Mechanisms Of Synaptic Plasticity In The Adult And Developing Nervous System

National Institute Of Environmental Health Sciences

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Abstract

A longstanding issue in environmental health is the need to understand the role the environment plays in human brain development. The brain of the neonate is particularly susceptible to disruption of the sensory environment, which can have profound effects on its physiology and morphology. Such susceptibility of the developing brain to environmental influence by sensory manipulation or to environmental toxicants is particularly pronounced during defined sensitive periods of postnatal life. On the one hand, the plasticity of the connections between neurons, or synapses, is critical for refining brain circuitry during postnatal development. However, on the other hand, it is this very plasticity that makes the developing brain particularly vulnerable to various insults. In addition, similar mechanisms for changing synapses are likely to serve the basis for learning in the adult. Our primary interest, therefore, has been to determine the molecular basis of regulating long-lasting synaptic plasticity. Toward our goal of learning how neuronal activity can induce lasting modifications in neurons, we use a diverse collection of molecular, biochemical, electrophysiological, and imaging techniques. Most of our studies are performed using neonatal and adult mice. To study synaptic plasticity, we use techniques that include whole-cell patch clamp recordings from hippocampal slices maintained in vitro, and we can stimulate them either electrically or pharmacologically to induce long-term potentiation (LTP) or long-term depression (LTD). In addition, we now use molecular and biochemical methods and in vivo recording and tracing techniques to address the role of the different hippocampal subregions in specific behaviors. One approach that we have taken to gain insight into the mechanisms of regulating synaptic plasticity has been to compare highly plastic brain areas, such as the CA1 area of hippocampus, with less plastic areas. From the expression pattern of some genes, we predicted, and found, that one area of the hippocampus, the CA2, would be more resistant to synaptic plasticity such as LTP and LTD, even though we found that synaptic responses in CA2 were very similar to those in the neighboring CA1 and CA3 areas. In addition, we have found that a regulator of G-protein signaling (RGS-14), and a number of G-protein coupled receptors including adenosine A1 receptors (A1Rs) and receptors for the social neuropeptides oxytocin and vasopressin are highly expressed in CA2 neurons and regulate plasticity there. Consistent with this cell signaling regulated by such ‘social neuropeptides’ is recent data by our lab and others showing that CA2 may play an important role in social behavior such as aggression. More recently, we had found that a specialized extracellular matrix, called perineuronal nets (PNNs), plays a role in limiting synaptic plasticity in area CA2 and that that they develop prematurely and to a greater degree in a mouse model of Rett Syndrome, a developmental disorder often accompanied by impairments in social cognition, among other abnormalities. In addition, we have begun to focus on mineralocorticoid receptors (MR; NR3C2), a ligand-dependent transcription factor stimulated by the stress hormone corticosterone in rodents (cortisol in humans) and is highly enriched in area CA2 in both rodents and humans. We found that expression of all of the markers we have for CA2 were disrupted in mice lacking the mineralocorticoid receptor. Notably, we observed that immunofluorescence for the vesicular glutamate transporter 2 (vGluT2), likely representing afferents from the supramammillary nucleus (SuM), was disrupted in the embryonic, but not postnatal, MR knockout mouse CA2. Prior studies using whole hippocampal isolates have uncovered much of the role of MRs in regulating gene expression, but as yet they have been unable to resolve CA2-specific functions due largely to CA2 being a relatively small portion of the total neurons. Successful approaches such as single-cell RNA sequencing are unlikely to be feasible for CA2-targeted analyses in MR knockout animals because of the loss of many known CA2 cell-type specific gene markers. To begin to bridge this gap, we therefore leveraged the advantages of spatial transcriptomic technology to conduct differential gene expression analysis across hippocampal subfields CA1, CA2, CA3, and the dentate gyrus (DG) of mice with MR conditionally deleted from forebrain neurons (Emx1-Cre: MRfl/fl; MR KO). As evident by expression of several ‘CA2 genes’ like Amigo2, Necab2, and Pcp4, the characteristic molecular profile of CA2 neurons was lost in the MR KO mice. That a ‘CA1 gene’, Wolfram syndrome-1 (Wfs1), but not a ‘CA3 gene’, iodotyrosine deiodinase (Iyd), appeared to be expressed in the area that should have been CA2 is strongly suggestive of the CA2 neurons acquiring a CA1-like profile, hinting at the idea that neurons that would have become CA2 neurons now resemble CA1 neurons, rather than CA3 neurons, in the MR KO mice. To assess the entire molecular profile of the area CA2 in WT and MR KO tissue, we took advantage of whole genome data that transcriptomics provided. In this case, we manually selected the hippocampal subregions for analysis, as automated clustering based on gene expression profiles would necessarily be altered. Using a principal component analysis (PCA), we found that, consistent with previous data using laser capture microscopy and RNA-seq, the spots in CA2 cluster closer to CA3 than to CA1 in the wild-type tissue. However, in the MR KO tissue, the expression profile in spots located in area CA2 no longer clustered with CA3, but instead clustered close to those from CA1. To investigate further whether MRs are required for the acquisition of structural features of CA2 neurons, we examined cell body density in the different hippocampal areas of WT and MRKO mice. In WT mice, neurons in CA2 and CA3 are larger than neurons in CA1 and so the cell density, as assessed by DAPI stain, is greatest in CA1. We found the normal relationship was altered in the MR KO (Cre-positive) tissue in that both distance to nearest neighbor and nuclear density in CA2 were intermediate between CA3 and CA1 (reduced distance between nuclei and increased nuclear density). We interpret these findings to indicate that CA2 neurons, without MRs, come to partially, but not entirely, resemble CA1 neurons at a cytoarchitectural level. To test whether pharmacological disruption of MR activity in utero similarly disrupts CA2 connectivity, we implanted slow-release pellets containing the MR antagonist spironolactone in mouse dams during mid-gestation. After confirming that at least one likely active metabolite crossed from the dams’ serum into the embryonic brains using mass spectrometry, we found that spironolactone treatment caused a significant reduction of CA2 axon fluorescence intensity in the CA1 stratum oriens, where CA2 axons preferentially project, and that vGluT2 staining was significantly decreased in both CA2 and dentate gyrus in spironolactone-treated animals. We also found that spironolactone-treated animals exhibited increased reactivity to novel objects, an effect similar to what is seen with embryonic or postnatal CA2-targeted MR knockout. However, we found no difference in preference for social novelty between the treatment groups. We infer these results to suggest that persistent or more severe disruptions in MR function may be required to interfere with this type of social behavior. These findings do indicate, though, that developmental disruption in MR signaling can have persistent effects on hippocampal circuitry and behavior. We think these results may be relevant to a different syndromic autism caused by disruption in the NR3C2 gene. PNNs are one of the unique identifiers of excitatory CA2 neurons in mice, but PNNs also exist surrounding parvalbumin (PV)-expressing inhibitory neurons. The most used approach for studying PNN function is through enzymatic degradation of the PNNs, and we have previously used this method to demonstrate the involvement of PNNs in restriction of plasticity in CA2. However, this method does not differentiate between PNNs on CA2 pyramidal cells and those on PV-expressing interneurons. As our goal is to identify the specific role of PNNs on CA2 neurons distinct from those on PV-expressing neurons in hippocampal-dependent memory functions we developed a conditional knockout strain for the primary chondroitin sulfate proteogylycan (CSPG) component of PNNs, aggrecan. To disentangle the specific roles of PNNs on CA2 pyramidal cells from those on PV-expressing neurons, we crossed the Acanfl/fl mice with each of an Amigo2-cre strain and a Pvalb-cre strain, resulting in Acan deletion from either CA2 pyramidal cells (CA2 Acan KO) or from PV cells (PV Acan KO). We tested these two strains for social memory, spatial memory and fear memory and found that PNNs on each cell type contribute to distinct forms of memory, with CA2 PNNs, but not PV PNNs, being important for social and spatial memory, specifically reversal learning. In contrast, PV PNNs, but not CA2 PNNs, were important for contextual fear memory. Related to our findings using a (prenatal) conditional MR KO mice, we also found that staining for vGluT2, a marker for specific inputs was reduced in CA2 Acan KOs. We think this reduction may be due to the mis-targeting or lack of stability of synaptic inputs in the absence of PNNs. To determine whether these differences between animals with and without CA2 PNNs effects were accompanied by neurophysiological changes during a test of social memory, we recorded local field potentials in freely moving animals. Remarkably, we found that theta oscillations, recorded in the local field potential (LFP), shifted to a higher frequency during investigation of a novel social stimulus, and this shift was not seen in CA2 Acan KOs. The findings from this study clarify the specific role of PNNs on each of CA2 pyramidal cells and PV cells. Further, our LFP findings suggest a functional relationship between CA2 PNNs, synaptic inputs, and an associated physiological signature of recognition of social novelty. Previous work by our lab and others has established initial CA2’s role in social memory. These previous findings, paired with our recent discovery that the stress hormone receptor, MR, controls molecular and synaptic properties of CA2 neurons, led us to search for a behavioral test that incorporates aspects of both social aggression and stress to test our hypothesis that CA2 plays a role in the response to, or memory for, stressful events, particularly those with social components. We found that a behavioral assay querying memory for a social defeat integrates aspects of social memory, aggression, and acute social stress. In this assay, acute social defeat (aSD) is used to test the degree to which an animal avoids another after only one aggressive encounter. We found that in control mice, aSD over the course of only 5 minutes typically resulted in long-lasting social avoidance of a novel mouse resembling the aggressor. To interrogate the role of CA2 in this form of social learning, we chemogenetically inhibited neuronal activity in vivo during an acute, socially-derived stressor and tested whether memory for the defeat was influenced. When CA2 pyramidal neuron activity was inhibited during the defeat, subject mice exhibited significantly greater amounts of social avoidance one day later when compared to control defeated littermates. Importantly, CA2 inhibition during defeat caused a reduction in submissive defense behaviors, such as standing up (vs. submissive-like fleeing and freezing) in response to aggression. These data are suggestive of a role for CA2 in behavior during the defeat in later avoidance. Taken together, these results indicate that CA2 neuronal activity is required to support behavioral resilience following an acute social stressor and that submissive defensive behavior during the defeat (vs. fleeing) is a predictor of future resilience to social stress. These studies have allowed us to test the idea that CA2 activity regulates aggression in many forms, including defensive behaviors. To summarize, we have identified several novel molecular mechanisms that contribute to what is a fundamental cellular process of the pre- and postnatal brain, the regulation of synaptic strength or plasticity, both acutely and over critical developmental periods. This understanding has contributed to an interest among neuroscientists in the circuit and behavioral functions of the CA2 subregion of the hippocampus. More importantly, although we have focused on biological factors that modify synaptic plasticity, our work provides several novel molecular targets that might be susceptible to disruption by exposures to environmental toxicants. Identifying the environmental exposures that contribute to the disorders will require further knowledge about the basic relevant molecular processes that must be assayed to reliably link the exposure to the disorder, and we have created mouse disease models to validate our initial identification of some of those targets. As we learn more about the mechanisms that control synaptic plasticity over large subregions for defined developmental periods, we will also gain new insights into individual susceptibility to exposure.