Doug Smith

A1 neurons and excitatory amino acid receptors in rat caudal medulla mediate vagal excitation of supraoptic vasopressin cells

Publisher: Elsevier BV

Date: 10-1992

DOI: 10.1016/0006-8993(92)91131-W

Abstract: Extracellular recordings from the supraoptic nucleus of the rat established that vasopressinergic neurosecretory cells were excited by stimulation of cervical but not abdominal vagal afferents. This response was absent or significantly attenuated after microinjection of gamma-aminobutyric acid into a region of the caudal medulla known to contain the A1 noradrenaline cell group. Consistent with the possible involvement of the A1 group, vagal stimulation approximately doubled the frequency of proto-oncogene expression in A1 noradrenaline neurons, as indicated by the occurrence of nuclear Fos-like immunoreactivity in tyrosine hydroxylase-positive neurons of the caudal ventrolateral medulla. Finally, A1 region microinjection of either the N-methyl-D-aspartic acid (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV), or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), significantly reduced vasopressin cell responses to vagal stimulation. These findings suggest that: (i) the A1 group is an essential component in a pathway which relays facilitatory vagal input of cardiopulmonary origin to neurosecretory vasopressin cells, and (ii) the activation of A1 neurons in this pathway involves both NMDA and non-NMDA excitatory amino acid receptors, an observation consistent with an input to A1 cells which generates 'mixed' excitatory postsynaptic potentials.

Nucleotide excision repair deficiency in melanoma in response to UVA

Publisher: Springer Science and Business Media LLC

Date: 12-2015

DOI: 10.1186/S40164-016-0035-4

Temporally specific miRNA expression patterns in the dorsal and ventral striatum of addiction-prone rats

Publisher: Wiley

Date: 13-06-2017

DOI: 10.1111/ADB.12520

Abstract: 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 in idual 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.

Dietary supplementation with resveratrol and/or docosahexaenoic acid alters hippocampal gene expression in adult C57Bl/6 mice

Publisher: Elsevier BV

Date: 10-2013

DOI: 10.1016/J.JNUTBIO.2013.03.002

Abstract: The hippoc us 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 hippoc al and therefore cognitive function. To understand the potential of diet for the preservation of hippoc al function, we investigated the effects of dietary supplementation with resveratrol (RES) or docosahexaenoic acid (DHA), or their combination, on hippoc al 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 hippoc i 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 PI3KR2 (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 hippoc al 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.

Advantages and limitations of current techniques for analyzing the biodistribution of nanoparticles

Publisher: Frontiers Media SA

Date: 14-08-2018

DOI: 10.3389/FPHAR.2018.00802

c-Fos expression in hypothalamic neurosecretory and brainstem catecholamine cells following noxious somatic stimuli

Publisher: Elsevier BV

Date: 02-1994

DOI: 10.1016/0306-4522(94)90453-7

Abstract: Noxious somatic stimuli elicit vasopressin secretion, an effect thought to result from activation of a facilitatory input from A1 catecholamine cells of the medulla oblongata. To better characterize the A1 cell response and effects on other neuroendocrine A1 projection targets, particularly within the paraventricular nucleus, we have now mapped c-fos expression in neurochemically identified catecholamine and neurosecretory cells following a noxious somatic stimulus. Unilateral hind paw pinch significantly increased c-fos expression in contralateral A1 cells whereas other brainstem catecholamine cell groups were unaffected. Expression of c-fos was also increased in the supraoptic nucleus, this effect being more pronounced for vasopressin than oxytocin neurosecretory cells and, as with A1 cells, primarily on the side contralateral to the stimulated paw. In contrast, the increase in the paraventricular nucleus was greater in oxytocin rather than in vasopressin cells. Additionally there was a significant rise in c-fos expression in medial parvocellular paraventricular nucleus cells of noxiously stimulated animals. Notably, the majority of tuberoinfundibular corticotropin-releasing factor cells are located in this medial parvocellular zone. These results are consistent with and expand on those previously reported from electrophysiological and anatomical studies. The finding of differing neurosecretory cell responses between supraoptic and paraventricular nuclei has interesting implications with regard to the afferent control of neurosecretory cell activity. For ex le, the substantially greater activation of supraoptic versus paraventricular nucleus vasopressin cells, despite being innervated by the same medullary noradrenergic cell group, raises the possibility of a differential input or differences in responsiveness. Furthermore, the activation of paraventricular nucleus parvocellular cells is consistent with suggestions that the A1 cell group provides an excitatory input to this population.

Initiation of rat vasopressin cell responses to simulated hypotensive hemorrhage

Publisher: American Physiological Society

Date: 10-1994

DOI: 10.1152/AJPREGU.1994.267.4.R1142

Abstract: Hypotensive hemorrhage is a major stimulus for vasopressin (VP) release, but in rats it is uncertain which receptors initiate this response. We have investigated this issue using transient occlusion of the inferior vena cava to simulate hypotensive hemorrhage. Single-unit recording experiments done in the supraoptic nucleus of pentobarbital-anesthetized rats demonstrated that severe caval occlusion, sufficient to drop mean arterial pressure (MAP) below 30 mmHg, excited 88% of putative VP neurosecretory cells and a similar proportion of putative oxytocin (OT) cells. Responsive VP cells increased their firing by 8.5 +/- 0.6 spikes/s within 11.2 +/- 0.8 s of the fall in MAP. This response was unrelated to the size of the fall in MAP and was unchanged by combined sinoaortic denervation (SAD) and vagal denervation, by T1 spinal section, or by administration of the angiotensin-converting enzyme inhibitor captopril, except that spinal section decreased the response latency. Moderate caval occlusion, sufficient to drop MAP to approximately 50 mmHg, did not excite any of the OT cells tested but did excite 65% of VP cells, causing a 3.8 +/- 0.3 spikes/s increase in firing after a delay of 9.0 +/- 1.3 s. This response was proportional to the size of the preceding fall in MAP, and after combined SAD and vagal denervation only 20% of VP cells still responded. Elimination of sinoaortic or vagal afferents alone had no effect on VP cell responses to moderate caval occlusion, except that SAD significantly increased the response latency. These data suggest that in rat the mechanisms that initiate the VP response to hypotensive hemorrhage depend on stimulus intensity.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Ageing on the Mitochondrial Genome in Rat Vestibular Organs

Publisher: Bentham Science Publishers Ltd.

Date: 07-02-2019

DOI: 10.2174/1874609811666180830143358

An emerging role for the mammalian target of rapamycin in pathological protein translation: relevance to cocaine addiction

Publisher: Frontiers Media SA

Date: 2012

DOI: 10.3389/FPHAR.2012.00013

Age-related gene expression changes in lumbar spinal cord: Implications for neuropathic pain

Publisher: SAGE Publications

Date: 2020

DOI: 10.1177/1744806920971914

Abstract: 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 ex le, 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.

Down-regulated striatal gene expression for synaptic plasticity-associated proteins in addiction and relapse vulnerable animals

Publisher: Oxford University Press (OUP)

Date: 09-2011

DOI: 10.1017/S1461145710001367

Age-related gene expression changes in substantia nigra dopamine neurons of the rat

Publisher: Elsevier BV

Date: 07-2015

DOI: 10.1016/J.MAD.2015.06.002

Abstract: Ageing affects most, if not all, functional systems in the body. For ex le, the somatic motor nervous system, responsible for initiating and regulating motor output to skeletal musculature, is vulnerable to ageing. The nigrostriatal dopamine pathway is one component of this system, with deficits in dopamine signalling contributing to major motor dysfunction, as exemplified in Parkinson's disease (PD). However, while the dopamine deficit in PD is due to degeneration of substantia nigra (SN) dopamine (DA) neurons, it is unclear whether there is sufficient loss of SN DA neurons with ageing to explain observed motor impairments. Instead, evidence suggests that age-related loss of DA neuron function may be more important than frank cell loss. To further elucidate the mechanisms of functional decline, we have investigated age-related changes in gene expression specifically in laser microdissected SN DA neurons. There were significant age-related changes in the expression of genes associated with neurotrophic factor signalling and the regulation of tyrosine hydroxylase activity. Furthermore, reduced expression of the DA neuron-associated transcription factor, Nurr1, may contribute to these changes. Together, these results suggest that altered neurotrophic signalling and tyrosine hydroxylase activity may contribute to altered DA neuron signalling and motor nervous system regulation in ageing.

Characterisation of mitochondrial DNA deletions by long-PCR in central nervous system regions of young, middle-and old-aged rats

Publisher: Bentham Science Publishers Ltd.

Date: 31-12-2013

DOI: 10.2174/1874609806666131204153812

Abstract: The causes of ageing remain poorly understood, although a role for mitochondria is widely accepted. These unique organelles that are responsible for a cell's energy, rely on their own small genome and translational machinery to produce proteins that, together with nuclear genome encoded proteins, form the electron transport chain complexes necessary for ATP production. Various forms of mitochondrial genome mutation and rearrangements are thought to be involved in the ageing process, particularly in post-mitotic cells, such as those of the nervous system. In the present study, we have characterised mitochondrial DNA (mtDNA) deletion mutations in five central nervous system (CNS) regions of the young, middle-aged, and old Fischer 344 (F344) rats. DNA was extracted from the cerebral cortex, striatum, midbrain, cerebellum and spinal cord, and long-PCR was used to detect mtDNA with deletions in the minor and major arcs. This approach has the advantage that all deletions can be detected without a priori knowledge of breakpoints. In the minor arc, we found evidence for deletions in the striatum of five out of six old animals and in the spinal cords from two of six old animals. In contrast, mtDNA deletions in the major arc appeared markedly more abundant, both in terms of affected CNS regions and number of deleted mtDNA molecules. Furthermore, major arc deletions were apparent earlier with a number of CNS regions showing deletions in the middle-aged group. The cerebral cortex, striatum and spinal cord were the most affected regions, while the midbrain and cerebellum were relatively spared. These findings are remarkably consistent with previous human brain data and further underscore the value of the rat model for investigation of ageing-related changes in the mitochondrial genome.

Advantages and limitations of current imaging techniques for characterizing liposome morphology

Publisher: Frontiers Media SA

Date: 06-02-2018

DOI: 10.3389/FPHAR.2018.00080

Discrepant effects of culture conditions on survival and function of dopaminergic neurons

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 04-2004

DOI: 10.1097/00001756-200404290-00018

Abstract: Primary midbrain cultures are valuable for probing the function of dopaminergic neurons and for elucidating the factors that cause their dysfunction and degeneration. To allow more effective control of the culture environment, we have characterized the survival, differentiation, and trophic factor response of dopaminergic neurons in the absence of serum. Combinations of media and supplements markedly affected all three indices measured. Combinations that produced maximal dopaminergic neuron survival are different from those that result in maximal differentiation and trophic factor response. Furthermore, antioxidant treatment was effective with only one medium/supplement combination, indicating that these neurons were not degenerating as a result of oxidative stress in the majority of culture conditions used in this study. These results demonstrate that dopaminergic neurons can be grown in serum-free conditions but that the choice of culture conditions has a marked influence on cell survival and function.

Role of neuronal nitric oxide in the dopamine deficit of HPRT-deficient mice

Publisher: Springer Science and Business Media LLC

Date: 31-01-2007

DOI: 10.1007/S11011-007-9044-7

Abstract: Lesch-Nyhan disease is a debilitating disorder caused by a lack of purine salvage activity. Basal ganglia dopamine deficits manifest in both patients and hypoxanthine phosphoribosyltransferase (HPRT) mutant mice. We previously reported decreased activity in an oxidant sensitive enzyme in the brain of HPRT-deficient mice. In the present study, we have investigated whether one source of free radicals, neuronal nitric oxide synthase (NOS1), contributes to the dopamine deficit associated with HPRT deficiency. HPRT knockout and wild-type mice were bred, either to lack, or to have the full complement of NOS1 alleles. Double mutant mice had striatal dopamine and dopamine metabolite levels indistinguishable from the HPRT single mutant counterparts. These results indicate that NOS1 produced nitric oxide does not contribute to the dopamine deficit seen in HPRT deficiency.

Orexin-1 receptor signalling within the ventral tegmental area, but not the paraventricular thalamus, is critical to regulating cue-induced reinstatement of cocaine-seeking

Publisher: Oxford University Press (OUP)

Date: 06-2011

DOI: 10.1017/S1461145711000423

Hypovolaemic and osmotic stimuli induce distinct patterns of c-Fos expression in the rat subfornical organ

Publisher: Elsevier BV

Date: 11-1995

DOI: 10.1016/0006-8993(95)00975-V

Abstract: Investigation of the effects of osmotic and hypovolaemic stimuli on the pattern of subfornical organ (SFO) c-fos expression yielded three distinct distributions of activated neurons. Hypertonic saline induced c-fos expression in peripheral SFO only. PEG/water induced c-fos in the central core of SFO and PEG/saline induced c-fos in both the central and peripheral regions. Isotonic saline failed to induce SFO c-fos expression. These results are consistent with the notion of functional segregation within the SFO.

Dietary resveratrol supplementation normalizes gene expression in the hippocampus of streptozotocin-induced diabetic C57Bl/6 mice

Publisher: Elsevier BV

Date: 03-2014

DOI: 10.1016/J.JNUTBIO.2013.11.005

Abstract: Diabetes is associated with cognitive impairment and brain aging, with alterations in hippoc al 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 hippoc us and genes of interest were confirmed by quantitative, real-time polymerase chain reaction. Genome-wide gene expression analysis of the hippoc us revealed that RES supplementation of the diabetic group resulted in 481 differentially expressed genes compared to non-supplemented diabetic mice. Intriguingly, gene expression that was previously found significantly altered in the hippoc us 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.

Aging alters signaling properties in the mouse spinal dorsal horn

Publisher: SAGE Publications

Date: 2019

DOI: 10.1177/1744806919839860

Alpha-9 nicotinic acetylcholine receptors mediate hypothermic responses elicited by provocative motion in mice

Publisher: Elsevier BV

Date: 05-2017

DOI: 10.1016/J.PHYSBEH.2017.03.012

Abstract: 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 α9 cholinoreceptor subunit predominantly expressed in the vestibular hair cells and CBA strain as a wild-type (WT) control. In WT mice, circular horizontal motion (1Hz, 4cm radius, 20min) 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.

Distinct miRNA expression in dorsal striatal subregions is associated with risk for addiction in rats

Publisher: Springer Science and Business Media LLC

Date: 03-02-2015

DOI: 10.1038/TP.2014.144

Rapamycin reduces motivated responding for cocaine and alters GluA1 expression in the ventral but not dorsal striatum

Publisher: Elsevier BV

Date: 08-2016

DOI: 10.1016/J.EJPHAR.2016.05.013

Abstract: 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 ex le, 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 (10mg/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.

Altered expression of histone and synaptic plasticity associated genes in the hippocampus of streptozotocin-induced diabetic mice

Publisher: Springer Science and Business Media LLC

Date: 06-07-2013

DOI: 10.1007/S11011-013-9418-Y

Abstract: Accumulating evidence indicates that hyper-glycaemia is deleterious to brain function, in particular to the hippoc us. It is thought this hippoc al dysfunction may contribute to hyperglycaemia related cognitive impairment, such as that which manifests with diabetes. In the present study, we investigated the effects of diabetes-related hyperglycaemia on hippoc al gene expression, in order to identify potential mechanisms that might be associated with the cognitive dysfunction that develops with diabetes mellitus. Genome-wide gene expression profiling was carried out on the hippoc i from streptozotocin (STZ)-induced diabetic mice, and from vehicle treated control mice. Genes of interest that satisfied expression fold-change and statistical criteria, and that were considered to be potentially associated with cognitive function, were further tested by real time, quantitative polymerase chain reaction (qPCR) analysis. We found that STZ-induced diabetes resulted in decreased hippoc al expression of genes involved in epigenetic regulation and synaptic plasticity, for ex le, histone deacetylases and glycogen synthase kinase 3 beta (HDACs and GSK3β). We also found increased expression of genes involved in signalling cascades related to cell growth, cell survival and energy metabolism, such as neurotropic tyrosine kinase receptor type 2, apolipoprotein E, and protein tyrosine phosphatase receptor type (Ntrk2, APOE, PTPRT). To our knowledge this is the first study to demonstrate a gene expression profile implicating epigenetic modifications and alterations of synaptic plasticity associated genes in diabetes mellitus. The present study will improve our understanding of the neural mechanisms that might underpin diabetes-related cognitive dysfunction.

Functional and Molecular Analysis of Proprioceptive Sensory Neuron Excitability in Mice

Publisher: Frontiers Media SA

Date: 05-05-2020

DOI: 10.3389/FNMOL.2020.00036

The origin and the role of mitochondrial DNA in postinjury inflammation

Publisher: Elsevier BV

Date: 12-2013

DOI: 10.1016/J.JCRC.2013.08.027

Increased mitochondrial DNA deletions in substantia nigra dopamine neurons of the aged rat

Publisher: Bentham Science Publishers Ltd.

Date: 02-03-2015

DOI: 10.2174/1874609808666150122150850

Abstract: The dopaminergic neurons of the substantia nigra (SN), which constitute the origin of the nigrostriatal system, are vulnerable to age-related degenerative processes. For ex le, in humans there is a relatively small age-related loss of neurons but a marked decline of the dopaminergic phenotype associated with impaired voluntary motor control. However, the mechanisms responsible for the dysfunction and degeneration of SN dopamine neurons remain poorly understood. One potential contributor is mitochondrial dysfunction, resulting from an increased abundance of mitochondrial DNA (mtDNA) mutations such as deletions. Human studies have identified relatively high levels of mtDNA deletions in these cells in both aging and Parkinson's disease (>35%), with a higher abundance of deletions (>60%) in in idual neurons with mitochondrial dysfunction. However, it is unknown whether similar mtDNA mutations occur in other species such as the rat. In the present study, we quantified mtDNA deletion abundance in laser microdissected SN dopaminergic neurons from young and old F344 rats. Our results indicate that mtDNA deletions accumulated with age, with approximately 20% more mtDNA deletions in SN dopaminergic neurons from old compared to young animals. Thus, while rat SN dopaminergic neurons do accumulate mtDNA deletions with aging, this does not reflect the deletion burden in humans, and other mechanisms may be operating to compensate for age-related mtDNA damage in the rat SN dopaminergic neurons.

Improved RNA preservation for immunolabeling and laser microdissection

Publisher: Cold Spring Harbor Laboratory

Date: 22-10-2009

DOI: 10.1261/RNA.1733509

Abstract: Microdissection techniques have the potential to allow for transcriptome analyses in specific populations of cells that are isolated from heterogeneous tissues such as the nervous system and certain cancers. Problematically, RNA is not stable under the labeling conditions usually needed to identify the cells of interest for microdissection. We have developed an immunolabeling method that utilizes a high salt buffer to stabilize RNA during prolonged antibody incubations. We first assessed RNA integrity by three methods and found that tissue incubated in high salt buffer for at least 20 h yielded RNA of similar quality to that for RNA extracted from fresh-frozen tissue, which is considered highest quality. Notably, the integrity was superior to that for RNA extracted from tissue processed using rapid immunolabeling procedures (5 min total duration). We next established that high salt buffer was compatible with immunolabeling, as demonstrated by immunofluorescent detection of dopamine neurons in the brain. Finally, we laser microdissected dopamine neurons that were immunolabeled using high salt buffer and demonstrated that RNA integrity was preserved. Our described method yields high quality RNA from immunolabeled microdissected cells, an essential requirement for meaningful genomics investigations of normal and pathological cells isolated from complex tissues.

Differential recruitment of hypothalamic neuroendocrine and ventrolateral medulla catecholamine cells by non-hypotensive and hypotensive hemorrhages

Publisher: Elsevier BV

Date: 07-1999

DOI: 10.1016/S0006-8993(99)01539-5

Abstract: We performed c-fos expression experiments in conscious rats to quantify the threshold and extent of activation of hypothalamic neuroendocrine cells in response to non-hypotensive and hypotensive hemorrhages allowing us to assess whether their pattern of recruitment corresponded to known oxytocin, vasopressin and ACTH release patterns. Also, because previous studies have implicated ventrolateral medulla catecholamine cells in the generation of certain hypothalamic neuroendocrine cell responses, we examined the response of ventrolateral medulla catecholamine cells to non-hypotensive and hypotensive hemorrhages and directly tested their role in regulating neuroendocrine cell responses to hypotensive hemorrhage. Animals were subjected to hemorrhages of 0, 4, 8, 12 or 16 ml/kg BW, the latter two levels being hypotensive. We found that only supraoptic nucleus vasopressin cells were significantly activated by the smallest non-hypotensive hemorrhage (4 ml/kg), which corresponds to reports that only vasopressin is released into the plasma after a small hemorrhage. Hypotensive hemorrhages resulted in significant recruitment of paraventricular and supraoptic oxytocin and vasopressin cells and parvocellular cells of the medial ision of the paraventricular nucleus. Vasopressin cells were recruited in much greater numbers than oxytocin cells, which is in agreement with previous findings that there is a greater release of vasopressin than oxytocin into the plasma after hypotensive hemorrhage. In addition, medial parvocellular cells of the paraventricular nucleus, most likely to be tuberoinfundibular-projecting corticotropin-releasing factor cells, were activated by hypotensive hemorrhage only when arterial pressure dropped below 60 mmHg which also corresponds well with the plasma release response of ACTH. Ventrolateral medulla catecholamine cells were only recruited by hypotensive hemorrhages. While caution must be exercised in interpreting an absence of response, this certainly suggests that catecholamine cells are unlikely to have a role in the activation of supraoptic neurosecretory cells in response to non-hypotensive hemorrhages. Unilateral lesions of the ventrolateral medulla catecholamine cell column, corresponding primarily to the location of A1 noradrenergic cells, significantly reduced the hypotensive hemorrhage-induced activation of hypothalamic vasopressin, oxytocin and medial parvocellular paraventricular nucleus cells. This suggests that A1 noradrenergic cells contribute to the activation of these neuroendocrine cell populations, including oxytocin cells, which is an unexpected finding. More significantly, however, because the reduction in responsiveness after A1 lesions was similar for all cell categories, it seems likely that other factors must determine the differential recruitment of hypothalamic neuroendocrine cells in response to a hypotensive hemorrhage.

Effects of dietary supplementation with docosahexaenoic acid (DHA) on hippocampal gene expression in streptozotocin induced diabetic C57Bl/6 mice

Publisher: Elsevier BV

Date: 08-2015

DOI: 10.1016/J.JNIM.2015.04.001

Oxidative stress and dopamine deficiency in a genetic mouse model of Lesch-Nyhan disease

Publisher: Elsevier BV

Date: 02-2002

DOI: 10.1016/S0165-3806(02)00280-8

Abstract: Lesch-Nyhan disease, a neurogenetic disorder caused by congenital deficiency of the purine salvage enzyme hypoxanthine guanine phosphoribosyl transferase, is associated with a prominent loss of striatal dopamine. The current studies address the hypothesis that oxidant stress causes damage or dysfunction of nigrostriatal dopamine neurons in a knockout mouse model of the disease, by assessing several markers of oxidative damage and free radical scavenging systems. Some of these measures provided evidence for an increase in oxidative stress in the mutant mice (aconitase activity, oxidized glutathione, and lipid peroxides), but others did not (superoxide dismutase, protein thiol content, carbonyl protein content, total glutathione, glutathione peroxidase, catalase, and thiobarbituric reducing substances). Immunolocalization of heme-oxygenase 1 provided no evidence for oxidative stress restricted to specific elements of the striatum or midbrain in the mutants. Striatal dopamine systems of the mutant mice were more vulnerable to a challenge with the neurotoxin 6-hydroxydopamine, but they were not protected by cross-breeding the mutants with transgenic mice over-expressing superoxide dismutase. Overall, these data provide evidence for increased oxidative stress, but the failure to protect the knockout mice by over-expressing SOD1 argues that oxidative stress is not the sole process responsible for the loss of striatal dopamine.

Purity and Enrichment of Laser-Microdissected Midbrain Dopamine Neurons

Publisher: Hindawi Limited

Date: 2013

DOI: 10.1155/2013/747938

Abstract: The ability to microdissect in idual cells from the nervous system has enormous potential, as it can allow for the study of gene expression in phenotypically identified cells. However, if the resultant gene expression profiles are to be accurately ascribed, it is necessary to determine the extent of contamination by nontarget cells in the microdissected s le. Here, we show that midbrain dopamine neurons can be laser-microdissected to a high degree of enrichment and purity. The average enrichment for tyrosine hydroxylase (TH) gene expression in the microdissected s le relative to midbrain sections was approximately 200-fold. For the dopamine transporter (DAT) and the vesicular monoamine transporter type 2 (Vmat2), average enrichments were approximately 100- and 60-fold, respectively. Glutamic acid decarboxylase (Gad65) expression, a marker for GABAergic neurons, was several hundredfold lower than dopamine neuron-specific genes. Glial cell and glutamatergic neuron gene expression were not detected in microdissected s les. Additionally, SN and VTA dopamine neurons had significantly different expression levels of dopamine neuron-specific genes, which likely reflects functional differences between the two cell groups. This study demonstrates that it is possible to laser-microdissect dopamine neurons to a high degree of cell purity. Therefore gene expression profiles can be precisely attributed to the targeted microdissected cells.

NTS catecholamine cell recruitment by hemorrhage and hypoxia

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 12-1999

DOI: 10.1097/00001756-199912160-00024

Abstract: Immunolabelling for Fos and tyrosine hydroxylase was used to determine the patterns of activation of nucleus tractus solitarius catecholamine cells in response to graded levels of hemorrhage (0, 4, 8, 12 and 16 ml/kg) and systemic hypoxia (21, 14, 12, 10 and 8% O2) in conscious rats. Both stimuli elicited graded catecholamine cell recruitment with thresholds of 8 ml/kg and 12% O2. The majority of responsive neurons were A2 noradrenergic rather than C2 adrenergic cells. After hemorrhage most Fos-positive catecholamine cells were found below obex whereas most hypoxia-responsive cells were rostral to obex. These distinctive patterns of catecholamine cell recruitment may explain the differences in neuroendocrine responses to these stimuli.

Perturbed cholesterol homeostasis in aging spinal cord

Publisher: Elsevier BV

Date: 09-2016

DOI: 10.1016/J.NEUROBIOLAGING.2016.05.017

Abstract: 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.

Reduced deoxyribonuclease enzyme activity in response to high postinjury mitochondrial DNA concentration provides a therapeutic target for Systemic Inflammatory Response Syndrome

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 08-2018

DOI: 10.1097/TA.0000000000001919

Abstract: Cell-free mitochondrial DNA (mtDNA) is proinflammatory and has been detected in high concentrations in trauma patients’ plasma. Deoxyribonuclease (DNAse) is the free plasma enzyme responsible for the digestion of extracellular DNA. The relationship between mtDNA and DNAse after major trauma is unknown. We hypothesized that DNAse activity would be elevated after injury and trauma surgery and would be associated with high concentrations of extracellular DNA. Two-year prospective study was performed on 103 consecutive trauma patients (male, 81% age, 38 years [interquartile range, 30–59 years] injury severity score, 18 [interquartile range, 12–26 years]) who underwent standardized major orthopedic trauma surgical interventions. Blood was collected at five perioperative time points (preoperative, postoperative, 7 hours, 24 hours, and 3 days postoperatively). Healthy control subjects (n = 20) were also s led. Cell-free mtDNA and nuclear DNA (nDNA) were measured using quantitative polymerase chain reaction. Deoxyribonuclease was also assayed in the same plasma s les. Increased levels of mtDNA (from preoperative 163 ± 86 ng/mL to 3 days 282 ± 201 ng/mL, p 0.0001) and nDNA (from preoperative 28 ± 20 ng/mL to 3 days 37 ± 27 ng/mL, p 0.05) were present in trauma patients at all perioperative time points compared with healthy controls (mtDNA: 4 ± 2 ng/mL nDNA: 10 ± 5 ng/mL). Deoxyribonuclease activity was lower in the trauma cohort (from preoperative 0.06 ± 0.04U/mL to 3 days 0.08 ± 0.04U/mL, p 0.0001) compared with healthy controls (DNAse: 0.17 ± 0.03U/mL). There was no correlation between DNAse and perioperative DNA concentrations. Elevated mtDNA (but not nDNA) correlated with the development of systemic inflammatory response syndrome (SIRS) ( p = 0.026) but not multiple organ failure. The significant perioperative elevation in plasma-free mtDNA concentration is associated with the development of SIRS. The fact that increased cell-free DNA concentrations present with significantly lower than healthy control DNAse activity suggests a potential therapeutic opportunity with DNAse administration to modulate postinjury severe SIRS. Prognostic/Epidemiological, level II.

Propensity to ‘relapse’ following exposure to cocaine cues is associated with the recruitment of specific thalamic and epithalamic nuclei

Publisher: Elsevier BV

Date: 12-2011

DOI: 10.1016/J.NEUROSCIENCE.2011.09.047

Abstract: The thalamus is considered an important interface between the ventral striatopallidum and the dorsal striatum, and may therefore contribute to compulsive drug-seeking behaviour. Recent evidence suggests that the paraventricular thalamus (PVT), a dorsal midline thalamic nucleus, and the mediodorsal thalamus (MD) are involved in drug self-administration and respond to drug-associated cues. At present, however, the role of these thalamic regions in mediating cue-induced reinstatement of cocaine-seeking is unclear. Similarly, the habenula complex, part of the epithalamus, has been implicated in nicotine self-administration and cue-induced reinstatement of heroin seeking, but the role of this region in cocaine reinstatement behaviour has received little attention. Rats (n=20) were trained to self-administer cocaine in the presence of discriminative stimuli associated with drug availability (S⁺) or drug non-availability (S⁻). Once a stable level of responding was reached, lever pressing was extinguished. Animals were then tested for reinstatement and sacrificed immediately following the presentation of either the S⁻ or S⁺ discriminative stimuli, and Fos-protein expression was assessed in thalamic and epithalamic regions. Interestingly, significant variation was observed in reinstatement behaviour, allowing a comparison between high-reinstating (HR), low-reinstating (LR) and control animals. Compared with LR animals, HR animals exhibited increased Fos-protein expression in the PVT, intermediodorsal thalamus and the medial and lateral isions of the habenula. Our data provide evidence that activation of thalamic and epithalamic nuclei is associated with propensity to reinstate to cocaine-seeking elicited by drug-related cues. We also build upon existing data highlighting the importance of the PVT in reinstatement behaviour.

Characterization of the dopamine defect in primary cultures of dopaminergic neurons from hypoxanthine phosphoribosyltransferase knockout mice

Publisher: Elsevier BV

Date: 05-2000

DOI: 10.1006/MTHE.2000.0057

Exercise reverses the effects of early life stress on orexin cell reactivity in male but not female rats

Publisher: Frontiers Media SA

Date: 23-07-2014

DOI: 10.3389/FNBEH.2014.00244

Region-specific changes in mitochondrial D-loop in aged rat CNS

Publisher: Elsevier BV

Date: 05-2009

DOI: 10.1016/J.MAD.2009.01.008

Abstract: Impaired mitochondrial oxidative phosphorylation (OXPHOS) is considered a cause of aging. A reduction in mitochondrial DNA (mtDNA) replication and/or transcription may contribute to this OXPHOS diminution. Impairments in the displacement (D) loop, or non-coding, region of the mitochondrial genome, or accumulation of mtDNA mutations, may affect mtDNA replication and transcription. We determined the effects of age on the D-loop and on mtDNA deletion mutations in the spinal cord, medulla, midbrain, cerebellum, striatum, and cerebral cortex of Fischer 344 rats. D-loop, 7S DNA levels were reduced by 3-fold in striatum, 2.5-fold in cortex, and 2-fold in the spinal cord of older animals. We did not detect a population of mtDNA affected by the most prevalent known (ND4-containing) deletions, indicating they do not comprise a significant portion of total mtDNA. However, we detected an age-related and region-specific increase in the common deletion, which comprised 0.0003-0.0007% of total mtDNA. Mitochondrial genome copy number varied between regions, in addition to an overall 18% decrease with age across the whole brain. These results suggest the age-related decline in OXPHOS may be related to a reduction in D-loop function.

Role of ventrolateral medulla catecholamine cells in hypothalamic neuroendocrine cell responses to systemic hypoxia

Publisher: Society for Neuroscience

Date: 12-1995

DOI: 10.1523/JNEUROSCI.15-12-07979.1995

Abstract: Systemic hypoxia stimulates the release of vasopressin (VP) and adrenocorticotropin hormone (ACTH). To examine the involvement of catecholamine cell groups of the ventrolateral medulla (VLM) in the neuroendocrine responses, we have used the c-fos activity mapping technique to compare the effects of hypoxia on VLM catecholamine cells to those on neurosecretory VP and putative corticotropin releasing factor (CRF) containing cells. A limited degree of catecholamine cell activation was evident at predominantly mid-VLM levels at 12% oxygen in the inspired air. Further reduction in inpsirate oxygen levels enhanced recruitment of caudally located VLM catecholamine cells considered to form part of the A1 noradrenergic cell group. Threshold for activation of VP and putative CRF cells occurred at the 10% oxygen level. Unexpectedly, this stimulus also activated neurosecretory oxytocin (OT) cells. With increasing hypoxic severity the number of activated supraoptic VP and OT cells was not significantly different to that observed at the 10% level. However, paraventricular neuroendocrine responses continued to increase with putative CRF containing cells of the medial parvocellular zone having nearly double the level of activity (as measured by the number of cells within this region displaying Fos-like immunoreactivity FLI) at 6% compared to that apparent to the 10% level of hypoxia. Paraventricular VP cells displaying FLI were also increased at the most severe levels of hypoxia but this effect was much less marked than the medial parvocellular response. Consistent with a role for VLM catecholamine cells in generation of neuroendocrine cell responses to hypoxia, unilateral VLM lesions, restricted to the caudal two thirds of the catecholamine cell column, resulted in significant reductions in the responses of all three cell types. These results, in addition to establishing a role for VLM catecholamine cells in neuroendocrine cell responses to systemic hypoxia, have important general implications for catecholamine cell group involvement in neuroendocrine regulation.

Cocaine- and Amphetamine-Regulated Transcript (CART) Signaling within the Paraventricular Thalamus Modulates Cocaine-Seeking Behaviour

Publisher: Public Library of Science (PLoS)

Date: 23-09-2010

DOI: 10.1371/JOURNAL.PONE.0012980

MTORC1 inhibition in the nucleus accumbens 'protects' against the expression of drug seeking and 'relapse' and is associated with reductions in GluA1 AMPAR and CAMKII levels

Publisher: Springer Science and Business Media LLC

Date: 28-01-2014

DOI: 10.1038/NPP.2014.16

Mitochondrial DNA neutrophil extracellular traps are formed after trauma and subsequent surgery.

Publisher: Elsevier BV

Date: 12-2014

DOI: 10.1016/J.JCRC.2014.07.013

Abstract: 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. 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. 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. 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.

Rat vasopressin cell responses to simulated hemorrhage: Stimulus-dependent role for A1 noradrenergic neurons

Publisher: American Physiological Society

Date: 05-1995

DOI: 10.1152/AJPREGU.1995.268.5.R1336

Abstract: c-fos expression mapping and electrophysiological recording experiments were done to clarify the role of the A1 noradrenergic cell group in the vasopressin response to hypotensive hemorrhage. In pentobarbital-anesthetized rats, moderate and severe hypotensive hemorrhages were simulated by brief occlusion of the inferior vena cava sufficient to reduce mean arterial pressure to approximately 50 or 30 mmHg, respectively. Both stimuli significantly increased the number of A1 region catecholamine cells displaying Fos-like immunoreactivity, this effect being most prominent at the level of the area postrema. Both stimuli also increased the number of supraoptic nucleus vasopressin cells displaying Fos-like immunoreactivity. Accordingly, electrophysiological studies involving separate animals confirmed that both moderate and severe caval occlusion significantly increased the firing of functionally identified vasopressin cells recorded in the supraoptic nucleus. However, although interruption of A1 region neuronal function by injection of gamma-aminobutyric acid at the level of the area postrema eliminated the increase in vasopressin cell firing elicited by moderate caval occlusion, it did not block the response to severe caval occlusion. These findings suggest that, in the rat, the vasopressin response to an acute reduction in central blood volume, such as that produced by hemorrhage, depends on the A1 projection only if the stimulus is of moderate intensity. Severe stimuli appear to involve activation of both the A1 projection and an additional vasopressin-stimulatory pathway that bypasses the A1 region.

Cell necrosis–independent sustained mitochondrial and nuclear DNA release following trauma surgery

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 02-2015

DOI: 10.1097/TA.0000000000000519

Fluoxetine prevents development of an early stress-related molecular signature in the rat infralimbic medial prefrontal cortex. Implications for depression?

Publisher: Springer Science and Business Media LLC

Date: 18-10-2012

DOI: 10.1186/1471-2202-13-125

Abstract: Psychological stress, particularly in chronic form, can lead to mood and cognitive dysfunction and is a major risk factor in the development of depressive states. How stress affects the brain to cause psychopathologies is incompletely understood. We sought to characterise potential depression related mechanisms by analysing gene expression and molecular pathways in the infralimbic medial prefrontal cortex (ILmPFC), following a repeated psychological stress paradigm. The ILmPFC is thought to be involved in the processing of emotionally contextual information and in orchestrating the related autonomic responses, and it is one of the brain regions implicated in both stress responses and depression. Genome-wide microarray analysis of gene expression showed sub-chronic restraint stress resulted predominantly in a reduction in transcripts 24 hours after the last stress episode, with 239 genes significantly decreased, while just 24 genes had increased transcript abundance. Molecular pathway analysis using DAVID identified 8 pathways that were significantly enriched in the differentially expressed gene list, with genes belonging to the brain-derived neurotrophic factor – neurotrophin receptor tyrosine kinase 2 (BDNF-Ntrk2) pathway most enriched. Of the three intracellular signalling pathways that are downstream of Ntrk2, real-time quantitative PCR confirmed that only the PI3K-AKT-GSK3B and MAPK/ERK pathways were affected by sub-chronic stress, with the PLCγ pathway unaffected. Interestingly, chronic antidepressant treatment with the selective serotonin reuptake inhibitor, fluoxetine, prevented the stress-induced Ntrk2 and PI3K pathway changes, but it had no effect on the MAPK/ERK pathway. These findings indicate that abnormal BDNF-Ntrk2 signalling may manifest at a relatively early time point, and is consistent with a molecular signature of depression developing well before depression-like behaviours occur. Targeting this pathway prophylactically, particularly in depression-susceptible in iduals, may be of therapeutic benefit.

Changes in spinal GDNF, BDNF, and NT-3 expression after transient spinal cord ischemia in the rat

Publisher: Wiley

Date: 21-10-2003

DOI: 10.1002/JNR.10760

Abstract: Previous studies have demonstrated that the expression of several growth factors including glial cell-derived neurotrophic factor (GDNF), brain-derived growth factor (BDNF), and neurotrophin-3 (NT-3) play an important role in defining neuronal survival after brain ischemia. In the present study, using a well-defined model of transient spinal ischemia in rat, we characterized the changes in spinal GDNF, BDNF, and NT-3 expression as defined by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence coupled with deconvolution microscopy. In control animals, baseline levels of GDNF, BDNF, and NT-3 (74 +/- 22, 3,600 +/- 270, 593 +/- 176 pg/g tissue, respectively) were measured. In the ischemic group, 6 min of spinal ischemia resulted in a biphasic response with increases in tissue GDNF and BDNF concentrations at the 2-hr and 72-hr points after ischemia. No significant differences in NT-3 concentration were detected. Deconvolution analysis revealed that the initial increase in tissue GDNF concentration corresponded to a neuronal upregulation whereas the late peak seen at 72 hr corresponded with increased astrocyte-derived GDNF synthesis. Increased expression of BDNF was seen in neurons, astrocytes, and oligodendrocytes. These data suggest that the early increase in neuronal GDNF/BDNF expression likely modulates neuronal resistance/recovery during the initial period of postischemic reflow. Increased astrocyte-derived BDNF/GDNF expression corresponds with transient activation of astrocytes and may play an active role in neuronal plasticity after non-injurious intervals of spinal ischemia.

Neurochemical identification of fos-positive neurons using two-colour immunoperoxidase staining

Publisher: Elsevier BV

Date: 04-1993

DOI: 10.1016/0165-0270(93)90023-K

Abstract: The discovery of immediate early genes (IEG) has provided neuroscientists with a new functional mapping technique. Labelling of neural tissue for the protein product of IEG provides an activity map with single-cell resolution. When combined with labelling for the chemical identity of the neuron, this provides a powerful tool for the investigation of specific cell populations along a neuraxis. Here we describe in detail a method which allows simultaneous bright-field visualization of neurochemically identified cells displaying increased IEG expression. This technique is evaluated in tissue from rats subjected to stimuli known to induce the expression of the IEG c-fos in various medullary catecholaminergic and hypothalamic neurosecretory cell groups. A 2-colour immunoperoxidase technique was used to visualize Fos, the nuclear protein product of c-fos, and the cytoplasmic antigens tyrosine hydroxylase (TH), phenylethanolamine N-methyl transferase (PNMT), oxytocin (OT) and vasopressin (VP). This involved simultaneous application of primary antibodies raised in different species followed by sequential application of appropriate biotinylated secondary antibodies and the avidin-biotin-peroxidase technique. Fos was visualized with nickel-intensified diaminobenzidine (Ni-DAB) in the first sequence while TH, PNMT, OT or VP were visualized with DAB alone, resulting in readily distinguishable black and amber reaction products, respectively. This dual immunoperoxidase technique is time saving compared to techniques using sequential application of primary antibodies and avoids the disadvantages associated with fluorescence techniques.

Catecholamine and oxytocin cells respond to hypovolaemia as well as hypotension

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 08-2003

DOI: 10.1097/00001756-200308060-00018

Abstract: Medullary catecholamine and hypothalamic neurosecretory oxytocin cells are activated by hypotension, but previous studies have provided uncertain outcomes concerning their ability to respond to a purely hypovolaemic stimulus. In the present study, injections of PEG/water and pentolinium were used to elicit non-hypotensive, isosmotic hypovolaemia and isovolaemic, isosmotic hypotension, respectively, in conscious rats. Animals were sacrificed 2 h after treatment. Immunolabelling for Fos, tyrosine hydroxylase and oxytocin established that these two stimuli activate almost identical populations of catecholamine neurons in the ventrolateral and dorsomedial medulla, and very similar populations of oxytocin cells in the supraoptic and paraventricular nuclei of the hypothalamus.

University Of Queensland

Location: Australia

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The University Of Newcastle

Location: Australia

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Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100008

Start Date: 2010

End Date: 12-2010

Amount: $350,000.00

Funder: Australian Research Council