Associate Professor Doug Smith

Clinically, pain has an uneven incidence throughout lifespan and impacts more on the elderly. In contrast, preclinical models of pathological pain have typically used juvenile or young adult animals to highlight the involvement of glial populations, proinflammatory cytokines, and chemokines in the onset and maintenance of pathological signalling in the spinal dorsal horn. The potential impact of this mismatch is also complicated by the growing appreciation that the aged central nervous system exists in a state of chronic inflammation because of enhanced proinflammatory cytokine/chemokine signalling and glial activation. To address this issue, we investigated the impact of aging on the expression of genes that have been associated with neuropathic pain, glial signalling, neurotransmission and neuroinflammation. We used qRT-PCR to quantify gene expression and focussed on the dorsal horn of the spinal cord as this is an important perturbation site in neuropathic pain. To control for global vs region-specific age-related changes in gene expression, the ventral half of the spinal cord was examined. Our results show that expression of proinflammatory chemokines, pattern recognition receptors, and neurotransmitter system components was significantly altered in aged (24¿32 months) versus young mice (2¿4 months). Notably, the magnitude and direction of these changes were spinal-cord region dependent. For example, expression of the chemokine, Cxcl13, increased 119-fold in dorsal spinal cord, but only 2-fold in the ventral spinal cord of old versus young mice. Therefore, we propose the dorsal spinal cord of old animals is subject to region-specific alterations that prime circuits for the development of pathological pain, potentially in the absence of the peripheral triggers normally associated with these conditions.

MicroRNAs (miRNAs) within the ventral and dorsal striatum have been shown to regulate addiction-relevant behaviours. However, it is unclear how cocaine experience alone can alter the expression of addiction-relevant miRNAs within striatal subregions. Further, it is not known whether differential expression of miRNAs in the striatum contributes to individual differences in addiction vulnerability. We first examined the effect of cocaine self-administration on the expression of miR-101b, miR-137, miR-212 and miR-132 in nucleus accumbens core and nucleus accumbens shell (NAcSh), as well as dorsomedial striatum and dorsolateral striatum (DLS). We then examined the expression of these same miRNAs in striatal subregions of animals identified as being ¿addiction-prone¿, either immediately following self-administration training or following extinction and relapse testing. Cocaine self-administration was associated with changes in miRNA expression in a regionally discrete manner within the striatum, with the most marked changes occurring in the nucleus accumbens core. When we examined the miRNA profile of addiction-prone rats following self-administration, we observed increased levels of miR-212 in the dorsomedial striatum. After extinction and relapse testing, addiction-prone rats showed significant increases in the expression of miR-101b, miR-137, miR-212 and miR-132 in NAcSh, and miR-137 in the DLS. This study identifies temporally specific changes in miRNA expression consistent with the engagement of distinct striatal subregions across the course of the addiction cycle. Increased dysregulation of miRNA expression in NAcSh and DLS at late stages of the addiction cycle may underlie habitual drug seeking, and may therefore aid in the identification of targets designed to treat addiction.

Background: Deterioration in vestibular function occurs with ageing and is linked to age-related falls. Sensory hair cells located in the inner ear vestibular labyrinth are critical to vestibular function. Vestibular hair cells rely predominantly on oxidative phosphorylation (OXPHOS) for energy production and contain numerous mitochondria. Mitochondrial DNA (mtDNA) mutations and perturbed energy production are associated with the ageing process. Objective: We investigated the effects of ageing on mtDNA in vestibular hair and support cells, and vestibular organ gene expression, to better understand mechanisms of age-related vestibular deficits. Methods: Vestibular hair and supporting cell layers were microdissected from young and old rats, and mtDNA was quantified by qPCR. Additionally, vestibular organ gene expression was analysed by microarray and gene set enrichment analyses. Results: In contrast to most other studies, we found no evidence of age-related mtDNA deletion mutations. However, we found an increase in abundance of major arc genes near the mtDNA control region. There was also a marked age-related reduction in mtDNA copy number in both cell types. Vestibular organ gene expression, gene set enrichment analysis showed the OXPHOS pathway was down regulated in old animals. Conclusion: Given the importance of mtDNA to mitochondrial OXPHOS and hair cell function, our findings suggest the vestibular organs are potentially on the brink of an energy crisis in old animals.

Animal models of maternal immune activation study the effects of infection, an environmental risk factor for schizophrenia, on brain development. Microglia activation and cytokine upregulation may have key roles in schizophrenia neuropathology. We hypothesised that maternal immune activation induces changes in microglia and cytokines in the brains of the adult offspring. Maternal immune activation was induced by injecting polyriboinosinic:polyribocytidylic acid into pregnant rats on gestational day (GD) 10 or GD19, with brain tissue collected from the offspring at adulthood. We observed no change in Iba1, Gfap, IL1-ß and TNF-a mRNA levels in the cingulate cortex (CC) in adult offspring exposed to maternal immune activation. Prenatal exposure to immune activation had a significant main effect on microglial IBA1-positive immunoreactive material (IBA1+IRM) in the corpus callosum; post-hoc analyses identified a significant increase in GD19 offspring, but not GD10. No change in was observed in the CC. In contrast, maternal immune activation had a significant main effect on GFAP+IRM in the CC at GD19 (not GD10); post-hoc analyses only identified a strong trend towards increased GFAP+IRM in the GD19 offspring, with no white matter changes. This suggests late gestation maternal immune activation causes subtle alterations to microglia and astrocytes in the adult offspring.

Prevailing evidence indicates a relatively late life decline in human vestibulo-ocular reflex (VOR) function. Although mice are commonly used in mechanistic studies of vestibular function, it remains unclear whether aging produces a corresponding decline in VOR function in mice. We sought to determine how the baseline VOR and its short-term adaptation were affected by aging. We tested 8 young (3-month old) and 8 aged (30-month old¿equivalent to a ~80-year old human) C57BL/6 mice. We measured their VOR response to whole-body static tilts and during 0.1¿10¿Hz whole-body sinusoidal and transient rotations before and after VOR adaptation training. Our data revealed minimal differences in static counter-tilt response between young and aged mice, but a significant deficit in baseline VOR gain in aged mice during transient rotations. Moreover, aged mice had a significant decrease in short-term VOR adaptation, particularly for training that sought to decrease the VOR response.

Hypothermic responses accompany motion sickness in humans and can be elicited by provocative motion in rats. We aimed to determine the potential role in these responses of the efferent cholinergic vestibular innervation. To this end, we used knockout (KO) mice lacking a9 cholinoreceptor subunit predominantly expressed in the vestibular hair cells and CBA strain as a wild-type (WT) control. In WT mice, circular horizontal motion (1¿Hz, 4¿cm radius, 20¿min) caused rapid and dramatic falls in core body temperature and surface head temperature associated with a transient rise in the tail temperature; these responses were substantially attenuated in KO mice; changes were (WT vs. KO): for the core body temperature¿-¿5.2¿±¿0.3 vs. -¿2.9¿±¿0.3¿°C; for the head skin temperature¿-¿3.3¿±¿0.2 vs. -¿1.7¿±¿0.2¿°C; for the tail skin temperature¿+¿3.9¿±¿1.1 vs¿+¿1.1¿±¿1.2¿°C. There was a close correlation in the time course of cooling the body and the surface of the head. KO mice also required 25% more time to complete a balance test. We conclude: i) that the integrity of cholinergic efferent vestibular system is essential for the full expression of motion-induced hypothermia in mice, and that the role of this system is likely facilitatory; ii) that the system is involvement in control of balance, but the involvement is not major; iii) that in mice, motion-induced body cooling is mediated via increased heat flow through vasodilated tail vasculature and (likely) via reduced thermogenesis. Our results support the idea that hypothermia is a biological correlate of a nausea-like state in animals.

The spinal cord is vital for the processing of sensorimotor information and for its propagation to and from both the brain and the periphery. Spinal cord function is affected by aging, however, the mechanisms involved are not well-understood. To characterize molecular mechanisms of spinal cord aging, microarray analyses of gene expression were performed on cervical spinal cords of aging rats. Of the metabolic and signaling pathways affected, cholesterol-associated pathways were the most comprehensively altered, including significant downregulation of cholesterol synthesis-related genes and upregulation of cholesterol transport and metabolism genes. Paradoxically, a significant increase in total cholesterol content was observed-likely associated with cholesterol ester accumulation. To investigate potential mechanisms for the perturbed cholesterol homeostasis, we quantified the expression of myelin and neuroinflammation-associated genes and proteins. Although there was minimal change in myelin-related expression, there was an increase in phagocytic microglial and astrogliosis markers, particularly in the white matter. Together, these results suggest that perturbed cholesterol homeostasis, possibly as a result of increased inflammatory activation in spinal cord white matter, may contribute to impaired spinal cord function with aging.

The mechanistic target of rapamycin complex 1 (mTORC1) regulates synaptic protein synthesis and therefore synaptic function and plasticity. A role for mTORC1 has recently been demonstrated for addiction-related behaviors. For example, central or intra-accumbal injections of the mTORC1 inhibitor rapamycin attenuates several indices of cocaine-seeking including progressive ratio (PR) responding and reinstatement. These behavioral effects are associated with decreased mTORC1 activity and synaptic protein translation in the nucleus accumbens (NAC) and point to a possible therapeutic role for rapamycin in the treatment of addiction. Currently, it is unclear whether similar behavioral and biochemical effects can be achieved by administering rapamycin systemically, which represents a more clinically-appropriate route of administration. Here, we assessed the effects of repeated, systemic administration of rapamycin (10 mg/kg, i.p.) on PR responding for cocaine. We also assessed whether systemic rapamycin was associated with changes in measures of mTORC1 activity and GluA1 expression in the ventral and dorsal striatum. We report that systemic rapamycin treatment reduced PR breakpoints to levels comparable to intra-NAC rapamycin. Systemic rapamycin treatment also reduced phosphorylated p70S6K and GluA1 AMPARs within the NAC but not dorsal striatum. Thus, systemic administration of rapamycin is as effective at reducing drug seeking behavior and measures of mTORC1 activity compared to direct accumbal application and may therefore represent a possible therapeutic option in the treatment of addiction. Possible caveats of this treatment approach are discussed.

Background: The causative link between UV exposure and melanoma development is well known, however the mechanistic relationship remains incompletely characterised. UVA and UVB components of sunlight are implicated in melanomagenesis; however the majority of studies have focused on the effects of UVB and UVC light. Interestingly, melanoma tumour sequencing has revealed an overrepresentation of mutations signature of unrepaired UV-induced DNA damage. Repair of UVA-induced DNA damage is thought to occur primarily through the Nucleotide Excision Repair (NER) pathway, which recognises and repairs damage either coupled to transcription (Transcription Coupled Repair; TCR), or through global genome scanning (Global Genome Repair; GGR). Current literature suggests NER is deficient in melanoma, however the cause of this remains unknown; and whether reduced NER activity in response to UVA may be involved in melanoma development remains uncharacterised. In this study we aimed to determine if melanoma cells exhibit reduced levels of NER activity in response to UVA. Methods: Melanocyte and melanoma cell lines were UVA-irradiated, and DNA damage levels assessed by immunodetection of Cyclobutane Pyrimidine Dimer (CPD) and (6-4) Photoproduct [(6-4)PP] lesions. Expression of NER pathway components and p53 following UVA treatment was quantified by qPCR and western blot. Results: UVA did not induce detectable induction of (6-4)PP lesions, consistent with previous studies. Repair of CPDs induced by UVA was initiated at 4 h and complete within 48 h in normal melanocytes, whereas repair initiation was delayed to 24 h and >40 % of lesions remained in melanoma cell lines at 48 h. This was coupled with a delayed and reduced induction of GGR component XPC in melanoma cells, independent of p53. Conclusion: These findings support that NER activity is reduced in melanoma cells due to deficient GGR. Further investigation into the role of NER in UVA-induced melanomagenesis is warranted and may have implications for melanoma treatment.

Recently, we published data using an animal model that allowed us to characterize animals into two groups, addiction vulnerable and addiction resilient, where we identified that addiction/relapse vulnerability was associated with deficits in synaptic plasticityassociated gene expression in the dorsal striatum (DS). Notable was the strong reduction in expression for activity-regulated cytoskeleton-associated protein (Arc) considered a master regulator of synaptic plasticity. In the present study, we confirmed that Arc messenger RNA was significantly decreased in the DS, but importantly, we identified that this reduction was restricted to the dorsomedial (DMS) and not dorsolateral striatum (DLS). There is recent evidence of microRNA (miRNA)-associated posttranscriptional suppression of Arc and animal models of addiction have identified a key role for miRNA in the regulation of addiction-relevant genes. In further support of this link, we identified several differentially expressed miRNA with the potential to influence addiction-relevant plasticity genes, including Arc. A key study recently reported that miR-212 expression is protective against compulsive cocaine-seeking. Supporting this hypothesis, we found that miR-212 expression was significantly reduced in the DMS but not DLS of addiction-vulnerable animals. Together, our data provide strong evidence that miRNA promote ongoing plasticity deficits in the DS of addiction-vulnerable animals.

A body of evidence has accumulated indicating diabetes is associated with cognitive impairments. Effective strategies are therefore needed that will delay or prevent the onset of these diabetes-related deficits. In this regard, dietary modification with the naturally occurring compound, docosahexaenoic acid (DHA), holds significant promise as it has been shown to have anti-inflammatory, anti-oxidant, and anti-apoptotic properties. The hippocampus, a limbic structure involved in cognitive functions such as memory formation, is particularly vulnerable to the neurotoxic effects related to diabetes, and we have previously shown that streptozotocin-induced diabetes alters hippocampal gene expression, including genes involved in synaptic plasticity and neurogenesis. In the present study, we explored the effects of dietary supplementation with DHA on hippocampal gene expression in C57Bl/6 diabetic mice. Diabetes was established using streptozotocin (STZ) and once stable, the dietary intervention group received AIN93G diet supplemented with DHA (50 mg/kg/day) for 6 weeks. Microarray based genome-wide expression analysis was carried out on the hippocampus of DHA supplemented diabetic mice and confirmed by real time polymerase chain reaction (RT-qPCR). Genome-wide analysis identified 353 differentially expressed genes compared to non-supplemented diabetic mice. For example, six weeks of dietary DHA supplementation resulted in increased hippocampal expression of Igf II and Sirt1 and decreased expression of Tnf-a, Il6, Mapkapk2 and ApoE, compared to non-supplemented diabetic mice. Overall, DHA supplementation appears to alter hippocampal gene expression in a way that is consistent with it being neuroprotective in the context of the metabolic and inflammatory insults associated with diabetes.

Introduction: Neutrophil extracellular traps (NETs) have not been demonstrated after trauma and subsequent surgery. Neutrophil extracellular traps are formed from pure mitochondrial DNA (mtDNA) under certain conditions, which is potently proinflammatory. We hypothesized that injury and orthopedic trauma surgery would induce NET production with mtDNA as a structural component. Methods: Neutrophils were isolated 8 trauma patients requiring orthopedic surgery postinjury and up to 5 days postoperatively. Four healthy volunteers provided positive and negative controls. Total hip replacement patients acted as an uninjured surgical control group. Neutrophil extracellular traps were visualized with DNA (Hoechst 33342TM/Sytox Green/MitoSox/MitoTracker) stains using live cell fluorescence microscopy with downstream quantitative polymerase chain reaction analysis of DNA composition. Results: Neutrophil extracellular traps were present after injury in all 8 trauma patients. They persisted for 5 days postoperatively. Delayed surgery resulted in NET resolution, but they reformed postoperatively. Total hip replacement patients developed NETs postoperatively, which resolved by day 5. Quantitative polymerase chain reaction analysis of NET-DNA composition revealed that NETs formed after injury and surgery were made of mtDNA with no detectable nuclear DNA component. Conclusions: Neutrophil extracellular traps formed after major trauma and subsequent surgery contain mtDNA and represent a novel marker of heightened innate immune activation. They could be considered when timing surgery after trauma to prevent systemic NET-induced inflammatory complications.

Diabetes is associated with cognitive impairment and brain aging, with alterations in hippocampal neurogenesis and synaptic plasticity implicated in these changes. As the prevalence of diabetes continues to rise, readily implemented strategies are increasingly needed in order to protect the brain's cognitive functions. One possibility is resveratrol (RES) (3,5,4- trihydroxystilbene), a polyphenol of the phytoalexin family that has been shown to be protective in a number of neuropathology paradigms. In the present study, we sought to determine whether dietary supplementation with RES has potential for the protection of cognitive functions in diabetes. Diabetes was induced using streptozotocin, and once stable, animals received AIN93G rodent diet supplemented with RES for 6 weeks. Genome-wide expression analysis was conducted on the hippocampus and genes of interest were confirmed by quantitative, real-time polymerase chain reaction. Genome-wide gene expression analysis of the hippocampus revealed that RES supplementation of the diabetic group resulted in 481differentially expressed genes compared to non-supplemented diabetic mice. Intriguingly, gene expression that was previously found significantly altered in the hippocampus of diabetic mice, and that is implicated in neurogenesis and synaptic plasticity (Hdac4, Hat1, Wnt7a, ApoE), was normalized following RES supplementation. In addition, pathway analysis revealed Jak-Stat signaling was the most significantly enriched pathway. The Jak-Stat pathway induces a pro-inflammatory signaling cascade, and we found most genes involved in this cascade (e.g. Il15, Il22, Socs2, Socs5) had significantly lower expression following RES supplementation. These data indicate RES could be neuroprotective and beneficial for the maintenance of cognitive function in diabetes. © 2014 Elsevier Inc.

The hippocampus is an important brain structure for multiple cognitive functions, including memory formation. It is particularly sensitive to insults, such as stress, ischemia, and aging; all of these can affect hippocampal and therefore cognitive function. To understand the potential of diet for the preservation of hippocampal function, we investigated the effects of dietary supplementation with resveratrol (RES) or docosahexaenoic acid (DHA), or their combination, on hippocampal gene expression in adult C57BL/6 mice. Animals in the supplemented group received either 50 mg/kg/day of RES or DHA, while the combination group received 50 mg/kg/day of each supplement. Dietary supplements were mixed with the AIN93G diet, and supplementation lasted 6 weeks. The control group received AIN93G diet alone for the same period. At the end of the experiment, the hippocampi were processed for genome-wide gene expression and pathway analyses. Most of the genes that were significantly altered were associated with inflammatory responses as determined by pathway analysis. RES-supplemented animals showed decreased expression of IL-6 ( P=001), MAPKapk2 ( P=015), and increased expression for PI3. KR2 ( P=034) and Wnt7a ( P=004) expression. DHA-supplemented animals showed a decreased IL-6 ( P=003) and an increased Wnt7a ( P=003) expression. Animals on the combination diet showed a decreased IL-6 ( P=005) and Apolipoprotien E ( ApoE) ( P=035) expression. Our findings demonstrate that hippocampal gene expression is significantly altered by all three dietary supplementation regimes. Moreover, our analysis indicates that RES and DHA likely exert their beneficial effects through antiinflammatory mechanisms. © 2013 Elsevier Inc.