Effects of Chronic Sleep Restriction during Early Adolescence on the Adult Pattern of Connectivity of Mouse Secondary Motor Cortex

Cortical circuits mature in stages, from early synaptogenesis and synaptic pruning to late synaptic refinement, resulting in the adult anatomical connection matrix. Because the mature matrix is largely fixed, genetic or environmental factors interfering with its establishment can have irreversible effects. Sleep disruption is rarely considered among those factors, and previous studies have focused on very young animals and the acute effects of sleep deprivation on neuronal morphology and cortical plasticity. Adolescence is a sensitive time for brain remodeling, yet whether chronic sleep restriction (CSR) during adolescence has long-term effects on brain connectivity remains unclear. We used viral-mediated axonal labeling and serial two-photon tomography to measure brain-wide projections from secondary motor cortex (MOs), a high-order area with diffuse projections. For each MOs target, we calculated the projection fraction, a combined measure of passing fibers and axonal terminals normalized for the size of each target. We found no homogeneous differences in MOs projection fraction between mice subjected to 5 days of CSR during early adolescence (P25–P30, ≥50% decrease in daily sleep, n=14) and siblings that slept undisturbed (n=14). Machine learning algorithms, however, classified animals at significantly above chance levels, indicating that differences between the two groups exist, but are subtle and heterogeneous. Thus, sleep disruption in early adolescence may affect adult brain connectivity. However, because our method relies on a global measure of projection density and was not previously used to measure connectivity changes due to behavioral manipulations, definitive conclusions on the long-term structural effects of early CSR require additional experiments

Brain structure and function in primary adrenal insufficiency

Individuals with primary adrenal insufficiency (PAI), i.e., congenital adrenal hyperplasia (CAH) and autoimmune Addison’s disease (AAD), suffer from impaired production of the adrenal gland hormones cortisol and aldosterone, and in the case of AAD, also androgens. Replacement medication for these hormones is sub-optimal due to the difficulties in replicating the natural rhythms of cortisol secretion. The hormones are known to affect brain function via many mechanisms, and both pre- and postnatal hormone dysregulation may affect cognitive functioning, brain structure and brain function. Therefore, studying brain health in PAI is of interest and is needed to optimise treatment and patient wellbeing. The present thesis investigated brain structure related to cognitive functioning in individuals with CAH, and cognitive functioning, brain structure and resting-state functional connectivity in individuals with AAD. We found that individuals with CAH have impairments in white matter microstructure, as well as cortical thinning of the frontoparietal network that was related to weaker performance on a visuospatial working memory task. On the other hand, individuals with AAD performed equally to control subjects on most measures of cognitive functions assessed with standardized tests during the lab-visit, but they self-reported executive function problems in daily life, which were related to experienced mental fatigue. As opposed to individuals with CAH, those with AAD did not have profound differences in the structure of the brain, apart from smaller total brain volumes. However, they displayed increased resting-state functional connectivity, particularly in primary visual regions and the orbitofrontal cortex. Our results suggest that the effects of adrenal hormone insufficiency affect individuals with CAH and AAD differently. This difference may be related to the onset of the disease, which is from conception for those with CAH and in adolescence or adulthood for those with AAD. Long-term follow-up studies are needed to assess whether the observed differences contribute to increased cognitive decline later in life and how to optimise replacement medication to sustain brain health

Effects of Chronic Sleep Restriction during Early Adolescence on the Adult Pattern of Connectivity of Mouse Secondary Motor Cortex

Cortical circuits mature in stages, from early synaptogenesis and synaptic pruning to late synaptic refinement, resulting in the adult anatomical connection matrix. Because the mature matrix is largely fixed, genetic or environmental factors interfering with its establishment can have irreversible effects. Sleep disruption is rarely considered among those factors, and previous studies have focused on very young animals and the acute effects of sleep deprivation on neuronal morphology and cortical plasticity. Adolescence is a sensitive time for brain remodeling, yet whether chronic sleep restriction (CSR) during adolescence has long-term effects on brain connectivity remains unclear. We used viral-mediated axonal labeling and serial two-photon tomography to measure brain-wide projections from secondary motor cortex (MOs), a high-order area with diffuse projections. For each MOs target, we calculated the projection fraction, a combined measure of passing fibers and axonal terminals normalized for the size of each target. We found no homogeneous differences in MOs projection fraction between mice subjected to 5 days of CSR during early adolescence (P25–P30, ≥50% decrease in daily sleep, n=14) and siblings that slept undisturbed (n=14). Machine learning algorithms, however, classified animals at significantly above chance levels, indicating that differences between the two groups exist, but are subtle and heterogeneous. Thus, sleep disruption in early adolescence may affect adult brain connectivity. However, because our method relies on a global measure of projection density and was not previously used to measure connectivity changes due to behavioral manipulations, definitive conclusions on the long-term structural effects of early CSR require additional experiments

Impact of ocular disease on circadian rhythms and brain connectivity

Investigation into the impact of ocular disease on sleep and mood has shown that in humans eyes have an important role, and that absence of eyes or interference with light reaching the retina can have deleterious effects. Light is the main zeitgeber ‘time-giver’ used by most species for the regulation of circadian rhythms and is detected by rods, cones and photosensitive retinal ganglion cells (pRGCs) in mammals. The aims of this research project were to investigate this from three different perspectives. Three prospective studies were undertaken. The first, studied the impact of ocular disease on the sleep/wake cycle in diabetic retinopathy (DR) and in bilateral anophthalmia. There was no significant difference found between the severity of DR and global sleep scores, however the acquired anophthalmic groups have significantly raised global sleep scores compared to controls and the congenital anophthalmic group. Both anophthalmic groups had varying sleep/wake cycles on their actograms depending on the lifestyle (independent of the urinary melatonin). All the anophthalmic participants showed a non 24 hour sleep-wake rhythm disorder after melatonin profiling. The second study investigates the evidence for the presence of extraocular circadian photoreceptors (EOCP) in participants with anophthalmia and sighted controls. Changes in brain activity using a functional MRI scan was assessed, when a bright white light is shining in different locations. This study did not reveal any evidence of EOCP.Finally, structural brain MRI differences in anophthalmic groups were investigated. While similar changes in structural reorganisation occur in all anophthalmic groups in the occipital cortex, the acquired anophthalmic groups show an inverse relation with the time since becoming anophthalmic and the volume of optic radiation and optic nerve volume. The acquired anophthalmic group did not show increase in hippocampal volume (memory areas) or in the precuneus (spatial navigation) contrast to the congenital anophthalmic groups

The effect of repetitive transcranial magnetic stimulation and the brain-derived neurotrophic factor genotype on resting-state functional network connectivity.

This thesis studied the interaction of neural stimulation and genotype on functional connectivity in 67 healthy subjects. Neural stimulation was performed using repetitive transcranial magnetic stimulation (rTMS) of the right dorsolateral prefrontal cortex (DLPFC). The effect of genotype was studied for a well-known polymorphism in the brain-derived neurotrophic factor (BDNF), which is implicated in neuronal plasticity. Functional connectivity was assessed as the degree of correlation between well-established functional networks during resting-state. In short, this thesis investigated the effect of rTMS and the genotype for a polymorphism in the BDNF on the connectivity between resting-state functional connectivity networks in 67 healthy subjects. Functional connectivity networks represent reproducible patterns of temporally correlated hemodynamic signal fluctuations in the human brain, which are involved in fundamental neurocognitive processes and show alterations in psychiatric disorders such as schizophrenia and depression. rTMS of the right DLPFC has been shown to produce lasting effects on functional connectivity and has emerged as an effective treatment in these disorders. Another mechanism affecting functional connectivity is the valine66methionine (val66met) polymorphism in the gene for the BDNF. Both mechanisms have been linked to neuronal plasticity. However, the combined effect of BDNF genotype and rTMS on functional connectivity is not known. To fill this gap, this thesis studied the interaction of rTMS and genotype on functional connectivity in a sample of 67 healthy subjects. Subjects received 5Hz stimulation of the right DLPFC during one data collection session and sham stimulation of the identical stimulation site during the other session. Following both true and sham stimulation, a resting-state functional magnetic resonance imaging scan was performed. Subjects were genotyped for the val66met single-nucleotide polymorphism (rs6265) in the 5’ proregion of the gene for the BDNF. Met66met homozygotes and val66met heterozygotes were grouped as met66 carriers for further analysis, due to the low number of homozygotes. The sample population consisted of 26 met66 allele carriers and 41 val66 homozygotes. Independent component analysis was used to generate independent components from the resting-state functional magnetic resonance imaging data. These independent components were spatially correlated with canonical samples of resting-state networks to determine best matches for the default-mode network (DMN), executive control network (ECN) and salience network (SLN). The DMN was represented by three independent components, comprising predominantly superior posterior, inferior posterior and anterior nodes respectively. The ECN was split into two components, corresponding to left-hemispheric and right-hemispheric network nodes, respectively. The SLN was covered by a single independent component. Functional connectivity between the networks was measured by the correlation of their voxel time series. Statistical analysis of the networks’ Fisher r-to-z-transformed correlation coefficients was performed using a mixed analysis of variance approach. The results of this study are as follows: rTMS did not result in significant changes in inter-network connectivity compared to sham stimulation. This concurs with published studies, which also reported a lack of effect of repetitive transcranial stimulation of the right DLPFC on inter-network connectivity. There was also no effect of the BDNF polymorphism on connectivity between the networks of interest, which contrasts with a publication, utilizing a different approach to functional connectivity analysis, that reported altered connectivity between nodes of two networks in met66 carriers. However, an interaction effect emerged which suggests that rTMS effects are influenced by the BDNF genotype. Following stimulation, met66 allele carriers showed stronger connectivity between superior posterior parts of the DMN and left-hemispheric parts of the ECN compared to the sham condition. This finding remained significant after correction for multiple comparisons and the effect was not observed in val66 homozygote individuals. This is the first study to demonstrate that the BDNF val66met genotype modulates rTMS effects on inter-network functional connectivity. A tentative interpretation could be that the observed stimulation effect may be implicated in previously observed adverse effects of rTMS in patients with schizophrenia involving increased severity of hallucinations, as it mirrors functional connectivity abnormalities observed in schizophrenic patients that correlate with symptom intensity. Variations in the therapeutic effectiveness of rTMS in major depressive disorder could also conceivably be associated to genotype-associated differences in functional connectivity modulation, although the observed effects did not align with published findings concerning the influence of this genotype on presumed therapeutic mechanisms of action of rTMS involving functional connectivity. The results from this investigation should be used to guide further research into the mechanisms of action underlying the therapeutic, and adverse, effects of rTMS and into the genotype for BDNF as a potential cause for interindividual differences in therapeutic response. These results also suggest that the BDNF val66met genotype of subjects should be routinely determined in rTMS studies, especially in those observing therapeutic effects of rTMS in patients suffering from major depressive disorder and schizophrenia

Neuroimaging investigations of the functional and structural changes of intrinsically connected brain networks in relation to habitual sleep status

This thesis uses fMRI and DTI neuroimaging modalities to investigate relationships between chronic habitual sleep status in waking control subjects and functional and structural changes in higher order intrinsically connected brain networks (ICN). Study one investigates methodologies; compares the use of deterministic and probabilistic tractography in combination with functional imaging to charaterise structural connectivity with respect to functional connectivity in a single ICN. The following chapter examines whether inter-individual differences in habitual sleep patterns are reflected in waking measurements of network functional connectivity (FC) between three ICNs. Subsequent work investigates group differences in structural connectivity with respect to habitual sleep duration and whole brain changes in white matter in relation to subjective habitual sleep quality using tract based spatial statistics (TBSS). The final chapter builds on the work from previous chapters examining a wider range of sleep features and overall network FC. Results presented in this thesis provide evidence of functional and structural brain connectivity changes, which are modulated by chronic habitual sleep status. This may help to elucidate the link between sleep, waking sleep status, cognition and explain individual differences in susceptibility to sleep deprivation, as well as potentially the networks and systems responsible for variations in sleep patterns themselves