A stereotaxic, population-averaged T1w ovine brain atlas including cerebral morphology and tissue volumes

Standard stereotaxic reference systems play a key role in human brain studies. Stereotaxic coordinate systems have also been developed for experimental animals including non-human primates, dogs, and rodents. However, they are lacking for other species being relevant in experimental neuroscience including sheep. Here, we present a spatial, unbiased ovine brain template with tissue probability maps (TPM) that offer a detailed stereotaxic reference frame for anatomical features and localization of brain areas, thereby enabling inter-individual and cross-study comparability. Three-dimensional data sets from healthy adult Merino sheep (Ovis orientalis aries, 12 ewes and 26 neutered rams) were acquired on a 1.5 T Philips MRI using a T1w sequence. Data were averaged by linear and non-linear registration algorithms. Moreover, animals were subjected to detailed brain volume analysis including examinations with respect to body weight (BW), age, and sex. The created T1w brain template provides an appropriate population-averaged ovine brain anatomy in a spatial standard coordinate system. Additionally, TPM for gray (GM) and white (WM) matter as well as cerebrospinal fluid (CSF) classification enabled automatic prior-based tissue segmentation using statistical parametric mapping (SPM). Overall, a positive correlation of GM volume and BW explained about 15% of the variance of GM while a positive correlation between WM and age was found. Absolute tissue volume differences were not detected, indeed ewes showed significantly more GM per bodyweight as compared to neutered rams. The created framework including spatial brain template and TPM represent a useful tool for unbiased automatic image preprocessing and morphological characterization in sheep. Therefore, the reported results may serve as a starting point for further experimental and/or translational research aiming at in vivo analysis in this species

Experimental models of focal neuroinflammation - Efficacy assessment of pharmaceuticals of multiple sclerosis using PET imaging

Neuroinflammation (NI) is a key player in neurodegenerative diseases, such as multiple sclerosis (MS). Magnetic resonance imaging is the gold standard imaging method for diagnosis of MS, however, better diagnostic tools are needed for the follow-up of disease progression, earlier diagnosis, and assessment of patients’ response to therapy. Positron emission tomography (PET) is a sensitive and selective functional imaging method for investigating mechanisms of diseases at a molecular level. NI can be visualised in PET by targeting the 18 kDa translocator protein (TSPO), which is upregulated during NI on the mitochondria of glial cells. This study aimed to evaluate the properties of [18F]GE-180, a 2nd generation TSPO PET radiotracer, in a unilateral model of acute NI and evaluate the efficacy of immunomodulatory drugs, anti-VLA-4 and dimethyl fumarate (DMF), in two different focal rat models of experimental autoimmune encephalomyelitis (EAE), the fDTH-EAE and fMOG-EAE models. The applied methods were in vivo PET imaging, digital autoradiography, and immunohistochemical (IHC) staining. Study I indicated improved binding potential of [18F]GE-180 over the 1st generation tracer [11C]PK11195 and showed that the unilateral model of acute NI is suitable for the evaluation of novel PET tracers of NI. Study II indicated that anti- VLA-4 had no short-term treatment effects in the fDHT-EAE-rat model. However, discontinuation of the treatment caused a rebound that could be detected with [18F]GE-180. Study III showed, that short-term, but not long-term, DMF treatment decreases uptake of [18F]GE-180 in the fDTH-EAE rat model, and no rebound effect was detected after halting the treatment for 10 weeks. Nevertheless, the efficacy of DMF was detected using IHC for CD4+ and CD8+ cells. No DMF treatment effect was observed in the fMOG-EAE model. In conclusion, focal animal models of NI are applicable for evaluating novel PET tracers. Furthermore, efficacy assessment of immunomodulatory drugs can be evaluated using TSPO PET when the tracer is binding to the same biomarker that the drug is affectingPesäkkeisen aivotulehduksen kokeelliset mallit – multippeliskleroosin lääkkeiden vaikuttavuuden arviointi käyttäen PETkuvantamista Keskushermoston tulehdus on tila, joka ilmenee useissa hermorappeumasairauksissa, kuten pesäkekovettumataudissa (MS-tauti). MS-taudin kuvantamisdiagnostiikan ensisijainen menetelmä on magneettikuvaus, mutta uusia diagnostisia menetelmiä tarvitaan taudin mahdollisimman varhaiseksi toteamiseksi, etenemisen seuraamiseksi, ja uusien lääkehoitojen tehon arvioimiseksi. Positroniemissiotomografia (PET) on kajoamaton kuvantamismenetelmä, jonka avulla on mahdollista seurata aivojen tulehdusreaktiota hyödyntämällä tulehduksen aikana yliilmentyvään translokaatioproteiiniin (TSPO) sitoutuvia PET-merkkiaineita. Tämän tutkimuksen tavoitteena oli verrata TSPO-molekyyliin sitoutuvan toisen sukupolven PET-merkkiaineen ([18F]GE-180) sekä jo käytössä olevan merkkiaineen ([11C]PK11195) ominaisuuksia akuutin tulehduksen rottamallissa. Lisäksi tavoitteena oli arvioida immuunivastetta muokkaavien lääkkeiden, anti-VLA-4:n ja dimetyylifumaraatin, tehokkuutta lyhyt- ja pitkäaikaishoidossa fDTH-EAE- ja fMOG-EAE-malleissa, joissa rotille aiheutetaan pesäkkeinen autoimmuunienkefalomyeliitti. Tulehdusreaktiossa tapahtuvia muutoksia seurattiin hyödyntäen in vivo [18F]GE-180-PET-kuvantamista, autoradiografiaa, sekä kudosvärjäyksiä. Ensimmäisessä osatyössä [18F]GE-180-merkkiaineen havaittiin soveltuvan [11C]PK11195-merkkiainetta paremmin aivotulehduksen kuvantamiseen eläinmalleissa: aivojen tulehdusalue oli merkittävästi suurempi ja merkkiaineen sitoutumiskyky ja spesifisyys parempi. Toisessa osatyössä anti-VLA-4-lääkehoidon vaikutusta ei ollut mahdollista seurata [18F]GE-180-PET-kuvantamisella fDTHEAE-mallin kroonisen tulehduksen vaiheessa. Hoidon lopettamisen jälkeen ilmenevä voimakas tulehduksen uudelleenaktivoituminen oli kuitenkin mahdollista havaita PET-kuvauksella. Kolmannessa osatyössä dimetyylifumaraatin havaittiin hillitsevän tulehdusta fDTH-EAE-mallissa lyhytaikaishoidossa yhden viikon hoidon jälkeen, mutta ei enää myöhemmissä aikapisteissä, eikä fMOG-EAE-mallissa. Tämä väitöskirjatutkimus osoitti, että pesäkkeisen tulehduksen rottamalleja voidaan hyödyntää uusien tulehdusta kuvantavien PET-merkkiaineiden, sekä immuunivastetta muokkaavien lääkkeiden tehokkuuden arvioinnissa

fMRI studies of amblyopia: Pediatric and adult perspectives

Functional magnetic resonance imaging (fMRI) is currently the technique of choice for mapping functional neuroanatomy in humans, and over the past 15 years there has been a dramatic growth in the number of studies that provide brain-behavior correlations in normal healthy adults. More recently, a few studies have begun to make such measures in healthy children. In addition, fMRI is increasingly being applied to study brain function in subjects with neurological disease. The overall aim of these studies was to apply fMRI methods to the study of amblyopia, the most prevalent developmental vision disorder. Amblyopia develops early in life, usually before 5 years old, and is most treatable during childhood. Our approach was to study both children and adults with either the strabismic or the anisometropic type of amblyopia. In our first experiment (Chapter 3), we applied fMRI techniques to map retinotopic visual organization in children. We conclude that cortical visual organization is measurable and highly mature in children aged 9 to 12 years. In our second experiment (Chapter 4), we applied similar techniques to adults with amblyopia. We conclude that visual field organization is abnormal in the brains of these adults. In our final experiment (Chapter 5), we applied these same techniques to children with amblyopia, and observed abnormalities similar to those seen in adults. These studies present a novel neurological characterization of amblyopia, and provide a basis for further studies of human visual development, in health and disease

MRI indices of functional recovery following intracerebral implantation of a human neural stem cell line in a pre-clinical model of ischaemic stroke

Stem cell therapy for ischaemic stroke is an emerging field in light of an increasing number of patients surviving with permanent disability. Several allogenic and autologous cells types are now in clinical trials with preliminary evidence of safety. Some clinical studies have reported functional improvements in some patients. After initial safety evaluation in a Phase 1 study, the conditionally immortalised human neural stem cell line CTX0E03 is currently in a Phase 2 clinical trial (PISCES-II). Previous pre-clinical studies conducted by ReNeuron Ltd, showed evidence of functional recovery in the Bilateral Asymmetry test up to 6 weeks following transplantation into rodent brain, 4 weeks after middle cerebral artery occlusion. Resting-state fMRI is increasingly used to investigate brain function in health and disease, and may also act as a predictor of recovery due to known network changes in the post-stroke recovery period. Resting-state methods have also been applied to non-human primates and rodents which have been found to have analogous resting-state networks to humans. The sensorimotor resting-state network of rodents is impaired following experimental focal ischaemia of the middle cerebral artery territory. However, the effects of stem cell implantation on brain functional networks has not previously been investigated. Prior studies assessed sensorimotor function following sub-cortical implantation of CTX0E03 cells in the rodent post-stroke brain but with no MRI assessments of functional improvements. This thesis presents research on the effect of sub-cortical implantation of CTX0E03 cells on the resting- state sensorimotor network and sensorimotor deficits in the rat following experimental stroke, using protocols based on previous work with this cell line. The work in this thesis identified functional tests of appropriate sensitivity for long-term dysfunction suitable for this laboratory, and investigated non-invasive monitoring of physiological variables required to optimize BOLD signal stability within a high-field MRI scanner. Following experimental stroke, rats demonstrated expected sensorimotor dysfunction and changes in the resting-state sensorimotor network. CTX0E03 cells did not improve post-stroke functional outcome (compared to previous studies) and with no changes in resting-state sensorimotor network activity. However, in control animals, we observed changes in functional networks due to the stereotaxic procedure. This illustrates the sensitivity of resting-state fMRI to stereotaxic procedures. We hypothesise that the damage caused by cell or vehicle implantation may have prevented functional and network recovery which has not been previously identified due to the application of different functional tests. The findings in this thesis represent one of few pre-clinical studies in resting-state fMRI network changes post-stroke and the only to date applying this technique to evaluate functional outcomes following a clinically applicable human neural stem cell treatment for ischaemic stroke. It was found that injury caused by stereotaxic injection should be taken into account when assessing the effectiveness of treatment

Differences in Activation of the Visual System in Mild Cognitive Impaired Subjects compared to Healthy Subjects measured using functional magnetic resonance imaging (fMRI)

Introduction: Mild Cognitive Impairment (MCI) is a cognitive stage between normal aging and Dementia. It is a heterogeneous group of patients, where most of them develop Alzheimer’s disease (AD), others stabilize, and a few revert to normal. AD’s first clinical symptoms are related to memory, but it has been shown that AD involves also a processing disorder in the visual sensory pathways. Accurate visual function facilitates memory, attention and executive functions, so that perceptual dysfunction contributes to the severity of cognitive impairment. Objective: The objective of the work is to measure changes in activation in the visual system between MCI patients and old Healthy Control (HC) subjects, using two different visual processing tasks with functional Magnet Resonance Imaging (fMRI). This is the first study which makes such a comparison between MCI and HC using fMRI. Methods: Brain activation was measured using fMRI. The MCI group was composed of 16 subjects and the HC group was composed of 19 subjects. All subjects performed two tasks: location matching (position of objects) and face matching (characteristics of the objects), which selectively activate one of the visual system pathways in healthy people. Answers were given by pressing a single button. Results: Performance of the task was not significantly different in both groups. The HC group selectively activated ventral pathway for face matching and the dorsal pathways for location matching. In contrast the MCI subjects did not selectively activate the ventral and dorsal pathways of the visual system. Additionally they showed higher activation in the left frontal lobe compared to HC when performing the location matching Task Conclusions: The results suggest that even when behavioural performance between groups is the same, the neural system which supports performance may differ. MCI subjects compensate their deficits using additional brain areas to help them to maintain performance. In this case MCI subjects used the left frontal lobe in addition to perform the location matching task. This work presents the usability of brain imaging techniques especially fMRI to better understand the underlying pathology of MCI and its subtypes as prodromal conditions of AD

Monitoring the Function of the P-glycoprotein Transporter at the Blood Brain Barrier

The P-glycoprotein (P-gp) transporter located at the blood-brain barrier (BBB) is an efflux transporter that pumps neurotoxic compounds out of the brain. Its main function is to protect the brain to ensure an appropriate neural function. Decreases in the P-gp function can result in increased accumulation of toxic compounds inside the brain such as beta-amyloid and this may cause the development of Alzheimer´s or other neurological disorders. By contrast, increases in the P-gp function can decrease the therapeutic drug concentration inside the brain and influence the efficacy of the treatment (drug resistance) as occurred in patients with intractable epilepsy. Thus, it is of interest to monitor the P-gp function in vivo to facilitate the early diagnosis of brain disorders and to monitor drug resistance. To this aim, we used Positron Emission Tomography (PET) imaging, a non-invasive technique that allows the quantification of biological processes in vivo, and the novel radiotracer [18F]MC225 which measures the P-gp function. The aim was to study the kinetic properties of the radiotracer in different species and prove its efficacy to measure increases and decrease in the P-gp function before its clinical evaluation. We conclude that the obtained results have broadened the knowledge of the P-gp function at the BBB. Moreover, the results highlight that [18F]MC225 may become the first radiofluorinated P-gp PET tracer able to measure both decreases and increases in the P-gp function in vivo. The radiolabeling with fluorine-18 would allow its distribution to other PET centers and improve the image quality

Transcranial magnetic stimulation combined with functional magnetic resonance imaging: From target identification to prediction of therapeutic effects in stroke patients

Repetitive transcranial magnetic stimulation (rTMS), particularly theta-burst stimulation (TBS), can be applied to modulate cortical excitability beyond the period of stimulation (Huang et al., 2005). Consequently, rTMS is regarded to have high therapeutic potential for treatment of various psychiatric and neurological diseases related to cortical hypo- or hyperexcitability such as stroke (Ridding & Rothwell, 2007). Whether rTMS induced effects are sufficiently robust to be useful in clinical settings is currently under intense investigation. The most challenging problem appears to be considerably high variability in rTMS induced effects both, across studies (Hoogendam et al., 2010) and individual patients (Ameli et al., 2009). Hence, the major goal of the present thesis was to improve rTMS intervention strategies in stroke patients suffering from chronic motor hand deficits by multimodal uses of (repetitive) TMS with state-of-the-art neuroimaging techniques. Sources of variance across studies are likely to be methodological in origin. They might result from different strategies to identify the cortical rTMS target position. Individual functional magnetic resonance (fMRI) data have been demonstrated to yield best spatial approximations of the most excitable TMS position compared to other techniques (Sparing et al., 2008). However, there is still a considerably large spatial mismatch between the cortical position showing highest movement-related fMRI signal and the cortical position yielding highest muscle responses when stimulated with TMS of up to 14 mm (Bastings et al., 1998; Boroojerdi et al., 1999; Herwig et al., 2002; Krings et al., 1997; Lotze et al., 2003; Sparing et al., 2008; Terao et al., 1998). The underlying cause of this spatial mismatch is unknown. Hence, the aim of the first study (Study I) of the present thesis was to test the hypothesis that the spatial mismatch between positions with highest fMRI signal change and positions with highest TMS excitability might be caused by the widely-used Gradient-Echo blood oxygenation level dependent (GRE-BOLD) fMRI technique. GRE-BOLD signal has been demonstrated to occur further downstream from the site of neural activity in large veins running on the cerebral surface (Uludag et al., 2009). Consequently, we tested the hypothesis that alternative fMRI sequences may localize neural activity (i) closer to the anatomical motor hand area, i.e. Brodmann Area 4 (BA4), and (ii) closer to the optimal TMS position than GRE-BOLD. The following alternative fMRI techniques were tested: (i) Spin-Echo (SE-BOLD) assessing blood oxygenation level dependent signal changes with decreased sensitivity for the macrovasculature at high magnetic fields (≥ 3 Tesla, Uludag et al., 2009) and (ii) arterial spin labelling (ASL), assessing local changes in cerebral blood flow (ASL-CBF) which have been shown to occur in close proximity to synaptic activity (Duong et al., 2000). GRE-BOLD, SE-BOLD, and ASL-CBF signal changes during right thumb abductions were obtained from 15 healthy young subjects at 3 Tesla. In 12 subjects, brain tissue at fMRI peak voxel coordinates was stimulated with neuronavigated TMS to investigate whether spatial differences between fMRI techniques are functionally relevant, i.e. impact on motor-evoked potentials (MEPs) recorded from a contralateral target muscle, which is involved in thumb abductions. A systematic TMS motor mapping was performed to identify the most excitable TMS position (i.e. the TMS hotspot) and the centre-of-gravity (i.e. the TMS CoG), which considers the spatial distribution of excitability in the pericentral region. Euclidean distances between TMS and fMRI positions were calculated for each fMRI technique. Results indicated that highest SE-BOLD and ASL-CBF signal changes occurred in the anterior wall of the central sulcus (BA4), whereas highest GRE-BOLD signal changes occurred significantly closer to the gyral surface where most large draining veins are located. fMRI techniques were not significantly different from each other in Euclidean distances to optimal TMS positions since optimal TMS positions were located considerably more anterior (and slightly surprisingly in premotor cortex (BA6) and not BA4). Stimulation of brain tissue at GRE-BOLD peak voxel coordinates with TMS resulted in significantly higher MEPs (compared to SE-BOLD and ASL-CBF coordinates). This was probably the case because GRE-BOLD positions tended to be located at the gyral crown, which was slightly (but not significantly) closer to the TMS hotspot position. Taken together, findings of Study I suggest that spatial differences between fMRI and TMS positions are not caused by spatial unspecificity of the widely-used GRE-BOLD fMRI technique. Hnece, other factors such as complex interactions between brain tissue and the TMS induced electric field (Opitz et al., 2011), could be the underlying cause. Identification of the cortical rTMS target position is particularly challenging in stroke patients since reorganization processes after stroke may shift both, fMRI and TMS positions in unknown direction and extend (Rossini et al., 1998). In the second study (Study II) of the present thesis, we therefore tested whether findings obtained from healthy young subjects in Study I do also apply to chronic stroke patients and older (i.e. age-matched) healthy control subjects. In this study, arterial spin labelling (ASL) was used to assess CBF and BOLD signal changes simultaneously during thumb abductions with the affected/non-dominant and the unaffected/dominant hand in 15 chronic stroke patients and 13 age-matched healthy control subjects at 3 Tesla. Brain tissue at fMRI peak voxel coordinates was stimulated with neuronavigated TMS to test whether spatial differences are functionally relevant and impact on MEPs. Systematic TMS motor mappings were performed for both hemispheres in overall 12 subjects (6 stroke patients and 6 healthy subjects). Euclidean distances between fMRI and TMS positions were calculated for each hemisphere and fMRI technique. In line with results of Study I, highest ASL-CBF signal changes were located in the anterior wall of the central sulcus (BA4), whereas highest ASL-BOLD signal changes occurred significantly closer to the gyral surface. In contrast to Study I, there were no significant differences between ASL-CBF and ASL-BOLD positions in MEPs when stimulated with neuronavigated TMS, which suggests that spatial differences (in depth) were not functionally relevant for TMS applications. In line with Study I, there were no significant differences between fMRI techniques in Euclidean distances to optimal TMS positions, since optimal TMS positions were located considerably more anterior than fMRI positions (in premotor cortex, i.e. BA6). Stroke patients showed overall larger displacements (between fMRI and TMS positions) on the ipsilesional (but not the contralesional) hemisphere compared to healthy subjects. However, none of the fMRI techniques yielded positions significantly closer to the optimal TMS position. Hence, functional reorganization may impact on spatial congruence between fMRI and TMS, but the effect is similar for ASL-CBF and ASL-BOLD. Pathomechanisms underlying stroke induced motor deficits are still poorly understood but a simplified model of hemispheric competition has been suggested, which proposes relative hypoexcitability of the ipsilesional hemisphere and hyperexcitability of the contralesional hemisphere leading to pathologically increased interhemispheric inhibition from the contralesional onto the ipsilesional hemisphere during movements of the paretic hand (Duque et al., 2005; Grefkes et al., 2008b, 2010; Murase et al., 2004). In line with the model of hemispheric competition, both increasing excitability of the ipsilesional hemisphere (Khedr et al., 2005; Talelli et al., 2007) as well as decreasing excitability of the contralesional hemisphere (Fregni et al., 2006; Di Lazzaro et al., 2008a) have been demonstrated to normalize cortical excitability towards physiological levels and/or ameliorate motor performance of the stroke affected hand. However, there is considerably high inter-individual variance and some patients may even show deteriorations of motor performance after rTMS (Ameli et al., 2009). Therefore, the aim of the third study (Study III) was to identify reliable predictors for TBS effects on motor performance of the affected hand in stroke patients, which appears essential for successful implementation of TBS in neurorehabilitation. Overall, 13 chronic stroke patients with unilateral motor hand deficit and 12 age-matched healthy control subjects were included in the study. All patients received 3 different TBS interventions on 3 different days: (i) intermittent TBS (iTBS, facilitatory) over the primary motor cortex (M1) of the ipsilesional hemisphere, (ii) continuous TBS (cTBS, inhibitory) over M1 of the contralesional hemisphere, and (iii) either iTBS or cTBS over a control stimulation site (to control for placebo effects). Motor performance was measured before and after each TBS session with 3 different motor tasks and an overall motor improvement score was calculated. All subjects participated in an fMRI experiment, in which they performed rhythmic fist closures with their affected/non-dominant and unaffected/dominant hand. A laterality index (LI), reflecting laterality of fMRI signal in cortical motor areas was calculated. Effective connectivity, i.e. the direct or indirect causal influence that activity in one area exerts on activity of another area (Friston et al., 1993a), was inferred from fMRI data by means of dynamic causal modelling (DCM). Due to relatively high inter-individual variance, neither iTBS nor cTBS was significantly different from control TBS in terms of average behavioural (or electrophysiological) changes over the group of patients. However, beneficial effects of iTBS over the ipsilesional hemisphere were predicted by a unilateral fMRI activation pattern during movements of the affected hand and by the integrity of the cortical motor network. The more pronounced the promoting influence from the ipsilesional supplementary motor area (SMA) onto ipsilesional M1 and the more pronounced the inhibitory effect originating from ipsilesional M1 onto contralesional M1, the better was the behavioural response to facilitatory iTBS applied to the ipsilesional hemisphere. No significant correlations were found for behavioural improvements following cTBS or behavioural changes of the unaffected hand. Taken together, Study III yielded promising results indicating that laterality of fMRI signal and integrity of the motor network architecture constitute promising predictors for response to iTBS. In patients in whom the connectivity pattern of the ipsilesional motor network resembled physiological network connectivity patterns (i.e. preserved inhibition of the contralesional hemisphere and supportive role of the SMA of the ipsilesional hemisphere), beneficial effects of iTBS over the ipsilesional hemisphere could be observed. In contrast, patients with severely disturbed motor networks did not respond to iTBS or even deteriorated

TorsinA and the Pathophysiology of DYT1 Dystonia

The goal of my dissertation work was to examine the systems biology of torsinA, a DYT1 dystonia-associated protein, by using rodent model systems. TorsinA is a putative ATPase associated with a variety of cellular activities (AAA+). Deletion of glutamic acid residue 302/303 in TOR1A is causally associated with many cases of early-onset primary dystonia. In our work, transient forebrain ischemia and sciatic nerve transection were used as central and peripheral neural perturbations, respectively, to gain insight into the in vivo role(s) of torsinA. Moreover, transgenic mouse models that overexpress either human mutant torsinA (hMT) or wild-type torsinA (hWT) were used to analyze the behavioral, morphological, neurochemical, and brain metabolical consequences of increased mutant torsinA burden. After transient forebrain ischemia and sciatic nerve transection, torsinA expression levels were temporally increased in both the central and peripheral nervous systems. In the hippocampus and dorsal root ganglion, increased torsinA immunoreactivity was found located in neuronal populations such as projection neurons, interneurons, and ganglion cells, and in glial elements such as reactive astrocytes and satellite cells. These results suggest that torsinA participates in the response of neural tissue to central and peripheral insults, and that limited recruitment of intact functional torsinA might contribute to the onset of DYT1 dystonia in TOR1A ΔGAG mutation carriers. The striking induction of torsinA in astrocytes and satellite cells points to the potential involvement of glial elements in the pathobiology of DYT1 dystonia. In the DYT1 transgenic mice, mutant torsinA burden resulted in prolonged traversal times and more slips on a raised-beam task; widened hind-base width; increased dopamine turnover in the striatum, and a shift in brain energy demand from the basal ganglia to olivocerebellar pathways. However, no morphological alterations were detected in the mutant mice with either light or electron microscopy. Our neurochemical findings in DYT1 transgenic mice are compatible with previous postmortem neurochemical studies of human DYT1 dystonia. Increased striatal dopamine turnover in torsinA mutant mice suggests that the nigrostriatal pathway may be a site of functional neuropathology in DYT1 dystonia. The relatively attenuated energy demand in basal ganglion output regions may be a manifestation of a primary functional abnormality in the nigrostriatal system of DYT1 mutation carriers and the relatively elevated energy demand in cerebellar cortex might be a compensatory response to dysfunction of the basal ganglia

Detour pathways of descending motor systems

The motor cortex makes a substantial contribution to contralateral limb function via the corticospinal tract (CST). The extent to which the motor cortex influences ipsilateral limb function is less clear. Interest in ipsilateral cortical control stems from studies of stroke survivors, demonstrating increased activation of the ipsilateral motor cortex during movement of the affected limb. This raises the possibility that ipsilateral pathways contribute to recovery of function following damage to the contralateral CST. The overarching aim of this thesis was to extend the knowledge of neural systems that might mediate ipsilateral actions of the motor cortex, both under normal circumstances and after stroke. In rodent models of stroke, there is evidence that CST axons originating from the non-ischaemic hemisphere sprout into the denervated (ipsilateral) side of the spinal cord, and the extent of sprouting correlates with the degree of motor recovery. However, it is yet to be confirmed whether the CST from the nonischaemic hemisphere establishes new terminals in the denervated (ipsilateral) side of the spinal cord to replace connections lost after stroke. Hence, the first major aim of this thesis was to assess for CST terminal remodelling between the non-ischaemic hemisphere and the denervated (ipsilateral) side of the cervical spinal cord following recovery from experimental stroke in the rat. Rats underwent 60 min middle cerebral artery occlusion (MCAo) or sham occlusion surgery. Behavioural testing was conducted prior to MCAo and postoperatively for 28 days to monitor functional deficit and recovery. At day 28, the anterograde tracer cholera toxin b (CTb) subunit was injected into the forelimb motor cortex of the non-ischaemic hemisphere. Spinal sections containing anterogradely labelled terminals were reacted with antibodies against CTb and immunoreactive terminals were quantified. MCAo was associated with loss of approximately 35% of CST axons originating from the ischaemic hemisphere and infarcts were localised to subcortical structures. Rats exhibited sensorimotor deficits in the early phase after MCAo but recovered over time such that there were no significant differences in sensorimotor performances between shamoperated and MCAo rats at post-operative day 28. Despite functional recovery demonstrated by MCAo rats, the number of CTb-labelled terminals in the II cervical spinal cord originating from the non-ischaemic hemisphere was not altered compared to shams. The results of this first study suggest that after subcortical stroke, the motor cortex from the non-ischaemic hemisphere does not contribute to recovery of the affected limb via increasing its direct CST connections to the denervated (ipsilateral) side of the spinal cord. If the motor cortex from the non-ischaemic hemisphere does take over control of ipsilateral spinal circuitry after stroke, it likely utilises an indirect route. In the intact animal, a number of indirect routes via which the motor cortex may gain access to ipsilateral spinal circuitry have been identified. These pathways are complex and involve intercalated neurons located in the brainstem and contralateral spinal cord. However, there are numerous putative indirect routes which have yet to be investigated. One such route involves contralaterally descending CST axons targeting spinal commissural interneurons (CINs), which in turn would either mono- or polysynaptically affect motor neurons on the opposite side of the spinal cord. CINs are a heterogeneous population of cells important for inter-limb coordination. Despite the importance of CINs to locomotion and their potential role in providing the motor cortex indirect access to ipsilateral spinal circuits, supraspinal input to CINs is poorly defined. Hence, the second major aim of this thesis was to characterise contacts to CINs from different supraspinal sources (the CST and reticulospinal tract (ReST)) in the cervical spinal cord of the intact rat. The CINs included i) those that issue longrange axonal projections to lumbar segments, termed long-descending propriospinal neurons (LDPNs), and ii) those that issue short-range axonal projections confined to a single segment, termed intrasegmental CINs. Axons were labelled anterogradely by injecting CTb into the forelimb motor cortex or medial longitudinal fasciculus (MLF), to label CST and ReST axons, respectively. Fluorogold (FG) was injected unilaterally into segments L1/L2 or C3/C4 in order to retrogradely label LDPNs or intrasegmental CINs, respectively. Spinal sections containing labelled cells and terminals were immunoreacted with various antibody combinations and were then examined with confocal microscopy. Both LDPNs and intrasegmental CINs received very few contacts from CST terminals but had significant numbers of contacts from ReST terminals. Use of vesicular glutamate and vesicular GABA transporters revealed that both cell types received approximately 80% of excitatory and 20% of inhibitory ReST contacts. III The results suggest that in the intact animal, the CST has a minimal direct influence on LDPNs and intrasegmental CINs but the ReST has a powerful direct influence. Therefore, following loss of CST axons (e.g. after stroke), the corticoreticulospinal- commissural pathway has the capacity to deliver information from the intact hemisphere to the denervated side of the spinal cord