The genetic causes of intellectual disability (ID) and autism spectrum disorder (ASD) are frequently associated with mutations in genes that encode synaptic proteins. A recent screen of ID patients has revealed that approximately 4% of individuals carry spontaneous autosomal-dominant de novo mutations in the SYNGAP1 gene. This gene encodes the synaptic GTPase activating protein (SYNGAP) a known regulator of Ras signalling. Investigations into the pathological consequences of Syngap1 haploinsufficiency (Syngap+/−) in mice have reported abnormalities in behaviour, synaptic plasticity and dendritic spine development. These are analogous to findings from the mouse model of fragile X syndrome (FXS; Fmr1-/y), the most common inherited form of ID. One of the prominent phenotypes reported in the mouse model of FXS is that a form of hippocampal long-term depression (LTD) mediated by the activation of Group 1 (Gp1) metabotropic glutamate (mGlu) receptors is enhanced and independent of new protein synthesis (Huber et al. 2002; Nosyreva et al. 2006). The cause of these synaptic plasticity deficits together with other cognitive abnormalities observed in FXS are thought to arise, in part, from excessive protein synthesis, the consequence of altered mGlu5 receptor signalling via the Ras-ERK1/2 signalling pathway. Enhanced protein synthesis rates in Fmr1-/y mice can be corrected by either inhibiting mGlu5 receptors or reducing Ras and subsequent ERK1/2 activity (Osterweil et al. 2013). In this thesis mGluR-dependent LTD was examined at Schaffer collateral/commissural inputs to CA1 pyramidal neurones in hippocampal slices obtained from Fmr1-/y, Syngap+/− and Fmr1-/ySyngap+/− double mutant mice. Extracellular field recordings reveal that acute application of the Gp1 mGluR agonist dihydroxyphenylglycine (DHPG) induces a form of mGluR-dependent LTD that is enhanced and independent of new protein synthesis in CA1 of Fmr1-/y mice. In Syngap+/− mice, the magnitude of mGluR-dependent LTD is also significantly increased relative to WT littermates and insensitive to protein synthesis inhibitors. Furthermore, in the Fmr1-/ySyngap+/− double mutant, Syngap haploinsufficiency occludes the increase in mGluR-dependent LTD caused by the loss of FMRP. In addition, metabolic labelling studies reveal basal protein synthesis rates to be modestly enhanced in the hippocampus of Fmr1-/y mice compared to WT mice. Importantly this phenotype translates to the rat model of FXS. In Syngap+/- hippocampal slices, basal protein synthesis rates are also significantly elevated compared to WT counterparts. Interestingly, elevated basal protein synthesis rates in Syngap+/- mice could be corrected in the hippocampus by similarly pharmacological strategies employed in Fmr1-/y mice. The comparable neuropathophysiology we observe between Syngap+/− and Fmr1-/y mice suggests that SYNGAP and fragile X mental retardation protein (FMRP) may converge on similar biochemical pathways raising the intriguing possibility that therapeutic strategies used in the treatment of FXS may also be of benefit in treating individuals with ID caused by mutations in SYNGAP1.

The ability of neurons to dynamically regulate their response to changing inputs is essential for the correct development and function of a nervous system capable of learning and memory. The post synaptic compartment of excitatory synapses contains a dense proteinaceous complex of molecules that link excitatory glutamatergic neurotransmission to downstream signalling pathways that ultimately result in modification of the synapse. One of the most abundant of such postsynaptic signalling molecules, synaptic GTPase activation protein, SynGAP, represents a key signalling link between the activation of the NMDA sensitive glutamate receptor to outcomes such as the structural rearrangement of synaptic sites and altered synaptic content of AMPA type glutamate receptors, molecular processes that underly learning and memory. The primary finding of this thesis is that different isoforms of SynGAP, which varies at it N terminus through alternative transcription start sites and at its C terminus through alternative splicing, can differentially affect the function of the synapse. Using primary murine neuronal cultures we show that despite being crucial for the survival of the mouse the absence of SynGAP does not effect mean dendritic spine morphology and density or miniature excitiatory post synaptic currents under a range of experimental conditions (days in vitro 10 – 14, with and without serum, high and low cell plating density). In order to examine the effects of different SynGAP isoforms we cloned two full length transcripts (SynGAP A-alpha-2 and SynGAP Ealpha- 1) which were used to construct a range of isoforms. Whole cell patch clamping of SynGAP transfected neurons revealed that the post synaptic expression of SynGAPs which terminate as an alpha-1 isoform can lead to the elimination of mEPSCs, while isoforms that terminate as an alpha-2 isoform can lead to synaptic strengthening. The magnitude of the effect in both cases is determined by the identity of the N terminus of the protein; SynGAP A-alpha-1 has the largest synaptic weakening effect and SynGAP B-and C alpha-2 strenghten the synapse. The changes in miniature electrophysiological properties are not mirrored by changes in dendritic spine morphology, whole cell AMPA/NMDA currents, or synaptic responsiveness to stimulation suggesting an undefined novel mechanism of action. SynGAPs A, B and C appear to be under the control of different promoters which are differentially regulated by development and synaptic activity, thus the differential function of SynGAP N and C terminal combinations could play a part in the activity dependent regulation of synaptic strength.

SYNGAP1 haploinsufficiency is one of the most common monogenic causes of nonsyndromic moderate to severe intellectual disability (NSID) and autism (Hamdan et al., 2009; Pinto et al., 2010). De novo truncating or frameshift mutations in the SYNGAP1 gene lead to the loss of the encoded protein Synaptic GTPase activating protein (SynGAP), one of the most abundant of postsynaptic proteins (Hamdan et al., 2011). SynGAP, present at excitatory and inhibitory synapses (Kim et al., 1998), acts as a key regulator of highly conserved signaling pathways linked to AMPA- and NMDA-receptor dependent plasticity at the post synaptic density (Krapivisky et al., 2004; Vazquez et al., 2004). The Syngap mouse model has been extensively used to understand the pathophysiology underlying abnormal SynGAP-mediated signaling. Syngap heterozygous (het) mice demonstrate a range of physiological and behavioural abnormalities from development to adulthood (Komiyama et al., 2002; Muhia et al., 2010). However, recent advances in techniques for genome manipulation have allowed for the generation of rat models of neurodevelopmental disorders, including Syngap; enabling phenotypes to be validated across species and to address cognitive and social dysfunction, using paradigms that are more difficult to assess in mice. In this study, we examined the pathophysiology associated with a heterozygous deletion of the C2 and catalytic GAP domain of the protein, in Long-Evans rats (het). In contrast with het mice, het rats do not present with hyperactivity and can be habituated to an open field environment. To examine associative recognition memory, we tested the rats in five spontaneous exploration tasks for short-term and long-term memory, object-recognition (OR), object-location (OL), object-place (OP), object-context (OC) and object-place-context (OPC). Both groups were able to perform short-term memory tasks, but only wild type rats performed above chance in OL with a 24hour delay, suggesting deficits in long- term spatial memory. We also tested if partial loss of the GAP domain in SynGAP affects social behaviour in rats and we found that het rats exhibited impaired short- term social memory, with no signs of social isolation. These findings do not fully recapitulate previous abnormalities reported in the mouse model of SYNGAP1 haploinsufficiency, suggesting that some key behavioural phenotypes may be species-specific. Furthermore, based on physiological deficits that Syngap het mice exhibit, such as alterations in mEPSC/mIPSC amplitude and frequency and evoked cortical hyperexcitability in vitro (Guo et al., 2009; Ozkan et al., 2014), we also aimed to test if in vivo neuronal activity and circuit properties are altered. Using two-photon calcium imaging in awake mice, we focused on two areas of the cortex; a primary sensory area, the binocular region of the visual cortex (V1), and an association area, the medial posterior parietal cortex (PPC). Both areas have been found to maintain activity during visual discrimination tasks but to present with divergent activity trajectories (Harvey et al., 2012; Goard et al., 2016). We found preliminary evidence that neurons in layer 2-3 of the PPC of Syngap mice are hypoactive in basal conditions when animals are still in the dark, compared to wild type controls. When we assessed whether that changes when animals are running, we found that during locomotion neurons of both genotypes increase their activity, consistent with previous findings in wild type mice (McGinley et al., 2015; Pakan et al., 2016). However, this response gain is exaggerated in Syngap het neurons of the PPC. In contrast to above findings in PPC, results in V1 show that layer 2-3 neurons are hyperactive during both behavioural states, suggesting seemingly different computations of these two cortical areas. This work provides the first evidence for a dysregulated neuronal circuit in vivo in both visual and parietal cortex of Syngap mice, two areas critical for sensory processing that has been found to be affected in individuals with NSID and autism (Joosten and Bundy, 2010). We also provide first evidence of the effect of loss of SynGAP activity in behaviour of rats, complimenting existing data in the literature in a species-specific manner and providing greater insight into sensory and cognitive dysfunction associated with dysregulation in SynGAP-mediated signaling.

Our ability to learn relies on the potential of neuronal networks to change through experience. The primary visual cortex (V1) has become a popular system for studying how experience shapes cortical neuronal networks. Experience-dependent plasticity in V1 has been extensively studied in young animals, revealing that experiences in early postnatal life substantially shape neuronal activity in the developing cortex. In contrast, less is known about how experiences modify the representation of visual stimuli in the adult brain. In addition, adult experience-dependent plasticity remains largely unexplored in neurodevelopmental disorders. To address this issue, we established a two-photon calcium imaging set-up, suitable for chronic imaging of neuronal activity in awake-behaving mice. We implemented protocols for the reliable expression of genetically encoded calcium indicators (GCaMP6), for the implantation of a chronic cranial window and for the analysis of chronic calcium imaging data. This approach enables us to monitor the activity of hundreds of neurons across days, and up to 4-5 weeks. We used this technique to determine whether the daily exposure to high-contrast gratings would induce experience-dependent changes in V1 neuronal activity. We monitored the activity of putative excitatory neurons and of three non-overlapping populations of inhibitory interneurons in layer 2/3 of adult mice freely running on a cylindrical treadmill. We compared the results obtained from mice that were exposed daily to either a high-contrast grating or to a grey screen and characterized their neuronal response properties. Our results did not reveal significant differences in neuronal properties between these two groups, suggesting a lack of stimulus-specific plasticity in our experimental conditions. However, we did observe and characterize, in both groups, a wide range of activity changes in individual cells over time. We finally applied the same method to investigate impairments in experience-dependent plasticity in a mouse model of intellectual disability (ID), caused by synaptic GTPase-activating protein (SynGAP) haploinsufficiency. SynGAP haploinsufficiency is a common de novo genetic cause of non-syndromic ID and is considered a Type1 risk for autism spectrum disorders. While the impact of Syngap gene mutations has been thoroughly studied at the molecular and cellular levels, neuronal network deficits in vivo remain largely unexplored. In this study, we compared in vivo neuronal activity before and after monocular deprivation in adult mutant mice and littermate controls. These results revealed differences in baseline network activity between both experimental groups. These impairments in cortical neuronal network activity may underlie sensory and cognitive deficits in patients with Syngap gene mutations.

Intellectual disability (ID) and autism spectrum disorders (ASDs) can have a devastating impact on an individual’s functioning and quality of life. Insights from pre-clinical models of monogenic forms of ID and ASD are now revealing the biochemical pathways and aberrations in cellular and synaptic functioning involved. One monogenic cause of ID, ASD and epilepsy is SYNGAP1 ID which results from mutations in the SYNGAP1 gene on human chromosome 6. Although a variety of symptoms have been reported, many affected individuals have moderate to severe intellectual impairment and severe seizure phenotypes. Previous pre-clinical studies have mainly focussed on the effects of altered SynGAP expression in mice. This thesis is therefore the first to explore altered SynGAP expression in a rat model. It also adds to the body of research exploring the roles of SynGAP isoforms in glutamatergic synaptic function. The SynGAP_GAP deletion rat was engineered to have a deletion encompassing the enzymatically active GTPase activating protein (GAP) domain of the protein, via which SynGAP regulates multiple biochemical pathways by enhancing the slow intrinsic hydrolysis of GTP by GTP-binding proteins. SyngapGAP/GAP rats appeared small and failed to thrive. As with Syngap-/- mice, this complete loss of WT SynGAP proved lethal, whereas Syngap+/GAP rats appeared to develop normally. The electrophysiological data obtained from this new model reveals a reduction in the frequency of miniature excitatory post-synaptic currents (mEPSCs) in Syngap+/GAP cultured neurons. However the exaggerated hippocampal long-term depression identified in Syngap+/- mice was not seen in the rats. There was also no evidence of differences in intrinsic cell properties, excitatory and inhibitory currents or ratios of AMPAR / GABAAR and AMPAR / NMDAR between WT and heterozygous rats. In addition to the characterisation of the SynGAP_GAP deletion rat, the impact of the previously unstudied Eα1 isoform on forebrain neuronal synaptic function was examined through mEPSC recordings. A trend towards lower mEPSC frequency was found which supports previous research showing that α1 isoforms reduce synaptic strength. This body of work therefore adds to published evidence of isoform specific functions and provides the first evidence of the impact of SynGAP alterations in rats, the results of which show some intriguing differences from previous work in mice.