialized regions of the extracellular matrix that often surround them and regulate their synaptic input and activity. Interestingly, the physiological maturation and integration of PV+ neurons, which involves the assembly of PNN, occurs during early postnatal life. Plasticity molecules associated to inhibitory neurons, such as PSA-NCAM, or NMDA receptors (NMDAR) can also influence the structure and function of these cells. Growing evidence also indicates that glial cells regulate the physiology of PV+ neurons by influencing their maturation and modulating their synaptic connectivity. To explore the impact of early-life aversive experiences and concomitant subtle neurodevelopmental alterations on diencephalic PV+ cells, we analyzed adult male mice subjected to a double-hit model (DHM) of schizophrenia, combining a single injection of an NMDAR antagonist at P7 and post-weaning social isolation. We observed that exploratory behavior, PV+ neurons and their associated PNN, as well as PSA-NCAM and NMDAR expression and glial cells, in the TRN and the habenula were affected by the DHM or one of its factors. To our knowledge, this is the first report on such alterations in these diencephalic structures in an animal model combining neurodevelopmental alterations and early-life stress during adolescence. Our findings complement previous work on PV+ neurons in cortical regions and underscore the importance of studying diencephalic inhibitory networks and their intricate interactions with aversive experiences and neurodevelopmental alterations during early life in the context of schizophrenia.

Fig. 1. Distribution of parvalbumin expressing (PV+) neurons (red) and perineuronal nets (PNN) (green, labeled with Wisteria floribunda agglutinin: WFA) in a control animal.

A Experimental procedure. B Scheme, based on Allen Brain Atlas, showing all studied regions: the thalamic reticular nucleus (TRN), lateral (LH) and medial (MH) subregions of habenula, the lateral posterior nucleus (LP), and the central (MDc), lateral (MDl) and medial (MDm) subregions of mediodorsal nucleus (C) Microphotograph showing the general distribution of PV+ neurons and PNN (WFA+) in a brain section, including the thalamic reticular nucleus (TRN). D High magnification of squared area in A showing the distribution of PV+ neurons and PNN in the TRN. Dotted squares indicate the 4 different ROIs used for analyses. Scale bar: 500 μm (A), 307 μm (B).

Fig. 2. Behavioral analysis.

Changes observed in exploratory behavior. Histograms representing the effects of treatment, isolation, and their interaction on the time spent exploring novel holes (seconds) (A) and time spent in all holes (seconds) (B).

Fig. 3. Analysis of parvalbumin expressing (PV+) neurons and perineuronal nets (PNN) in the thalamic reticular nucleus (TRN).

A1–A4 Representative confocal planes of PV+ cells (red) and PNN (green, labeled with Wisteria floribunda agglutinin: WFA) in the TRN of animals from vehicle social (A1), vehicle isolation (A2), MK-801 social (A3) and MK-801 isolation (A4) groups. B–E Histograms representing the effects of treatment, isolation, and their interaction on the density of PV+ neurons (number of cells/mm²) (B), PV fluorescence intensity (values normalized to control) (C), WFA fluorescence intensity in PNN (values normalized to control) (D), and the ratio between the fluorescence intensities of PV and WFA (E). Horizontal black lines represent the statistically significant effect of rearing. Dashed lines indicate the presence of significant interactions in the two-way ANOVA. *p < 0.05, **p < 0.01. Scale bar: 70 µm.

Fig. 4. Analysis of parvalbumin expressing neurons and perineuronal nets in the lateral habenula.

A1–A4 Representative confocal planes of PV+ neurons (red) and PNN (green, labeled with Wisteria floribunda agglutinin [WFA]) in the habenula of animals from vehicle social (A1), vehicle isolation (A2), MK-801 social (A3) and MK-801 isolation (double-hit mice, A4) groups. The upper right panel shows a high magnification of WFA staining surrounding the cell soma and proximal dendrites of a cell lacking PV expression. B–F Histograms representing the effects of treatment, isolation, and their interaction on the total number of PV+ neurons (B), total number of PNN (C), total number of PV+ cells not surrounded by PNN (D), total number of PV+ cells surrounded by PNN (E) and the ratio of PV + PNN+ cells in relation to the total number of PV+ cells (F). Horizontal black lines represent statistically significant effects of MK-801 treatment in a two-way ANOVA. #0.1 > p > 0.05, *p < 0.05, **p < 0.01. Scale bar: 70 µm (A1–A4) and 10 µm (A5).

Fig. 5. Analysis of the expression of the NMDA receptor subunit 1 (GluN1) in parvalbumin expressing (PV+) cells of the thalamic reticular nucleus (TRN).

A1–A4 Representative confocal planes of PV+ somata (green) and GluN1+ puncta (red) in the TRN of animals from vehicle social (A1), vehicle isolation (A2), MK-801 social (A3) and MK-801 isolation (double-hit mice, A4) groups. B–D Histograms representing the effects of treatment, isolation, and their interaction on GluN1 fluorescence intensity (values normalized to control) (B), percentage of area covered by GluN1+ puncta (C), and density of GluN1+ puncta (number of puncta/µm²) (D). Horizontal lines represent statistically significant effects of MK-801 treatment (black) or rearing (blue) in a two-way ANOVA. Dashed black lines indicate the presence of a significant interaction between the 2 factors in the two-way ANOVA. *p < 0.05, **p < 0.01. Scale bar” 10 μm.