Characterisation of genetic risk factors for mental illness in rodent models, impact of Map2k7+/- and Fxyd6-/- mice on neural systems and working memory

Even in wealthy and seemingly prosperous countries like the United Kingdom, the spectre of mental illness and psychiatric disorders remains highly prevalent. These disorders present a huge economic burden to societies, where in the UK alone, mental disorders cost the economy an estimated €134 billion a year; along with the unmeasurable societal and human costs. This has led to an intense debate over the past few decades just as to what factors contribute to these illnesses. It is now understood that a number of biological and non-biological factors contribute. These include socio-economic pressures, early-life trauma, gestational and peri-natal infections; genetic and familial factors, and molecular and cellular factors. However, while the definitions and diagnostic criteria of mental disorders remain based in the subjective realms of the DSM and ICD, treatment and understanding of psychiatric illness has had little chance to progress over the last fifty years. As a result, neuroscientists are starting to direct psychiatric disorder research from the bottom-up; where genetic, cognitive and neuroconnectivity factors are being investigated to serve as a future basis for diagnosis and treatment. One of the most complex and debilitating psychiatric disorders, schizophrenia, exhibits a complex array of genetic, cognitive and neuroconnectivity abnormalities. Current challenges in schizophrenia research is to understand how identified genetic abnormalities contribute to neuroconnectivity and cognitive impairments which are prominent in schizophrenia. Recently, genetic association studies have implicated two genes as risk factors for schizophrenia - FXYD6 and MAP2K7. Currently it is unclear exactly how these genes contribute to schizophrenia pathology, particularly cognitive symptoms and neural circuitry.;This thesis investigates these two genes by utilising two mouse models, first a heterozygous mouse line of Map2k7+/- and second, a gene knock-out line of Fxyd6-/-. MAP2K7 is a gene that expresses a kinase that is involved in the c-Jun N-terminal kinase (JNK) pathway, which is implicated in neuronal activity, receptor function, and cortical and hippocampal plasticity. Recent studies have found a decreased expression of MA2PK7 in the PFC, ACC and hippocampal regions in schizophrenia patients; regions associated with memory and decision making. A component of the cognitive profile of MAP2K7 was therefore investigated using Map2k7+/- mouse lines in a working memory paradigm in the radial arm maze. This test is known as the n-back test or the retention interval test. For the first time this investigation reveals that Map2k7+/- mice exhibit a subtle yet significant spatial working memory deficit compared to WT mice; as judged by their average performance over the whole experiment. WT mice exhibited an overall average performance of 70% and MAP2K7+/- mice 66% (p<0.001). This indicates that MAP2K7 may play a subtle role in working memory function in rodents, and may represent a component of the aberrations in the genetic architecture that gives rise to working memory impairments in psychiatric disorders, particularly schizophrenia. This experiment also backs up previous evidence for this radial arm maze paradigm as a robust behavioural test for testing rodent working memory.;FXYD6 belongs to a group of proteins that are known to be involved in modulating NaKATPase activity. Previously, NaKATPase has been associated with bipolar disorder and depression, but has now also been implicated in schizophrenia. Previous studies have found that FXYD6 is also abnormally expressed in the PFC of schizophrenia patients, and therefore may contribute to the cognate symptoms of the disorder. This experiment, therefore, investigated how Fxyd6 contributes to local brain activation, particularly in neural systems relevant to cognition, using gene knockout Fxyd6-/- mouse models and semi quantitative 2DG autoradiographic imaging. Three regions showed a significant deviation in activity in Fxyd6-/- mice compared to WT mice. The subiculum, medial septum and lateral septum all exhibited significant reductions in activity in Fxyd6-/- mice compared to WT mice. Notably the subiculum is heavily implicated with memory functions, particularly working memory and disambiguation of previously learned memory. Indicating a possible role for FXYD6 and NaKATPase in working memory processing and memory disambiguation in the subiculum. Finally, the role of glutamate in relation to FXYD6 function and brain activity was assessed by administering the NMDA receptor antagonist ketamine and analysing regional brain activity using semi quantitative 2DG autoradiographic imaging. Generally, regions which were affected by ketamine in WT mice including PFC, thalamic and septal regions, were not affected in Fxyd6-/- mice. It is hypothesized that this may be down to a compensatory effect that knocking-out Fxyd6 may have on glutamate reuptake. Because NaKATPase is involved in glutamate reuptake into glia and neurons, the blockage of NMDA receptors may have less effect due to a reduction in glutamate reuptake, and therefore higher than normal postsynaptic glutamate concentrations. In conclusion, this investigation highlights two genes which may have roles in working memory functioning and neural circuitry that contribute to cognitive processes. While the evidence from this investigation does not explicitly associate these genes with symptoms of schizophrenia and other psychiatric disorders; the evidence does provide indication that they are involved in cognitive processes in rodents, and possibly humans. This investigation provides an interesting path of investigation for the potential roles of these genes regardless of their relationship to psychiatric disorders and will inform future research into the genetic architecture of neural circuits and cognition.Even in wealthy and seemingly prosperous countries like the United Kingdom, the spectre of mental illness and psychiatric disorders remains highly prevalent. These disorders present a huge economic burden to societies, where in the UK alone, mental disorders cost the economy an estimated €134 billion a year; along with the unmeasurable societal and human costs. This has led to an intense debate over the past few decades just as to what factors contribute to these illnesses. It is now understood that a number of biological and non-biological factors contribute. These include socio-economic pressures, early-life trauma, gestational and peri-natal infections; genetic and familial factors, and molecular and cellular factors. However, while the definitions and diagnostic criteria of mental disorders remain based in the subjective realms of the DSM and ICD, treatment and understanding of psychiatric illness has had little chance to progress over the last fifty years. As a result, neuroscientists are starting to direct psychiatric disorder research from the bottom-up; where genetic, cognitive and neuroconnectivity factors are being investigated to serve as a future basis for diagnosis and treatment. One of the most complex and debilitating psychiatric disorders, schizophrenia, exhibits a complex array of genetic, cognitive and neuroconnectivity abnormalities. Current challenges in schizophrenia research is to understand how identified genetic abnormalities contribute to neuroconnectivity and cognitive impairments which are prominent in schizophrenia. Recently, genetic association studies have implicated two genes as risk factors for schizophrenia - FXYD6 and MAP2K7. Currently it is unclear exactly how these genes contribute to schizophrenia pathology, particularly cognitive symptoms and neural circuitry.;This thesis investigates these two genes by utilising two mouse models, first a heterozygous mouse line of Map2k7+/- and second, a gene knock-out line of Fxyd6-/-. MAP2K7 is a gene that expresses a kinase that is involved in the c-Jun N-terminal kinase (JNK) pathway, which is implicated in neuronal activity, receptor function, and cortical and hippocampal plasticity. Recent studies have found a decreased expression of MA2PK7 in the PFC, ACC and hippocampal regions in schizophrenia patients; regions associated with memory and decision making. A component of the cognitive profile of MAP2K7 was therefore investigated using Map2k7+/- mouse lines in a working memory paradigm in the radial arm maze. This test is known as the n-back test or the retention interval test. For the first time this investigation reveals that Map2k7+/- mice exhibit a subtle yet significant spatial working memory deficit compared to WT mice; as judged by their average performance over the whole experiment. WT mice exhibited an overall average performance of 70% and MAP2K7+/- mice 66% (p<0.001). This indicates that MAP2K7 may play a subtle role in working memory function in rodents, and may represent a component of the aberrations in the genetic architecture that gives rise to working memory impairments in psychiatric disorders, particularly schizophrenia. This experiment also backs up previous evidence for this radial arm maze paradigm as a robust behavioural test for testing rodent working memory.;FXYD6 belongs to a group of proteins that are known to be involved in modulating NaKATPase activity. Previously, NaKATPase has been associated with bipolar disorder and depression, but has now also been implicated in schizophrenia. Previous studies have found that FXYD6 is also abnormally expressed in the PFC of schizophrenia patients, and therefore may contribute to the cognate symptoms of the disorder. This experiment, therefore, investigated how Fxyd6 contributes to local brain activation, particularly in neural systems relevant to cognition, using gene knockout Fxyd6-/- mouse models and semi quantitative 2DG autoradiographic imaging. Three regions showed a significant deviation in activity in Fxyd6-/- mice compared to WT mice. The subiculum, medial septum and lateral septum all exhibited significant reductions in activity in Fxyd6-/- mice compared to WT mice. Notably the subiculum is heavily implicated with memory functions, particularly working memory and disambiguation of previously learned memory. Indicating a possible role for FXYD6 and NaKATPase in working memory processing and memory disambiguation in the subiculum. Finally, the role of glutamate in relation to FXYD6 function and brain activity was assessed by administering the NMDA receptor antagonist ketamine and analysing regional brain activity using semi quantitative 2DG autoradiographic imaging. Generally, regions which were affected by ketamine in WT mice including PFC, thalamic and septal regions, were not affected in Fxyd6-/- mice. It is hypothesized that this may be down to a compensatory effect that knocking-out Fxyd6 may have on glutamate reuptake. Because NaKATPase is involved in glutamate reuptake into glia and neurons, the blockage of NMDA receptors may have less effect due to a reduction in glutamate reuptake, and therefore higher than normal postsynaptic glutamate concentrations. In conclusion, this investigation highlights two genes which may have roles in working memory functioning and neural circuitry that contribute to cognitive processes. While the evidence from this investigation does not explicitly associate these genes with symptoms of schizophrenia and other psychiatric disorders; the evidence does provide indication that they are involved in cognitive processes in rodents, and possibly humans. This investigation provides an interesting path of investigation for the potential roles of these genes regardless of their relationship to psychiatric disorders and will inform future research into the genetic architecture of neural circuits and cognition

Neural circuitry of acoustic startle habituation and prepulse inhibition in the context of sex steroid hormones using innovative silent functional MRI and electromyography techniques

This project aimed to explore the neural basis of acoustic startle reflex (ASR) plasticity, namely startle habituation and prepulse inhibition (PPI), and the influence of sex steroid hormones on startle habituation, PPI, and their associated neural substrates. Startle habituation and PPI are two forms of startle plasticity which result in the attenuation of the startle reflex response; however, these two processes reflect different attentional and sensorimotor processing. Moreover, sex differences have been observed in both startle habituation and PPI, with animal and human studies illustrating more rapid habituation and more PPI in males, compared to females. Sex differences in startle modulation can be explored through sex steroid hormones, namely oestrogens, progesterone, and androgens, in the brain, of which these hormone levels will naturally fluctuate, such as during puberty, menstrual cycle, menopause and andropause. In addition, pharmaceutical intervention can affect endogenous sex steroid hormone levels, such as hormonal contraception use or hormonal replacement therapy (HRT). Consequently, the observed sex differences in startle habituation and PPI, may reflect the contribution of sex steroid hormones on its underlying neural circuitry.Neural circuitry underpinning the primary acoustic startle pathway and ASR plasticity has stemmed from animal models which are acoustic-focused. Functional neuroimaging studies in male-only and mixed-sex human studies have corroborated these findings, illustrating cortico-striatal-pallido-thalamic (CSPT) circuitry in PPI, and subcortical, cortical and brainstem activity during startle habituation. Yet existing human functional magnetic resonance imaging (fMRI) studies of ASR, startle habituation and PPI have been challenging due to the loud scanner noise caused by rapid switching of gradient coils in conventional fMRI sequences. In this context, Looping Star, a multi-echo, zero echo time (ZTE), near-silent pulse sequence offers a novel approach to fMRI research. This will encourage the use of auditory startle paradigms in functional neuroimaging studies of startle habituation and PPI, allowing for better translation from animal models of the neurofunctional basis of these forms of ASR plasticity.This project aimed to demonstrate the applicability of Looping Star with simultaneous electromyography (EMG) to an auditory experimental paradigm to study startle habituation and PPI. Before exploring sex steroid hormone differences on PPI and startle habituation neural circuitry, the project aimed to map the neural correlates of startle habituation and PPI in a healthy mixed-sex adult sample. To explore sex differences observed in PPI and startle habituation, the influence of biological sex and hormonal contraception was investigated. Our goal was to explore whether there were differences in neural activity during startle habituation and PPI between sexes, and between non-hormonal contraceptive users and hormonal contraceptive users.To aid in the development of the fMRI study, the published EMG study was conducted to systematically explore the effect of task and population parameters on prepulse-induced startle modulation. This would ensure a reliable and valid paradigm was developed for future imaging and drug research. Stimulus onset asynchrony (SOA), and task order were shown to affect prepulse-induced modulation (PPI and prepulse facilitation, PPF). Sex differences in PPI were observed, with males showing more PPI than females. A follow-up of the influence of hormonal contraception on PPI also showed greater PPI in males, compared to females not on hormonal contraception. Consequently, task and population parameters should be considered in the design of future research.The simultaneous EMG-silent fMRI study was designed based on the EMG study. The auditory startle paradigm consisted of acoustic startle probes to elicit ASR, and repetition of this stimulus was expected to produce habituation effects. PPI trials used SOA based on the EMG study to elicit PPI, but PPF was not examined in the fMRI study as more investigative work was required for robust PPF research. Startle habituation and PPI were analysed separately, and to derive stronger inferences about brain-behaviour correlations, EMG-assessed measures of startle habituation (regression slope) or PPI (percentage) were modelled at the group level as a covariate. In addition, raw ASR amplitude was modelled at the individual level for parametric modulation to better characterise changes in neural activity with expected decrease in ASR during startle habituation and PPI. Hypothesised PPI neural circuitry were derived from the published systematic review of functional neuroimaging research on PPI and PPF. Functional neuroimaging studies of startle habituation studies are few.Neural activity in thalamic (right), striatal, and insula regions of interest, which decreased in line with startle habituation, was observed across the whole group. Brainstem and thalamic (left) activity were also observed and decreased with more startle habituation, but this was not significant after corrections for multiple comparisons. There were no group differences in startle habituation. Startle habituation neural activity was consistent across sexes and hormonal contraception use, suggesting consistent neural recruitment in our sample. PPI, on the other hand, showed group differences in neural activity, as males and females not on hormonal contraception showed significantly more globus pallidus activity than females on hormonal contraception during PPI trials with SOA 60 ms, compared to pulse-only trials. This finding was not replicated on trials with SOA 120 ms, and this may indicate sensitivity of PPI to SOA when illustrating group differences. In line with the EMG study, PPI scores differed with males and females on hormonal contraception showing greater PPI than females not on hormonal contraception, but these differences were not shown in neural activity. This may result from differences in exploring PPI in a laboratory vs MRI environment.This project sheds light on sensory information processing mechanisms in healthy populations and can be used to inform clinical studies investigating these processes in clinical disorders with aberrant PPI and startle habituation, such as Parkinson’s disease, Huntington’s disease, and schizophrenia. In addition, sex differences associated with these disorders can be examined in line with the current findings

Assessment of the visual thalamic circuitry in hallucinations in dementia with Lewy bodies

PhD ThesisBackground Visual hallucinations occur in 70-90% of patients with dementia with Lewy bodies (DLB) and are related to decreased quality of life for patients. However, the underlying neuropathological changes that promote the manifestation of visual hallucinations in DLB are not known. Several hypotheses of visual hallucinations in DLB have either directly implicated the lateral geniculate nucleus (LGN), pulvinar and superior colliculus or suggested impairments in their putative functions. Methods Post-mortem LGN, pulvinar and superior colliculus tissue was obtained from DLB cases with a clinical history of visual hallucinations and compared to cognitively normal control and Alzheimer’s disease (AD) cases without visual hallucinations. Neuropathological lesions were quantified in individual cases using densitometry and neuronal and glial cell populations were quantified with stereology. RNA sequencing and subsequent bioinformatics analysis of biological pathway alterations was performed by a collaborator on pulvinar tissue from DLB and non-hallucinating control cases. The bioinformatics data was used to identify protein targets based on pathway alterations, which were then investigated using western blot analysis. Results Lewy body pathology and neuronal loss was specifically found in the pulvinar and superior colliculus of DLB cases, particularly in regions implicated in visual attention and target selection. In contrast, AD cases had more widespread degenerative changes. Molecular analysis of the pulvinar demonstrated reduced expression of several synaptic markers, concomitant with elevated expression of several astrocytic markers in DLB. Conclusion ii The relative specificity of changes in visual thalamic regions may contribute to the occurrence of visual hallucinations in DLB. Synaptic degeneration in the pulvinar likely further impedes visual attentional function in DLB. The present results may indicate DLB patients have impairments in directing visual attention to external stimuli, thus facilitating visual hallucinations by an over-reliance upon expectations and experience rather than stimulus-driven perception.Yvonne Emily Mairy, whose generous donation to the National Institute for Health Research Biomedical Research Unit in Lewy body dementia at Newcastle University funded this project. I a

IMAGING SPECIFIC ABSORPTION RATE WITH MR THERMOMETRY USING PARAMAGNETIC LANTHANIDE COMPLEXES AND IN VIVO GABA MR SPECTROSCOPY IN MOVEMENT DISORDERS

Magnetic Resonance Imaging (MRI) is a popular imaging modality due to its ability to provide excellent soft tissue contrast without exposure to ionizing radiation. It can be used for temperature monitoring (thermometry) as well as for assessing the biochemistry in vivo (MRS). This dissertation focuses separately on the development, application and quantitation issues of these two aspects of MRI

Investigation into the mechanisms of depressive illness

Functional and structural brain abnormalities have been reported in many imaging studies of depressive illness. However, the mechanisms by which these abnormalities give rise to symptoms remain unknown. The work described in this thesis focuses on such mechanisms, particularly with regard to neural predictive error signals. Recently, these signals have been reported to be present in many studies on animals and healthy humans. The central hypothesis explored in this thesis is that depressive illness comprises a disorder of associative learning. Chapter 2 reviews the brain regions frequently reported as abnormal in imaging studies of depressive illness, and the normal function of these particular brain regions. It is concluded that such regions comprise the neural substrate for associative learning and emotion. However, confidence in this conclusion is limited by considerable variability in the human imaging literature. Therefore, chapter 3 describes a meta-analysis, which tests the hypothesis that, consistent with the non-imaging literature, the ventromedial prefrontal cortex is most active during emotional experience. The results of the meta-analysis were clearly consistent with this hypothesis. Chapter 4 provides an introduction to neural predictive error signals from the general perspective of homeostatic physiological regulation. Both experimental evidence supporting the error signals, and various formal mathematical theories describing the error signals, are summarised. This provides the background to chapter 5, which describes an original fMRI study which tested the hypothesis that patients with depressive illness would exhibit abnormal predictive error signals in response to unexpected motivationally significant stimuli. Evidence of such abnormality was found. Chapter 6 describes a further original study using transcranial ultrasound and diffusion tensor imaging of the brainstem, which investigated reports of a subtle structural abnormality in depressed patients. If present, it might give rise to abnormal error signals. However, no structural abnormality was found. Finally, chapter 7 discusses the significance of these findings in the context of clinical features of depressive illness and a wide range of treatments, ranging from psychotherapy through antidepressants to physical treatments. A number of potential future studies are identified, which could clarify understanding of depressive illness

Neural Mechanisms and Psychology of Psychedelic Ego Dissolution

Neuroimaging studies of psychedelics have advanced our understanding of hierarchical brain organization and the mechanisms underlying their subjective and therapeutic effects. The primary mechanism of action of classic psychedelics is binding to serotonergic 5-HT2A receptors. Agonist activity at these receptors leads to neuromodulatory changes in synaptic efficacy that can have a profound effect on hierarchical message-passing in the brain. Here, we review the cognitive and neuroimaging evidence for the effects of psychedelics: in particular, their influence on selfhood and subject-object boundaries—known as ego dissolution—surmised to underwrite their subjective and therapeutic effects. Agonism of 5-HT2A recep-tors, located at the apex of the cortical hierarchy, may have a particularly powerful effect on sentience and consciousness. These effects can endure well after the pharmacological half-life, suggesting that psychedelics may have effects on neural plasticity that may play a role in their therapeutic efficacy. Psychologi-cally, this may be accompanied by a disarming of ego resistance that increases the repertoire of perceptual hypotheses and affords alternate pathways for thought and behavior, including those that undergird selfhood. We consider the interaction between serotonergic neuromodulation and sentience through the lens of hierarchical predictive coding, which speaks to the value of psychedelics in understanding how we make sense of the world and specific predictions about effective connectivity in cortical hierarchies that can be tested using functional neuroimaging. Significance Statement——Classic psychedelics bind to serotonergic 5-HT2A receptors. Their agonist activity at these receptors leads to neuromodulatory changes in synaptic efficacy, resulting in a profound effect on information processing in the brain. Here, we synthesize an abundance of brain imaging research with pharmacological and psychological interpretations informed by the framework of predictive coding. Moreover, predictive coding is suggested to offer more sophisticated interpretations of neuroimaging find-ings by bridging the role between the 5-HT2A receptors and large-scale brain networks

Role of the Transcription Factor CREB in the NMDA Receptor Antagonist-Induced Attention Deficits

This thesis is the result of a multidisciplinary approach which combines behavioral, biochemical and neurochemical assays to identify and delineate the molecular mechanisms involved in the control of attention. Blockade of prefrontocortical glutamate NMDAR is associated with attentional performance deficits as assessed in a task capable of measuring selective attention and response control such as the five choice serial reaction time (5-CSRT) task. I investigated whether phosphorylation of proteins linked to the PKA/CREB pathway in the prefrontal cortex (PFC) and in subcortical regions may be affected by NMDAR blockade. Pharmacological experiments using western blot and immunohistochemical techniques clearly demonstrated that increased CREB phosphorylation (p-CREB) in the PFC but not in subcortical regions may be associated to deficits in attention performance caused by the blockade of prefrontocortical NMDAR with the selective antagonist CPP. Attempts has been made to identify the intracellular pathways responsible for CPP-induced p-CREB changes by examining different protein kinases upstream to CREB such as protein kinase A (PKA), extracellular regulated protein kinases 1/2 (ERK1/2) and calcium/calmodulin kinase II (CaMKII). However, the precise role of these kinases in NMDAR antagonist induced changes in p-CREB is unclear and deserves further studies. The hypothesis that attention deficits induced by NMDAR blockade in the PFC might reflect excessive glutamate (GLU) release in the PFC was investigated by assessing the effects of intracortical CPP on attention, GLU release and p-CREB under basal conditions and in rats pre-treated with the mGlu2/3 receptor agonist LY379268. The results provide evidence that enhanced GLU release in the PFC and CREB phosphorylation are associated to attention deficit. As there are evidences that drug responses may be modulated by the behavioural state of animals, I evaluated the effect of blockade of NMDAR in the PFC on p-CREB in rats performing the 5-CSRT task. Intriguingly, I found that in these rats CPP-induced p-CREB in the PFC was decreased as opposed to the increase found in behaviourally naive rats at the same time point, suggesting that the direction of CPP-induced p-CREB changes critically depends on the behavioural state of animals. Moreover CPP-induced cognitive impairments were prevented by the intracortical administration of Sp-cAMP suggesting that the activation of the PKA/CREB cascade in the PFC is required for the correct performance of the task. In conclusion the present thesis support the role of CREB and provide new information on the mechanisms involved in the control of attention and executive functions associated with some neuropsychiatric disorders