Florey Department of Neuroscience and Mental Health - Research Publications
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ItemMGLU5 receptors are necessary for extinction of drug associated cues and contexts(Wiley, 2017-08-01)Drug-associated cues and contexts are strong predictors of relapse. We used complex behavioural preparations to examine whether extinction of such cues reduces their capacity to trigger drug-seeking. We also examined whether the mGlu5 receptor is necessary for extinction learning. In Experiment 1, rats were trained to lever press for cocaine. Once stable responding was established, the context was extinguished by replacing the rats in the chambers, but with no opportunity to respond (levers were retracted). Control group remained in their home cage. An mGlu5 receptor negative allosteric modulator (MTEP) or vehicle was administered immediately after context extinction sessions. During subsequent drug-induced reinstatement, rats responded less if they had received context extinction; however, this effect was attenuated where MTEP had been applied. In Experiment 2, rats were trained to lever press for cocaine, now paired with a cue light. To extinguish the cue, half of the rats were placed in the chambers and given non-reinforced presentations of the cue, but with the levers retracted. Control rats remained in home cage. All rats received either MTEP or vehicle 20 minutes prior. Cue-induced reinstatement was tested the following day by re-pairing the lever with the light. Rats gave fewer drug-seeking responses following cue extinction. This effect was attenuated by MTEP. Experiment 3 followed the same protocol as Experiment 2, except that a positive allosteric modulator CDPPB or vehicle was administered 20 minutes before CS extinction. At reinstatement, cue-elicited cocaine seeking was lower for the animals that had previously been administered CDPPB, regardless of extinction condition. This study highlights the important role cues and contexts play in driving drug-seeking behaviour during reinstatement. It also shows that mGlu 5 signalling is necessary for extinction of drug-cue associations, and that mGlu5 positive allosteric modulators are promising targets for treating cocaine addiction.
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ItemBioenergetic failure, mitochondrial dysfunction and mitophagy(SPRINGER INTERNATIONAL PUBLISHING AG, 2015-06-12)
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ItemNo Preview AvailableEvaluating retinal biomarkers in a mouse model of Parkinson's disease(Association for Research in Vision and Ophthalmology, 2019-07-01)Purpose : The retina, an accessible outpouching of the central nervous system, may manifest cortical changes that occur with Parkinson’s disease (PD), lending itself as a potential biomarker. PD is characterised by reduced dopamine levels, a neurotransmitter found in amacrine cells. Human PD patients have also shown structural changes in the outer retina. This work aims to determine if retinal function and structure are altered in a murine model of PD and whether deficits can be ameliorated with L-DOPA treatment. Methods : A PD model was induced in adult C57BL6/J mice using MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 4x i.p. injections, 20mg/kg) and vehicle control and examined at day 21 and 45. Another MPTP group was administered L-DOPA (L-3,4-dihydroxyphenylalanine 0.2 mg/ml) or control in their drinking water and assessed at day 45 (n=12–15/group). In ketamine:xylazine anaesthetised (80:10mg/kg) mice full-field dark- and light-adapted electroretinography (ERG) was assessed to target dopamine-related responses. Optical coherence tomography (OCT) was used to quantify thickness of retinal layers. Retinal and cortical tissue were collected for immunohistochemical assessment of changes in tyrosine hydroxylase (TH)and imaged using confocal microscopy. Data (mean±SEM) were compared using unpaired ANOVA and t-tests as appropriate. Results : At day 21 no retinal changes were found. At day 45 dark and light adapted ERGs showed slower amacrine cell responses (oscillatory potential, p<0.05), a finding which reversed with L-DOPA treatment (p<0.05). Other components of the ERG were unchanged. TH staining showed a trend towards decreased retinal levels in MPTP mice but this did not reach significance (p=0.10). Reduced levels of TH were found in the ventral hippocampus of MPTP mice compared with control (p<0.05). OCT revealed thinning of the outer plexiform layer at day 45, and the L-DOPA group exhibited a thinning of the outer nuclear layer (p<0.05). Conclusions : This study shows for the first time that the MPTP model recapitulates key dopaminergic changes previously reported in humans. In particular, electroretinographic changes that correspond with dopaminergic retinal cells occur in the Parkinson’s model and reverse with therapeutic treatment. Structural thinning of the outer retinal layers also occur, which parallels some human findings. This work paves the way for retinal measures as preclinical screening tools in drug development.
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ItemNo Preview AvailableMETHYLOME-WIDE ASSOCIATION STUDIES FOR MAJOR DEPRESSIVE DISORDER IN BLOOD OVERLAP WITH METHYLATION RESULTS FROM BRAIN AND LARGE-SCALE GWAS(ELSEVIER SCIENCE BV, 2019-01-01)Background: Epigenetic modifications such as DNA methy- lation provide stability and diversity to the cellular phenotype and aberrant methylation has been implicated in processes underlying psychiatric disorders. Therefore, studies combining DNA methylation and genotype information provide a promis- ing approach to study disorders where genotype information alone has failed to reveal the full etiology. Methods: We applied an optimized MBD-seq protocol to assay the complete CpG methylome in cases with Major Depressive Disorder (MDD) and controls using blood samples (N=1,132) from Netherlands Study of Depression and Anxiety and brain samples (N=64) from the Victorian Brain Bank Network. Data were analyzed with RaMWAS, a novel Biocon- ductor package specifically designed for Methylome-Wide Association Studies (MWAS). To study the overlap between top MWAS findings in blood and brain, we used a permutation based enrichment test (shiftR) that accounted for the depen- dency between adjacent CpG sites. Furthermore, we utilized the methylation data in combination with existing genotype information from the same individuals in a MWAS of CpGs created or destroyed by SNPs. Next, we tested whether top results from this CpG-SNP MWAS overlapped with recent large- scale GWAS to identify robust associations with genomic loci of importance for MDD etiology Results: The MWAS in blood identified five methylome- wide significant sites (P o 5 10-8) from three distinct loci and 472 nominally significant (P o 1 10-5) CpG sites. To study the robustness of the overall MWAS signal, we used an “in-sample” replication based on k-fold cross validation. Results showed that the findings replicated (P = 4.0 10- 10). When we compared blood and brain we found that top blood MWAS findings were significantly enriched for top CpGs in the brain MWAS (P = 5.4 10-3). The MWAS of CpG-SNPs identified 32 nominally significant sites and in- sample replication showed that the signal replicated (P = 2.2 10-8). Finally, the top CpG-SNP MWAS showed a consistent trend towards enrichment in all tested large- scale GWAS, with the most significant enrichment observed for the 23andMe study (P = 4.9 10-3). This overlap involved 55 genes that were overrepresented (P o 0.01) in 12 level-5 gene ontology terms, of which a major portion was related to neuronal regulation, function and development. Discussion: This work involves the largest MWAS for MDD performed to date. Our integrated analysis with brain tissue, genotype information and GWAS results highlighted biological functions of potential value for MDD etiology. Part of the associated methylation marks in blood overlapped with MWAS finding in brain. As blood can easily be collected in a clinical setting, these loci may be of direct value as potential diagnostic biomarkers for MDD.
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ItemCerebrovascular disease, Alzheimer's disease biomarkers and longitudinal cognitive decline(WILEY-BLACKWELL, 2016-06-01)
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ItemBottom-of-sulcus dysplasia: the role of F-18-FDG PET in identifying a focal surgically remedial epileptic lesion(SPRINGERNATURE, 2020-12-15)
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ItemEarly detection of amyloid load using F-18-florbetaben PET(BMC, 2021-03-27)BACKGROUND: A low amount and extent of Aβ deposition at early stages of Alzheimer's disease (AD) may limit the use of previously developed pathology-proven composite SUVR cutoffs. This study aims to characterize the population with earliest abnormal Aβ accumulation using 18F-florbetaben PET. Quantitative thresholds for the early (SUVRearly) and established (SUVRestab) Aβ deposition were developed, and the topography of early Aβ deposition was assessed. Subsequently, Aβ accumulation over time, progression from mild cognitive impairment (MCI) to AD dementia, and tau deposition were assessed in subjects with early and established Aβ deposition. METHODS: The study population consisted of 686 subjects (n = 287 (cognitively normal healthy controls), n = 166 (subjects with subjective cognitive decline (SCD)), n = 129 (subjects with MCI), and n = 101 (subjects with AD dementia)). Three categories in the Aβ-deposition continuum were defined based on the developed SUVR cutoffs: Aβ-negative subjects, subjects with early Aβ deposition ("gray zone"), and subjects with established Aβ pathology. RESULTS: SUVR using the whole cerebellum as the reference region and centiloid (CL) cutoffs for early and established amyloid pathology were 1.10 (13.5 CL) and 1.24 (35.7 CL), respectively. Cingulate cortices and precuneus, frontal, and inferior lateral temporal cortices were the regions showing the initial pathological tracer retention. Subjects in the "gray zone" or with established Aβ pathology accumulated more amyloid over time than Aβ-negative subjects. After a 4-year clinical follow-up, none of the Aβ-negative or the gray zone subjects progressed to AD dementia while 91% of the MCI subjects with established Aβ pathology progressed. Tau deposition was infrequent in those subjects without established Aβ pathology. CONCLUSIONS: This study supports the utility of using two cutoffs for amyloid PET abnormality defining a "gray zone": a lower cutoff of 13.5 CL indicating emerging Aβ pathology and a higher cutoff of 35.7 CL where amyloid burden levels correspond to established neuropathology findings. These cutoffs define a subset of subjects characterized by pre-AD dementia levels of amyloid burden that precede other biomarkers such as tau deposition or clinical symptoms and accelerated amyloid accumulation. The determination of different amyloid loads, particularly low amyloid levels, is useful in determining who will eventually progress to dementia. Quantitation of amyloid provides a sensitive measure in these low-load cases and may help to identify a group of subjects most likely to benefit from intervention. TRIAL REGISTRATION: Data used in this manuscript belong to clinical trials registered in ClinicalTrials.gov ( NCT00928304 , NCT00750282 , NCT01138111 , NCT02854033 ) and EudraCT (2014-000798-38).
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ItemNo Preview AvailableDescending forebrain projections targeting respiratory control areas in the midbrain and brainstem of rats(WILEY, 2020-04-01)We locally microinjected the conventional retrograde tracer cholera toxin subunit B (CT‐B, 100–150nL) into the BötC, KF, the pre‐Bötzinger complex (pre‐BötC), the midline raphé nuclei and the midbrain periaqueductal gray (PAG). Twelve days after unilateral CT‐B injections, brains were sectioned (40μm) and immunohistochemically stained with an anti‐CT‐B antibody. The strength of descending projections was qualitatively assessed: as strong (+++), moderate (++) or weak (+) numbers of CT‐B labeled cell bodies. Retrogradely labelled neurons after unilateral injections into the lateral PAG confirmed the predominantly ipsilateral location of strong and moderate descending projection neurons in the cingulate (+++), pre‐limbic (+++), ectorhinal (++), motor (+++) and insular (++) cortices, the lateral septum (++), amygdala (+++) and hypothalamus (+++). In comparison, retrogradely labeled neurons after unilateral KF injection were also found ipsilaterally in the motor (++), prelimbic (++) and insular cortices (+++), the amygdala (++) and hypothalamus (+++). However, amongst all analysed descending target areas, only the KF receives substantial inputs from the ectorhinal (+++) and endopiriform (++) cortices. In addition, the medullary BötC receives weaker inputs from prelimbic (+) and insular (+) cortices and receives moderate inputs from the amygdala and hypothalamus. Descending projection neurons to the pre‐BötC were in accordance with the literature: motor (+) and insular (+) cortices, amygdala (+++) and hypothalamus (++). Finally, descending inputs to the medullary raphé obscurus and raphé magnus nuclei also arose from motor, prelimbic and insular cortices, amygdala and hypothalamus. However, these projections were significantly weaker compared to KF or PAG. The results suggest that descending forebrain projections into respiratory control areas are organized in general pathways that originate from motor, prelimbic and insular cortices as well as the amygdala and hypothalamus. However, only the KF, a key area for the gating of post‐inspiratory activity and respiratory plasticity, receives projections arising from the endopiriform and ectorhinal cortex. The functional implications of these descending control pathways need to be explored in future studies.
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ItemNo Preview AvailableRelaxin-3 receptor (RXFP3) mediated modulation of central respiratory activity(WILEY, 2020-04-01)The neuropeptide, relaxin‐3, is expressed by pontine nucleus incertus (NI) neurons. Relaxin‐3 and synthetic agonist peptides modulate arousal and cognitive processes via activation of the rela xin‐ family peptide 3 receptor (RXFP3). We recently demonstrated that a double‐chain RXFP3 peptidomimetic (RXFP3‐A2) in the nucleus of the solitary tract (NTS) triggered a mild stimulation of respiration and augmented the chemoreceptor reflex in an in situ perfused brainstem preparation ( ). In the present study, we assessed the central respiratory effects of systemic application and local microinjection into the NTS, Kölliker‐Fuse nucleus (KF) or NI of a single chain RXFP3 peptidomimetic (B18) in the perfused brainstem preparation. Systemic application of B18 (2 μM) triggered a dose‐dependent increase in respiratory rate by 22 ± 8%. At this concentration of B18, the NaCN‐evoked (0.1% w/v, 100 μl, bolus injection) tachycardia of the arterial chemoreceptor reflex was augmented by 95 ± 14% compared to control (p<0.001, n=4). Local microinjections into the NTS also increased respiratory frequency (28 ± 5%, p<0.05, n=6) and enhanced the NaCN‐evoked tachycardia by 59%. Microinjections into the KF only triggered a mild increase in respiratory frequency (18 ± 7%, p<0.05, n=6) but had no effect on the NaCN‐evoked chemoreceptor reflex. Finally, microinjections into the NI (n=6) had no effect on either stationary breathing activity, or on the chemoreceptor reflex. We conclude that relaxin‐3 neurons target RXFP3 in respiratory control areas and acts as a general respiratory stimulant, causing mild increases in respiratory frequency. Importantly, RXFP3 stimulation significantly enhanced the respiratory response of arterial chemoreceptor reflex, implicating a major neuromodulatory role in this specific reflex pathway.
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ItemNo Preview AvailableSpinal Oxygen Sensors (SOS): A Novel Oxygen Sensing Mechanism Involved in Cardiovascular Responses to Hypoxia(WILEY, 2020-04-01)Objective: To study the cellular oxygen sensing mechanism and contribution of the SOS in responses to cardiorespiratory crisis. Methods: We investigated the cellular mechanism of oxygen sensing in artificially‐perfused (in situ) and slice (in vitro) thoracic spinal cord preparations, recording sympathetic nerve root and single cell responses to hypoxia during pharmacological interrogation. To determine if the SOS are involved in cardiorespiratory responses to asphyxia, we also used an in situ rat spinal cord – carotid body ‐ brainstem preparation in which each oxygen sensitive compartment is separately perfused while recording phrenic (respiratory) and splanchnic (sympathetic) nerve activity. Results: Our data suggest the SOS use a novel oxygen sensing mechanism. This mechanism involves two interacting NADPH and oxygen‐dependent enzymes: Neuronal Nitric Oxide Synthase (NOS1) and NADPH oxidase (NOX2). NOS1 is expressed in surprising abundance in the SOS and is oxygen sensitive across the entire physiological range. Hence, in the presence of oxygen, NOS1 is likely to utilize most of the available NADPH in the cell. When oxygenation falls during hypoxia, NOS1 activity is reduced, increasing NADPH availability for NOX2. NOX2 produces Reactive Oxygen Species (ROS) which in turn, activate ROS‐dependent internal Ca2+ stores and/or Ca2+ channels leading to increased intracellular Ca2+, neuronal firing and, consequently, SOS responses to hypoxia. Functionally, during hypoxia, the SOS enhance sympathetic and breathing activity, while shortening apnea and gasping towards recovery, and are capable of triggering brief periods of sympathetic and respiratory‐like activity in the brainstem’s absence. Conclusions: The results provide critical new knowledge required to unlock the cellular mechanisms involved in how the body mounts emergency responses to conditions that involve chronic and acute hypoxia.