Role of the GLUT1 Glucose Transporter in Postnatal CNS Angiogenesis and Blood-Brain Barrier Integrity.

RATIONALE: Endothelial cells (ECs) are highly glycolytic and generate the majority of their energy via the breakdown of glucose to lactate. At the same time, a main role of ECs is to allow the transport of glucose to the surrounding tissues. GLUT1 (glucose transporter isoform 1/Slc2a1) is highly expressed in ECs of the central nervous system (CNS) and is often implicated in blood-brain barrier (BBB) dysfunction, but whether and how GLUT1 controls EC metabolism and function is poorly understood. OBJECTIVE: We evaluated the role of GLUT1 in endothelial metabolism and function during postnatal CNS development as well as at the adult BBB. METHODS AND RESULTS: Inhibition of GLUT1 decreases EC glucose uptake and glycolysis, leading to energy depletion and the activation of the cellular energy sensor AMPK (AMP-activated protein kinase), and decreases EC proliferation without affecting migration. Deletion of GLUT1 from the developing postnatal retinal endothelium reduces retinal EC proliferation and lowers vascular outgrowth, without affecting the number of tip cells. In contrast, in the brain, we observed a lower number of tip cells in addition to reduced brain EC proliferation, indicating that within the CNS, organotypic differences in EC metabolism exist. Interestingly, when ECs become quiescent, endothelial glycolysis is repressed, and GLUT1 expression increases in a Notch-dependent fashion. GLUT1 deletion from quiescent adult ECs leads to severe seizures, accompanied by neuronal loss and CNS inflammation. Strikingly, this does not coincide with BBB leakiness, altered expression of genes crucial for BBB barrier functioning nor reduced vascular function. Instead, we found a selective activation of inflammatory and extracellular matrix related gene sets. CONCLUSIONS: GLUT1 is the main glucose transporter in ECs and becomes uncoupled from glycolysis during quiescence in a Notch-dependent manner. It is crucial for developmental CNS angiogenesis and adult CNS homeostasis but does not affect BBB barrier function.

Regional changes in the type 1 cannabinoid receptor are associated with cognitive dysfunction in Parkinson's disease.

PURPOSE: The endocannabinoid system plays a regulatory role in a number of physiological functions, including motor control but also mood, emotion, and cognition. A number of preclinical studies in Parkinson's disease (PD) models demonstrated that modulating the type 1 cannabinoid receptor (CB1R) may improve motor symptoms and components of cognitive processing. However, the relation between CB1R, cognitive decline and behavioral symptoms has not been investigated in PD patients so far. The aim of this study was to examine whether CB1R availability is associated with measures of cognitive and behavioral function in PD patients. METHODS: Thirty-eight PD patients and ten age- and gender-matched controls underwent a [18F]MK-9470 PET scan to assess CB1R availability, as well as volumetric MR imaging. Neuropsychological symptoms were evaluated using an extensive cognitive and behavioral battery covering the five cognitive domains, depression, anxiety, apathy, and psychiatric complications, and were correlated to CB1R availability using vowel-wise regression analysis (P < 0.05, corrected for familywise error). RESULTS: PD patients with poorer performance in episodic memory, executive functioning, speed and mental flexibility (range P 0.003-0.03) showed lower CB1R availability in predominantly the midcingulate cortex and middle to superior frontal gyrus (Tpeak-level > 4.0). Also, PD patients with more severe visuospatial dysfunction showed decreased CB1R availability in the precuneus, midcingulate, supplementary motor cortex, inferior orbitofrontal gyrus and thalamus (Tpeak-level = 5.5). These correlations were not related to cortical gray matter atrophy. No relationship was found between CB1R availability and mood or behavioral symptom scores. CONCLUSIONS: Decreased CB1R availability in the prefrontal and midcingulate cortex in PD patients is strongly correlated with disturbances in executive functioning, episodic memory, and visuospatial functioning. Further investigation of regional CB1R expression in groups of PD patients with mild cognitive impairment or dementia is warranted in order to further investigate the role of CB1R expression in different levels of cognitive impairment in PD.

Effects of alcohol exposure on the glutamatergic system: a combined longitudinal 18 F-FPEB and 1 H-MRS study in rats.

In a longitudinal rat model of alcohol consumption, we showed that exposure to alcohol decreased the concentration of glutamate in the prefrontal cortex, whereas a normalization occurred during abstinence. 18F-FPEB PET scans revealed that pre-exposure mGluR5 availability in the nucleus accumbens was associated with future alcohol preference. Finally, alcohol exposure induced a decrease in mGluR5 availability in the bilateral hippocampus and amygdala compared with baseline, and in the hippocampus and striatum compared with saccharin (Figure).

Positron emission tomography imaging of cerebral glucose metabolism and type 1 cannabinoid receptor availability during temporal lobe epileptogenesis in the amygdala kindling model in rhesus monkeys.

OBJECTIVE: We investigated changes in the endocannabinoid system and glucose metabolism during temporal lobe epileptogenesis. METHODS: Because it is rarely possible to study epileptogenesis in humans, we applied the electrical amygdala kindling model in nonhuman primates to image longitudinal changes in type 1 cannabinoid receptor (CB1R) binding and cerebral glucose metabolism. Two rhesus monkeys received [18 F]-MK-9470 and fluorodeoxyglucose-positron emission tomography ([18 F]-FDG -PET) scans in each of the 4 kindling stages to quantify relative changes over time of CB1R binding and cerebral glucose metabolism in vivo. We constructed z-score images relative to a control group (n = 8), and considered only those changes measured in both kindled animals by calculating the binary conjunction image per kindling stage. RESULTS: The seizure-onset zone exhibited an increased CB1R binding and a decreased glucose metabolism, which both aggravated gradually in extent and intensity throughout kindling. The ipsilateral thalamus and insula showed hypometabolism that coincided with an increase and a decrease in CB1R binding, respectively. These changes also became gradually more severe throughout kindling and overlapped with ictal perfusion changes during the final stage of amygdala kindling, with hyperperfusion in the ipsilateral thalamus and hypoperfusion in the ipsilateral insula. SIGNIFICANCE: The observed changes in CB1R binding may reflect a combination of a protective mechanism of neurons against seizure activity that becomes stronger over time to combat more severe seizures, and on the other hand, a process of epileptogenesis that facilitates seizure activity and generalization, depending on the cell type involved in those specific regions. This study provides unique evidence that the CB1R is dynamically and progressively involved from the start of mesial temporal lobe epileptogenesis.

Quantification of TSPO overexpression in a rat model of local neuroinflammation induced by intracerebral injection of LPS by the use of [(18)F]DPA-714 PET.

PURPOSE: [(18)F]DPA-714 is a radiotracer with high affinity for TSPO. We have characterized the kinetics of [(18)F]DPA-714 in rat brain and evaluated its ability to quantify TSPO expression with PET using a neuroinflammation model induced by unilateral intracerebral injection of lipopolysaccharide (LPS). METHODS: Dynamic small-animal PET scans with [(18)F]DPA-714 were performed in Wistar rats on a FOCUS-220 system for up to 3 h. Both plasma and perfused brain homogenates were analysed using HPLC to quantify radiometabolites. Full kinetic modelling of [(18)F]DPA-714 brain uptake was performed using a metabolite-corrected arterial plasma input function. Binding potential (BPND) calculated as the distribution volume ratio minus one (DVR-1) between affected and healthy brain tissue was used as the outcome measure and evaluated against reference tissue models. RESULTS: The percentage of intact [(18)F]DPA-714 in arterial plasma samples was 92 ± 4 % at 10 min, 75 ± 8 % at 40 min and 52 ± 6 % at 180 min. The radiometabolite fraction in brain was negligible (<3 % at 30 min). Among the models investigated, the reversible two-tissue (2T) compartment model best described [(18)F]DPA-714 brain kinetics. BPND values obtained with a simplified and a multilinear reference tissue model (SRTM, MRTM) using the contralateral striatum as the reference region correlated well (Spearman's r = 0.96, p ≤ 0.003) with 2T BPND values calculated as DVR-1, and showed comparable bias (bias range 17.94 %, 20.32 %). Analysis of stability over time suggested that the acquisition time should be at least 90 min for SRTM and MRTM. CONCLUSION: Quantification of [(18)F]DPA-714 binding to TSPO with full kinetic modelling is feasible using a 2T model. SRTM and MRTM can be suggested as reasonable substitutes with the contralateral striatum as the reference region and a scan duration of at least 90 min. However, selection of the reference region depends on the disease model used.

Kinetic modeling and long-term test-retest reproducibility of the mGluR5 PET tracer 18F-FPEB in human brain.

(18)F-FPEB is a promising PET tracer for studying the metabotropic glutamate subtype 5 receptor (mGluR5) expression in neuropsychiatric disorders. To assess the potential of (18)F-FPEB for longitudinal mGluR5 evaluation in patient studies, we evaluated the long-term test-retest reproducibility using various kinetic models in the human brain. Nine healthy volunteers underwent consecutive scans separated by a 6-month period. Dynamic PET was combined with arterial sampling and radiometabolite analysis. Total distribution volume (V(T)) and nondisplaceable binding potential (BP(ND)) were derived from a two-tissue compartment model without constraints (2TCM) and with constraining the K(1)/k(2) ratio to the value of either cerebellum (2TCM-CBL) or pons (2TCM-PONS). The effect of fitting different functions to the tracer parent fractions and reducing scan duration were assessed. Regional absolute test-retest variability (aTRV), coefficient of repeatability (CR) and intraclass correlation coefficient (ICC) were computed. The 2TCM-CBL showed best fits. The mean 6-month aTRV of V(T) ranged from 8 to 13% (CR < 25%) with ICC > 0.6 for all kinetic models. BPND from 2TCM-CBL with a sigmoid fit for the parent fractions showed the best reproducibility, with aTRV ≤ 7% (CR < 16%) and ICC > 0.9 in most regions. Reducing the scan duration from 90 to 60 min did not affect reproducibility. These results demonstrate for the first time that (18)F-FPEB brain PET has good long-term reproducibility, therefore validating its use to monitor mGluR5 expression in longitudinal clinical studies. We suggest a 2TCM-CBL with fitting a sigmoid function to the parent fractions to be optimal for this tracer.

Changes in cerebral CB1 receptor availability after acute and chronic alcohol abuse and monitored abstinence.

Involvement of the type 1 cannabinoid receptor (CB1R) in the effects of alcohol on the brain is supported by animal experiments, but how in vivo CB1R levels are altered in alcoholic patients is still unclear. To assess the short-time effects of a binge drinking episode on CB1R availability, 20 healthy social drinkers underwent [(18)F]MK-9470-positron emission tomography (PET) at baseline and after intravenous ethanol administration (ALC ACU). Moreover, 26 alcoholic patients underwent sequential CB1R PET after chronic heavy drinking (ALC CHR) and after 1 month of abstinence (ALC ABST). Seventeen healthy subjects served as controls. Compared with baseline, ALC ACU resulted in a global increase of CB1R availability (+15.8%). In contrast, a global decreased CB1R availability was found in ALC CHR patients (-16.1%) compared with controls, which remained unaltered after abstinence (-17.0%). Voxel-based analysis showed that ALC CHR patients had reduced CB1R availability, especially in the cerebellum and parieto-occipital cortex. After abstinence, reduced CB1R availability extended also to other areas such as the ventral striatum and mesotemporal lobe. In conclusion, whereas the acute alcohol effect is an increase in CB1R availability, chronic heavy drinking leads to reduced CB1R availability that is not reversible after 1 month of abstinence. Longer follow-up is required to differentiate whether this is a compensatory effect of repeated endocannabinoid overstimulation or an enduring trait-like feature. An enhanced CB1R signaling may offer a new therapeutic direction for treatment of the negative affective state produced by alcohol withdrawal and abstinence, which is critical for the maintenance of alcohol addiction.

Templated misfolding of Tau by prion-like seeding along neuronal connections impairs neuronal network function and associated behavioral outcomes in Tau transgenic mice.

Prion-like seeding and propagation of Tau-pathology have been demonstrated experimentally and may underlie the stereotyped progression of neurodegenerative Tauopathies. However, the involvement of templated misfolding of Tau in neuronal network dysfunction and behavioral outcomes remains to be explored in detail. Here we analyzed the repercussions of prion-like spreading of Tau-pathology via neuronal connections on neuronal network function in TauP301S transgenic mice. Spontaneous and GABA(A)R-antagonist-induced neuronal network activity were affected following templated Tau-misfolding using synthetic preformed Tau fibrils in cultured primary neurons. Electrophysiological analysis in organotypic hippocampal slices of Tau transgenic mice demonstrated impaired synaptic transmission and impaired long-term potentiation following Tau-seed induced Tau-aggregation. Intracerebral injection of Tau-seeds in TauP301S mice, caused prion-like spreading of Tau-pathology through functionally connected neuroanatomical pathways. Electrophysiological analysis revealed impaired synaptic plasticity in hippocampal CA1 region 6 months after Tau-seeding in entorhinal cortex (EC). Furthermore, templated Tau aggregation impaired cognitive function, measured in the object recognition test 6 months post-seeding. In contrast, Tau-seeding in basal ganglia and subsequent spreading through functionally connected neuronal networks involved in motor control, resulted in motoric deficits reflected in clasping and impaired inverted grid hanging, not significantly affected following Tau-seeding in EC. Immunostaining, biochemical and electron microscopic analysis in the different models suggested early pathological forms of Tau, including Tau-oligomers, rather than fully mature neurofibrillary tangles (NFTs) as culprits of neuronal dysfunction. We here demonstrate for the first time using in vitro, ex vivo and in vivo models, that prion-like spreading of Tau-misfolding by Tau seeds, along unique neuronal connections, causes neuronal network dysfunction and associated behavioral dysfunction. Our data highlight the potential relevance of this mechanism in the symptomatic progression in Tauopathies. We furthermore demonstrate that the initial site of Tau-seeding thereby determines the behavioral outcome, potentially underlying the observed heterogeneity in (familial) Tauopathies, including in TauP301 mutants.

Ictal perfusion changes associated with seizure progression in the amygdala kindling model in the rhesus monkey.

OBJECTIVE: Amygdala kindling is a widely used animal model for studying mesial temporal lobe epileptogenesis. In the macaque monkey, electrical amygdala kindling develops slowly and provides an opportunity for investigating ictal perfusion changes during epileptogenesis. METHODS: Two rhesus monkeys were electrically kindled through chronically implanted electrodes in the right amygdala over a period of 16 and 17 months. Ictal perfusion single photon emission computed tomography (SPECT) imaging was performed during each of the four predefined clinical stages. RESULTS: Afterdischarge duration increased slowly over 477 days for monkey K and 515 days for monkey S (18 ± 8 s in stage I; 52 ± 13 s in stage IV). During this time, the animals progressed through four clinical stages ranging from interrupting ongoing behavior to bilateral convulsions. Ictal SPECT perfusion imaging showed well-localized but widely distributed regions of hyperperfusion and hypoperfusion, in both cortical and subcortical structures, at every seizure stage. A large portion of the ictal network was involved in the early stages of epileptogenesis and subsequently expanded over time as seizure severity evolved. SIGNIFICANCE: Our data indicate that the different mesial temporal lobe seizure types occur within a common network affecting several parts of the brain, and that seizure severity may be determined by seizure-induced epileptogenesis within a bihemispheric network that is implicated from the start of the process.

Small animal PET imaging of the type 1 cannabinoid receptor in a rodent model for anorexia nervosa.

PURPOSE: Several lines of evidence strongly implicate a dysfunctional endocannabinoid system (ECS) in eating disorders. Using [(18)F]MK-9470 and small animal positron emission tomography (PET), we investigated for the first time cerebral changes in type 1 cannabinoid (CB1) receptor binding in vivo in the activity-based rat model of anorexia (ABA), in comparison to distinct motor- and food-related control conditions and in relation to gender and behavioural variables. METHODS: In total, experiments were conducted on 80 Wistar rats (23 male and 57 female). Male rats were assigned to the cross-sectional conditions: ABA (n = 12) and CONTROL (n = 11), whereas female rats were divided between two settings: (1) a cross-sectional design using ABA (n = 13), CONTROL (n = 9), and two extra control conditions for each of the variables manipulated in ABA, i.e. DIET (n = 8) and WHEEL (n = 9), and (2) a longitudinal one using ABA (n = 10) and CONTROL (n = 8) studied at baseline, during the model and upon recovery. The ABA group was subjected to food restriction in the presence of a running wheel, the DIET group to food restriction without wheel, the WHEEL group to a normal diet with wheel and CONTROL animals had a normal diet and no running wheel. Parametric CB1 receptor images of each group were spatially normalized to Paxinos space and analysed voxel-wise. RESULTS: In the ABA model, absolute [(18)F]MK-9470 binding was significantly increased in all cortical and subcortical brain areas as compared to control conditions (male +67 %; female >51%, all p cluster < 6.3×10(-6)) that normalized towards baseline values after weight gain. Additionally, relative [(18)F]MK-9470 binding was increased in the hippocampus, inferior colliculus and entorhinal cortex of female ABA (+4.6%; p cluster < 1.3×10(-6)), whereas no regional differences were observed in male subjects. Again, relative [(18)F]MK-9470 binding values normalized upon weight gain. CONCLUSION: These data point to a widespread transient disturbance of the endocannabinoid transmission, specifically for CB1 receptors in the ABA model. Our data also suggest (1) gender effects on regional CB1 receptor binding in the hippocampus and (2) add further proof to the validity of the ABA model to mimic aspects of human disease.

Micro-positron emission tomography imaging of rat brain metabolism during expression of contextual conditioning.

Using (18)F-fluorodeoxyglucose microPET imaging, we investigated the neurocircuitry of contextual anxiety versus control in awake, conditioned rats (n = 7-10 per group). In addition, we imaged a group expressing cued fear. Simultaneous measurements of startle amplitude and freezing time were used to assess conditioning. To the best of our knowledge, no neuroimaging studies in conditioned rats have been conducted thus far, although visualizing and quantifying the metabolism of the intact brain in behaving animals is clearly of interest. In addition, more insight into the neurocircuitry involved in contextual anxiety may stimulate the development of new treatments for anxiety disorders. Our main finding was hypermetabolism in a cluster comprising the bed nucleus of the stria terminalis (BST) in rats expressing contextual anxiety compared with controls. Analysis of a subset of rats showing the best behavioral results (n = 5 per subgroup) confirmed this finding. We also observed hypermetabolism in the same cluster in rats expressing contextual anxiety compared with rats expressing cued fear. Our results provide novel evidence for a role of the BST in the expression of contextual anxiety.

Transient changes in the endocannabinoid system after acute and chronic ethanol exposure and abstinence in the rat: a combined PET and microdialysis study.

PURPOSE: Recent biochemical and post-mortem evidence suggests involvement of the endocannabinoid system in alcohol drinking behaviour and dependence. Using [(18)F]MK-9470 small-animal PET imaging, our primary objective was to evaluate in vivo type 1 cannabinoid receptor (CB1R) binding changes in rats subjected to several ethanol conditions: (1) at baseline, (2) after acute intraperitoneal administration of ethanol (4 g/kg) or saline, (3) after 7 days of forced chronic ethanol consumption, and (4) after abstinence for 7 and 14 days. Secondly, levels of anandamide (AEA) in the nucleus accumbens (NAcc) were investigated in the same animals using in vivo microdialysis and correlated with the changes in CB1R binding. METHODS: In total, 28 male Wistar rats were investigated. Small-animal PET was done on a FOCUS-220 tomograph with [(18)F]MK-9470. Parametric images of [(18)F]MK-9470 binding based on standard uptake values (SUV, as a measure of CB1R binding) were generated. Images were normalized to Paxinos space and analysed voxel-wise using SPM8 (p(height) = 0.005; k(ext) = 200). The AEA content was quantified using HPLC with tandem mass spectrometry detection. RESULTS: Acute ethanol administration increased relative CB1R binding in the NAcc that was positively correlated with the change in AEA levels of that region. In contrast, compared to rats at baseline, AEA levels in the NAcc were not significantly different in rats after chronic ethanol consumption or after a 14-day abstinence period. Chronic ethanol consumption decreased relative CB1R binding in the hippocampus and caudate-putamen, whereas same regions showed increased relative CB1R binding after 7 and 14 days of abstinence compared to the baseline condition. After 7 and 14 days of abstinence, relative CB1R binding additionally decreased in the orbitofrontal cortex. The magnitude of the hippocampal and frontal changes was highly correlated with daily ethanol intake. CONCLUSION: This study provides in vivo evidence that acute ethanol consumption is associated with enhanced endocannabinoid signalling in the NAcc, indicated by an increased CB1R binding and AEA content. In addition, chronic ethanol exposure leads to regional dysfunctions in CB1R levels, involving the hippocampus and caudate-putamen that are reversible within 2 weeks in this animal model.

Preclinical evaluation and quantification of [¹8F]MK-9470 as a radioligand for PET imaging of the type 1 cannabinoid receptor in rat brain.

PURPOSE: [(18)F]MK-9470 is an inverse agonist for the type 1 cannabinoid (CB1) receptor allowing its use in PET imaging. We characterized the kinetics of [(18)F]MK-9470 and evaluated its ability to quantify CB1 receptor availability in the rat brain. METHODS: Dynamic small-animal PET scans with [(18)F]MK-9470 were performed in Wistar rats on a FOCUS-220 system for up to 10 h. Both plasma and perfused brain homogenates were analysed using HPLC to quantify radiometabolites. Displacement and blocking experiments were done using cold MK-9470 and another inverse agonist, SR141716A. The distribution volume (V(T)) of [(18)F]MK-9470 was used as a quantitative measure and compared to the use of brain uptake, expressed as SUV, a simplified method of quantification. RESULTS: The percentage of intact [(18)F]MK-9470 in arterial plasma samples was 80 ± 23 % at 10 min, 38 ± 30 % at 40 min and 13 ± 14 % at 210 min. A polar radiometabolite fraction was detected in plasma and brain tissue. The brain radiometabolite concentration was uniform across the whole brain. Displacement and pretreatment studies showed that 56 % of the tracer binding was specific and reversible. V (T) values obtained with a one-tissue compartment model plus constrained radiometabolite input had good identifiability (≤10 %). Ignoring the radiometabolite contribution using a one-tissue compartment model alone, i.e. without constrained radiometabolite input, overestimated the [(18)F]MK-9470 V(T), but was correlated. A correlation between [(18)F]MK-9470 V(T) and SUV in the brain was also found (R(2) = 0.26-0.33; p ≤ 0.03). CONCLUSION: While the presence of a brain-penetrating radiometabolite fraction complicates the quantification of [(18)F]MK-9470 in the rat brain, its tracer kinetics can be modelled using a one-tissue compartment model with and without constrained radiometabolite input.

Small-animal PET imaging of the type 1 and type 2 cannabinoid receptors in a photothrombotic stroke model.

PURPOSE: Recent ex vivo and pharmacological evidence suggests involvement of the endocannabinoid system in the pathophysiology of stroke, but conflicting roles for type 1 and 2 cannabinoid receptors (CB(1) and CB(2)) have been suggested. The purpose of this study was to evaluate CB(1) and CB(2) receptor binding over time in vivo in a rat photothrombotic stroke model using PET. METHODS: CB(1) and CB(2) microPET imaging was performed at regular time-points up to 2 weeks after stroke using [(18)F]MK-9470 and [(11)C]NE40. Stroke size was measured using MRI at 9.4 T. Ex vivo validation was performed via immunostaining for CB(1) and CB(2). Immunofluorescent double stainings were also performed with markers for astrocytes (GFAP) and macrophages/microglia (CD68). RESULTS: [(18)F]MK-9470 PET showed a strong increase in CB(1) binding 24 h and 72 h after stroke in the cortex surrounding the lesion, extending to the insular cortex 24 h after surgery. These alterations were consistently confirmed by CB(1) immunohistochemical staining. [(11)C]NE40 did not show any significant differences between stroke and sham-operated animals, although staining for CB(2) revealed minor immunoreactivity at 1 and 2 weeks after stroke in this model. Both CB (1) (+) and CB (2) (+) cells showed minor immunoreactivity for CD68. CONCLUSION: Time-dependent and regionally strongly increased CB(1), but not CB(2), binding are early consequences of photothrombotic stroke. Pharmacological interventions should primarily aim at CB(1) signalling as the role of CB(2) seems minor in the acute and subacute phases of stroke.

Automated quantitative gait analysis in animal models of movement disorders.

BACKGROUND: Accurate and reproducible behavioral tests in animal models are of major importance in the development and evaluation of new therapies for central nervous system disease. In this study we investigated for the first time gait parameters of rat models for Parkinson's disease (PD), Huntington's disease (HD) and stroke using the Catwalk method, a novel automated gait analysis test. Static and dynamic gait parameters were measured in all animal models, and these data were compared to readouts of established behavioral tests, such as the cylinder test in the PD and stroke rats and the rotarod tests for the HD group. RESULTS: Hemiparkinsonian rats were generated by unilateral injection of the neurotoxin 6-hydroxydopamine in the striatum or in the medial forebrain bundle. For Huntington's disease, a transgenic rat model expressing a truncated huntingtin fragment with multiple CAG repeats was used. Thirdly, a stroke model was generated by a photothrombotic induced infarct in the right sensorimotor cortex. We found that multiple gait parameters were significantly altered in all three disease models compared to their respective controls. Behavioural deficits could be efficiently measured using the cylinder test in the PD and stroke animals, and in the case of the PD model, the deficits in gait essentially confirmed results obtained by the cylinder test. However, in the HD model and the stroke model the Catwalk analysis proved more sensitive than the rotarod test and also added new and more detailed information on specific gait parameters. CONCLUSION: The automated quantitative gait analysis test may be a useful tool to study both motor impairment and recovery associated with various neurological motor disorders.

The effect of anaesthesia on [(18)F]MK-9470 binding to the type 1 cannabinoid receptor in the rat brain.

PURPOSE: Small animal PET can be applied to study molecular processes in animal models of a variety of human diseases. In order to keep the animals in a restricted position during imaging, anaesthesia is in many instances inevitable. Using small animal PET and ex vivo autoradiography, we examined the influence of pentobarbital and isoflurane anaesthesia on the rat brain uptake of [(18)F]MK-9470, a radioligand for the type 1 cannabinoid receptor. METHODS: PET imaging was performed on adult Wistar rats under pentobarbital (n = 6) and isoflurane anaesthesia (n = 7), and under control conditions (free moving during tracer uptake, n = 8). Parametric PET images were generated, anatomically standardized and analysed by voxel-based Statistical Parametric Mapping and a predefined volume of interest approach. Immediately after in vivo PET, brains were processed for ex vivo autoradiography using manually placed regions of interest. An extra group (n = 6) was included ex vivo, in which animals were intravenously injected without the use of anaesthetics. RESULTS: Using in vivo and ex vivo molecular imaging techniques, no significant changes in absolute [(18)F]MK-9470 uptake were present in the brain of pentobarbital and isoflurane rats as compared to control conditions. Relative [(18)F]MK-9470 uptake PET values obtained applying global scaling were, however, decreased in the cortex under both anaesthetics (pentobarbital: -13.3+/-1.4%; isoflurane -8.7 +/- 3.1%), while an increase was seen in the cerebellum by 13.5 +/- 4.0% and 13.9 +/- 4.1% under pentobarbital and isoflurane, respectively. Ex vivo results were in agreement with in vivo findings. CONCLUSION: These findings suggest a similar, regionally specific interference of pentobarbital and isoflurane anaesthesia with in vivo CB1 receptor imaging using [(18)F]MK-9470.

Type 1 cannabinoid receptor mapping with [18F]MK-9470 PET in the rat brain after quinolinic acid lesion: a comparison to dopamine receptors and glucose metabolism.

PURPOSE: Several lines of evidence imply early alterations in metabolic, dopaminergic and endocannabinoid neurotransmission in Huntington's disease (HD). Using [18F]MK-9470 and small animal PET, we investigated cerebral changes in type 1 cannabinoid (CB1) receptor binding in the quinolinic acid (QA) rat model of HD in relation to glucose metabolism, dopamine D2 receptor availability and amphetamine-induced turning behaviour. METHODS: Twenty-one Wistar rats (11 QA and 10 shams) were investigated. Small animal PET acquisitions were conducted on a Focus 220 with approximately 18 MBq of [18F]MK-9470, [18F]FDG and [11C]raclopride. Relative glucose metabolism and parametric CB1 receptor and D2 binding images were anatomically standardized to Paxinos space and analysed voxel-wise using Statistical Parametric Mapping (SPM2). RESULTS: In the QA model, [18F]MK-9470 uptake, glucose metabolism and D2 receptor binding were reduced in the ipsilateral caudate-putamen by 7, 35 and 77%, respectively (all p<2.10(-5)), while an increase for these markers was observed on the contralateral side (>5%, all p<7.10(-4)). [18F]MK-9470 binding was also increased in the cerebellum (p=2.10(-5)), where it was inversely correlated to the number of ipsiversive turnings (p=7.10(-6)), suggesting that CB1 receptor upregulation in the cerebellum is related to a better functional outcome. Additionally, glucose metabolism was relatively increased in the contralateral hippocampus, thalamus and sensorimotor cortex (p=1.10(-6)). CONCLUSION: These data point to in vivo changes in endocannabinoid transmission, specifically for CB1 receptors in the QA model, with involvement of the caudate-putamen, but also distant regions of the motor circuitry, including the cerebellum. These data also indicate the occurrence of functional plasticity on metabolism, D2 and CB1 neurotransmission in the contralateral hemisphere.

Influence of chronic bromocriptine and levodopa administration on cerebral type 1 cannabinoid receptor binding.

OBJECTIVES: The endocannabinoid system is an important modulatory system in the brain. Complex interactions with brain dopaminergic circuits have been demonstrated. The aim of this study was to investigate the in vivo effect of the commonly used antiparkinsonian drugs, levodopa (L-DOPA) and bromocriptine, on type 1 cannabinoid (CB1) receptors, using the PET radioligand [(18)F]MK-9470. EXPERIMENTAL APPROACH: Seventeen female Wistar rats were studied at baseline and after chronic exposure to either L-DOPA (6 mg/kg/day with 1.5 mg/kg/day carbidopa; n = 6), bromocriptine (4 mg/kg/day; n = 5), or saline (n = 6). [(18)F]MK-9470 binding was assessed in vivo using small animal PET imaging. [(18)F]MK-9470 parametric images were generated, anatomically standardized to Paxinos space and analyzed by voxel-based statistical parametric mapping (SPM2) and a predefined volume-of-interest (VOI) approach. RESULTS: In a 2 x 2 analysis design (condition vs. treatment), no significant changes in absolute or relative [(18)F]MK-9470 binding were present upon chronic exposure to L-DOPA or bromocriptine as compared to saline treatment. The post hoc comparison of chronic scans to baseline within each treatment modality showed regional increases in relative [(18)F]MK-9470 binding in the thalamus (peak average value +6.3%) and in the sensorimotor cortex and hippocampus (peak average value +10.2%) after bromocriptine exposure, while no changes were found for L-DOPA. CONCLUSION: Chronic administration of L-DOPA and bromocriptine at the applied doses does not produce major cerebral changes in in vivo cannabinoid CB1 receptor binding of [(18)F]MK-9470 in the rat brain. These results also suggest that similar chronic L-DOPA and bromocriptine usage is unlikely to interfere with human PET imaging in healthy conditions using this radioligand.

Morphologic and functional changes in the unilateral 6-hydroxydopamine lesion rat model for Parkinson's disease discerned with microSPECT and quantitative MRI.

OBJECT: In the present study, we aimed to evaluate the impact of neurodegeneration of the nigrostriatal tract in a rodent model of Parkinson's disease on the different MR contrasts (T(2), T(1), CBF and CBV) measured in the striatum. MATERIAL AND METHODS: Animals were injected with 6-hydroxydopamine (6OHDA) in the substantia nigra resulting in massive loss of nigrostriatal neurons and hence dopamine depletion in the ipsilateral striatum. Using 7T MRI imaging, we have quantified T(2), T(1), CBF and CBV in the striata of 6OHDA and control rats. To validate the lesion size, behavioral testing, dopamine transporter muSPECT and tyrosine hydroxylase staining were performed. RESULTS: No significant differences were demonstrated in the absolute MRI values between 6OHDA animals and controls; however, 6OHDA animals showed significant striatal asymmetry for all MRI parameters in contrast to controls. CONCLUSIONS: These PD-related asymmetry ratios might be the result of counteracting changes in both intact and affected striatum and allowed us to diagnose PD lesions. As lateralization is known to occur also in PD patients and might be expected in transgenic PD models as well, we propose that MR-derived asymmetry ratios in the striatum might be a useful tool for in vivo phenotyping of animal models of PD.

Non-invasive characterization of amyotrophic lateral sclerosis in a hTDP-43A315T mouse model: A PET-MR study.

Currently TAR DNA binding protein 43 (TDP-43) pathology, underlying Amyotrophic Lateral Sclerosis (ALS), remains poorly understood which hinders both clinical diagnosis and drug discovery efforts. To better comprehend the disease pathophysiology, positron emission tomography (PET) and multi-parametric magnetic resonance imaging (mp-MRI) provide a non-invasive mode to investigate molecular, structural, and neurochemical abnormalities in vivo. For the first time, we report the findings of a longitudinal PET-MR study in the TDP-43A315T ALS mouse model, investigating disease-related changes in the mouse brain. 2-deoxy-2-[18F]fluoro-D-glucose [18F]FDG PET showed significantly lowered glucose metabolism in the motor and somatosensory cortices of TDP-43A315T mice whereas metabolism was elevated in the region covering the bilateral substantia nigra, reticular and amygdaloid nucleus between 3 and 7 months of age, as compared to non-transgenic controls. MR spectroscopy data showed significant changes in glutamate + glutamine (Glx) and choline levels in the motor cortex and hindbrain of TDP-43A315T mice compared to controls. Cerebral blood flow (CBF) measurements, using an arterial spin labelling approach, showed no significant age- or group-dependent changes in brain perfusion. Diffusion MRI indices demonstrated transient changes in different motor areas of the brain in TDP-43A315T mice around 14 months of age. Cytoplasmic TDP-43 proteinaceous inclusions were observed in the brains of symptomatic, 18-month-old mice, but not in non-symptomatic transgenic or wild-type mice. Our results reveal that disease- and age-related functional and neurochemical alterations, together with limited structural changes, occur in specific brain regions of transgenic TDP-43A315T mice, as compared to their healthy counterparts. Altogether these findings shed new light on TDP-43A315T disease pathogenesis and may prove useful for clinical management of ALS.