Effects of acute altered gravity during parabolic flight and/or vestibular loss on cell proliferation in the rat dentate gyrus.

Both parabolic flight, i.e. a condition of altered gravity, and loss of vestibular function, have been suggested to affect spatial learning and memory, which is known to be influenced by neurogenesis in the hippocampus. In this study we investigated whether short alternated micro- and hyper-gravity stimulations during parabolic flight and/or loss of vestibular function, would alter cell proliferation in the hippocampal dentate gyrus of rats, by measuring the number of bromodeoxyuridine (BrdU)-incorporated cells. Rats were randomly allocated to the following experimental groups: (1) sham transtympanic saline injection only (n=5); (2) bilateral vestibular deafferentation (BVD) by sodium arsanilate transtympanic injection only (n=5); (3) sham treatment and parabolic flight (n=5); (4) BVD and parabolic flight (n=6). Forty-two days following transtympanic injection, the animals were subjected to parabolic flight in an awake restrained condition after habituation. A modified Airbus A300 aircraft was flown on a parabolic path, creating 20s of 1.8G during both climbing and descending and 22s of 0G at the apex of each parabola. The no flight animals were subjected to the same housing for the same duration. Immediately after the parabolic flight or control ground condition, animals were injected with BrdU (300mg/kg, i.p). Twenty-four hs after BrdU injection, rats were sacrificed. BrdU immunolabelling was performed and the number of BrdU+ve cells in the dentate gyrus of the hippocampus was quantified using a modified fractionator method. BVD caused a large and significant reduction in the number of BrdU-positive cells compared to sham animals (P≤0.0001); however, flight and all interactions were non-significant. These results indicate that BVD significantly decreased cell proliferation irrespective of the short exposure to altered/modified gravity.

Psychoactive Substances Bill and Act of New Zealand: A Chance to Engage Undergraduate Scientists with Society using a Transfer Learning Paradigm.

Our aim was to develop a teaching paradigm that connected undergraduate's neuropharmacological/toxicological knowledge to that of government policy. One goal of undergraduate education should be to help develop scientists that can use their scientific knowledge to critique government policy. There is little research, however, on whether democratization of science occurs: nor how to achieve this. Our work focused on a semi-structured workshop designed around the Psychoactive Substances Bill (PSB). Third year science students were given a questionnaire that was designed to address whether participating in the workshop enhanced their understanding of the PSB and its relationship to their established knowledge (i.e., transfer learning). Furthermore, whether they felt that they had enough expertise to consider making a submission (i.e., societal engagement). Results showed that the students appreciated the opportunity to explore potential application of their knowledge and delve into a socio-scientific issue. However, our findings suggested they felt uncomfortable discussing their ideas outside the classroom: nor, did they identify themselves as having sufficient knowledge to contribute to a submission. In conclusion, this study highlights two points. First, that discussion based transfer learning can be used in the tertiary sector and students value the opportunity to apply their knowledge to socio-scientific issue. Second, if social participation and democratization of science is a goal, then more emphasis should be placed on how students can realistically and confidently apply their learning to change social policy. In order to achieve this, education programs need to focus on legitimate real-life processes such as the PSB for engagement.

White matter tract and glial-associated changes in 5-hydroxymethylcytosine following chronic cerebral hypoperfusion.

White matter abnormalities due to age-related cerebrovascular alterations is a common pathological hallmark associated with functional impairment in the elderly which has been modeled in chronically hypoperfused mice. 5-Methylcytosine (5mC) and its oxidized derivative 5-hydroxymethylcytosine (5hmC) are DNA modifications that have been recently linked with age-related neurodegeneration and cerebrovascular pathology. Here we conducted a pilot investigation of whether chronic cerebral hypoperfusion might affect genomic distribution of these modifications and/ or a Ten-Eleven Translocation protein 2 (TET2) which catalyses hydroxymethylation in white and grey matter regions of this animal model. Immunohistochemical evaluation of sham and chronically hypoperfused mice a month after surgery revealed significant (p<0.05) increases in the proportion of 5hmC positive cells, Iba1 positive inflammatory microglia, and NG2 positive oligodendroglial progenitors in the hypoperfused corpus callosum. In the same white matter tract there was an absence of hypoperfusion-induced alterations in the proportion of 5mC, TET2 positive cells and CC1 positive mature oligodrendrocytes. Correlation analysis across animals within both treatment groups demonstrated a significant association of the elevated 5hmC levels with increases in the proportion of inflammatory microglia only (p=0.01) in the corpus callosum. In vitro studies revealed that 5hmC is lost during oligodendroglial maturation but not microglial activation. Additionally, TET1, TET2, and TET3 protein levels showed dynamic alterations during oligodendroglial development and following oxidative stress in vitro. Our study suggests that 5hmC exhibits white matter tract and cell type specific dynamics following chronic cerebral hypoperfusion in mice.

The effects of bilateral vestibular loss on hippocampal volume, neuronal number, and cell proliferation in rats.

Previous studies in humans have shown that bilateral loss of vestibular function is associated with a significant bilateral atrophy of the hippocampus, which correlated with the patients' spatial memory deficits. More recently, patients who had recovered from unilateral vestibular neuritis have been reported to exhibit a significant atrophy of the left posterior hippocampus. Therefore, we investigated whether bilateral vestibular deafferentation (BVD) would result in a decrease in neuronal number or volume in the rat hippocampus, using stereological methods. At 16 months post-BVD, we found no significant differences in hippocampal neuronal number or volume compared to sham controls, despite the fact that these animals exhibited severe spatial memory deficits. By contrast, using bromodeoxyuridine (BrdU) as a marker of cell proliferation, we found that the number of BrdU-labeled cells significantly increased in the dentate gyrus of the hippocampus between 48 h and 1 week following BVD. Although a substantial proportion of these cells survived for up to 1 month, the survival rate was significantly lower in BVD animals when compared with that in sham animals. These results suggest a dissociation between the effects of BVD on spatial memory and hippocampal structure in rats and humans, which cannot be explained by an injury-induced increase in cell proliferation.

Proteomic analysis of mitochondria in APOE transgenic mice and in response to an ischemic challenge.

Apolipoprotein E (APOE)-ɛ4 is associated with a deleterious outcome after ischemic brain injury, which may involve abnormal regulation of mitochondrial function. We have assessed the mitochondrial proteomic response of APOE-ɛ3 and APOE-ɛ4 transgenic mice to transient global ischemic injury in the hippocampus. A genotype-dependent increase in ApoE levels in mitochondria was observed after ischemia, with APOE-ɛ4 mice showing significantly greater increases than APOE-ɛ3 mice. Quantitative analysis of the mitochondria-enriched fractions was performed using liquid-chromatography mass spectrometry coupled to label-free analysis. Of the 1,067 identified proteins, 274 were mitochondria associated. Mitochondrial protein expression was significantly different between genotypes under basal conditions as well as in response to global ischemia. A total of 12 mitochondrial proteins (including respiratory chain proteins NDUFA11, NDUFS3, NDUF5B, ATP5J, as well as ETFA, CYB5B, ATP6V1A, HSPA1B, OXR1, GLUL, IARS2, and PHYHIPL) were significantly altered with respect to genotype, global ischemia, or their interaction (P<0.01). A compelling interactome, created using proteins found to be significantly modulated by global ischemia (P<0.05), involved proteins that regulate energy production and oxidative stress. Thus, APOE genotype has a differential effect on the mitochondrial protein expression in the absence and presence of an injury, which may underlie the differing genotype susceptibility.

Rapid disruption of axon-glial integrity in response to mild cerebral hypoperfusion.

Myelinated axons have a distinct protein architecture essential for action potential propagation, neuronal communication, and maintaining cognitive function. Damage to myelinated axons, associated with cerebral hypoperfusion, contributes to age-related cognitive decline. We sought to determine early alterations in the protein architecture of myelinated axons and potential mechanisms after hypoperfusion. Using a mouse model of hypoperfusion, we assessed changes in proteins critical to the maintenance of paranodes, nodes of Ranvier, axon-glial integrity, axons, and myelin by confocal laser scanning microscopy. As early as 3 d after hypoperfusion, the paranodal septate-like junctions were damaged. This was marked by a progressive reduction of paranodal Neurofascin signal and a loss of septate-like junctions. Concurrent with paranodal disruption, there was a significant increase in nodal length, identified by Nav1.6 staining, with hypoperfusion. Disruption of axon-glial integrity was also determined after hypoperfusion by changes in the spatial distribution of myelin-associated glycoprotein staining. These nodal/paranodal changes were more pronounced after 1 month of hypoperfusion. In contrast, the nodal anchoring proteins AnkyrinG and Neurofascin 186 were unchanged and there were no overt changes in axonal and myelin integrity with hypoperfusion. A microarray analysis of white matter samples indicated that there were significant alterations in 129 genes. Subsequent analysis indicated alterations in biological pathways, including inflammatory responses, cytokine-cytokine receptor interactions, blood vessel development, and cell proliferation processes. Our results demonstrate that hypoperfusion leads to a rapid disruption of key proteins critical to the stability of the axon-glial connection that is mediated by a diversity of molecular events.

Cellular distribution of the neutral amino acid transporter subtype ASCT2 in mouse brain.

ASCT2 is an ASC (alanine-, serine-, cysteine-preferring) neutral amino acid exchanger that may regulate CNS function by transporting amino acid substrates including L-serine, L-cysteine, L-glutamine, L-glutamate and D-serine. Despite the potentially important role of ASCT2 in influencing metabolic and signaling functions of these amino acids in brain, there has been little description of its distribution in brain tissue. We employed a commercially available human ASCT2 antibody in immunohistochemistry studies in adult mouse brain and found a wide regional distribution for ASCT2 that was limited to dendrites labeled by anti-microtubule-associated protein-2 in cortex, hippocampus and striatum. No ASCT2 immunoreactivity was observed in areas labeled by antibodies against a neuronal cell body marker (NeuN), or either of the astrocyte markers, glial fibrillary acidic protein or S100beta. In cerebellum both Purkinje cell bodies and dendrites were positive for ASCT2 immunoreactivity. In support of a dendritic localization for ASCT2 in cortex, low affinity (K(T) > 1 mM), Na(+)-dependent D-serine and L-glutamine uptake characteristic of ASCT2-mediated transport was observed in P2 synaptosomal preparations. These results suggest that ASCT2 may be an important neuronal neutral amino acid transporter and highlight a discrepancy between findings of astrocyte ASCT2 function in tissue culture and brain in situ.

GABA(A) receptor subunit expression in the guinea pig vestibular nucleus complex during the development of vestibular compensation.

The aim of this experiment was to investigate whether vestibular compensation following unilateral vestibular deafferentation (UVD) is associated with changes in the expression of GABA(A) receptor subunits in the guinea pig vestibular nuclear complex (VNC) at 2, 10, and 30 h post-surgery. Using Western blotting, the alpha1 and gamma2 subunits (but not the beta2 subunit) were detected in the VNC of labyrinthine-intact animals. However, there were no significant differences in the protein expression of the alpha1 and gamma2 subunits within the ipsilateral or contralateral VNC at any time post-UVD compared to sham and anesthetic controls. Furthermore, UVD did not induce the expression of the beta2 protein. These results suggest that vestibular compensation in guinea pig, as in the rat, is not associated with changes in the protein levels of the GABA(A) receptor subunits alpha1, beta2, and gamma2 in the VNC. However, a limitation of this study is that the Western blotting technique can detect only changes that are larger than 30% and therefore small changes cannot be excluded.

Effects of chronic infusion of a GABAA receptor agonist or antagonist into the vestibular nuclear complex on vestibular compensation in the guinea pig.

The aim of this study was to determine the effects of chronic infusion of a GABA(A) receptor agonist/antagonist into the ipsilateral or contralateral vestibular nuclear complex (VNC) on vestibular compensation, the process of behavioral recovery that occurs after unilateral vestibular deafferentation (UVD). This was achieved by a mini-osmotic pump that infused, over 30 h, muscimol or gabazine into the ipsilateral or contralateral VNC. Spontaneous nystagmus (SN), yaw head tilt (YHT), and roll head tilt (RHT) were measured. Infusion of muscimol or gabazine into either the ipsilateral or the contralateral VNC had little effect on SN compensation. In contrast, infusion of muscimol (250, 500, and 750 ng) into the contralateral VNC and gabazine (31.25, 62.5, and 125 ng) into the ipsilateral VNC significantly affected YHT and RHT (p < 0.05), but not their rate of compensation (p > 0.05). Interestingly, the effects of muscimol and gabazine on YHT and RHT were consistent throughout the first 30 h post-UVD. Infusion of muscimol (62.5, 125, and 250 ng) into the ipsilateral VNC and gabazine (125, 375, and 750 ng) into the contralateral VNC had little effect on YHT and RHT or their rate of compensation. These results suggest that the ipsilateral gabazine and contralateral muscimol infusions are modifying the expression of the symptoms without altering the mechanism of compensation. Furthermore, the neurochemical mechanism responsible for vestibular compensation can cope with the both the GABA(A) receptor-mediated and the UVD-induced decrease in resting activity.

GABAergic systems in the vestibular nucleus and their contribution to vestibular compensation.

GABA and the GABAA and GABAB receptors play a pivotal role in the coordination of the central vestibular pathways. The commissural inhibition, which exists between the two vestibular nucleus complexes (VNCs) and which is responsible for enhancing the dynamic sensitivity of VNC neurons to head acceleration, is known to be substantially mediated by GABA acting on GABAA and GABAB receptors. After unilateral vestibular deafferentation (UVD), the large asymmetry in spontaneous resting activity between the two VNCs is reinforced and exacerbated by the GABAergic interaction between the ipsilateral and contralateral sides. Although it has been suggested that reduced GABAergic inhibition of the ipsilateral VNC may be partially responsible for the recovery of resting activity that underlies vestibular compensation of the static symptoms of UVD, at present there are few data available to test this hypothesis systematically. There is some evidence that GABA concentrations change in the ipsilateral VNC during the development of compensation; however, it is unclear whether these changes relate to GABA release or to metabolic pools of GABA. Most biochemical studies of GABA receptors have been conducted at the gene expression level. Therefore, it is unclear whether changes in the receptor protein also occur, although the most recent data suggest that changes in GABAA and GABAB receptor density in the VNC are unlikely. The few radioligand binding data relate to GABAA receptors with benzodiazepine binding sites only. A decrease in the sensitivity of ipsilateral VNC neurons from compensated animals to GABA receptor agonists has been reported; however, these studies have employed brainstem slices and therefore the functional identity of the neurons involved has been unclear. Although it seems likely that some changes in central GABAergic systems accompany the recovery of resting activity in the ipsilateral VNC during the development of vestibular compensation, at the present stage there is no compelling evidence that these changes have a causal role in the compensation process.

Cytosolic glucocorticoid receptor expression in the rat vestibular nucleus and hippocampus following unilateral vestibular deafferentation.

It has been suggested that vestibular compensation, the process of behavioural recovery that occurs following peripheral vestibular damage, might be partially dependent on the release of glucocorticoids (GC) during the early stages of recovery from the lesion. One possibility is that glucocorticoid receptors (GRs) in the vestibular nucleus complex (VNC) might change following the lesion, altering their response to GCs. We sought to test this hypothesis by quantifying the expression of cytosolic GRs in the bilateral VNCs at 10 h, 58 h and 2 weeks following unilateral vestibular deafferentation (UVD) in rat, using western blotting. We also examined GR expression in the CA1, CA2/3 and dentate gyrus (DG) subregions of the hippocampus and measured serum corticosterone levels. Compared with sham surgery and anaesthetic controls, we found no significant changes in GR expression in the ipsilateral or contralateral VNCs at any time post-UVD. However, we did find a significant decrease in GR expression in the ipsilateral CA1 at 2 weeks post-UVD. Serum corticosterone levels were significantly lower in all groups at 58 h post-op. compared to 10 h and 2 weeks; however, there were no significant differences between the UVD and control groups at any time point. These results suggest that changes in GR expression in the VNC are unlikely to contribute to the development of vestibular compensation. However, long-term changes in GR expression in CA1 might be related to chronic deficits in hippocampal function and spatial cognition following vestibular damage.

Rapid vestibular compensation in guinea pig even with prolonged anesthesia.

The results of previous studies have suggested that prolonged anesthesia following unilateral labyrinthectomy (UL) results in a retardation of vestibular compensation, the process of behavioral recovery that occurs following the lesion. In this study we investigated the effects of short-term (25 min) and long-term (4 h) anesthesia with isoflurane on the time course of vestibular compensation following UL in guinea pig. Although there were significant differences in the frequency of spontaneous nystagmus (SN) (p < 0.05) and its rate of compensation (p < 0.05) between the 25 min and 4h isoflurane groups, these differences appeared to be due largely to the 5, 9 and 13 h time points. There was also a significant difference in the rate of yaw head tilt (YHT) compensation, largely due to the 5 h time point. When exponential regression analysis was performed to evaluate the overall pattern of compensation, there was no significant difference in the time required to reach 100% SN or YHT compensation between the 25 min and 4 h isoflurane groups. Furthermore, there were no significant differences in roll head tilt (RHT) compensation between the two groups. These results suggest that the time course of vestibular compensation is largely independent of the duration of the anesthesia used for UL surgery.

Nitric oxide synthase and arginase expression changes in the rat perirhinal and entorhinal cortices following unilateral vestibular damage: a link to deficits in object recognition?

Previous studies have shown that peripheral vestibular damage causes long-term neurochemical changes in the hippocampus which may be related to spatial memory deficits. Since recent studies have also demonstrated deficits in non-spatial object recognition memory following vestibular lesions, the aim of the present study was to extend these investigations into the perirhinal cortex (PRC), which is known to be important for object recognition, and the related entorhinal cortex (EC). We examined the effects of unilateral vestibular deafferentation (UVD) on the expression of four enzymes associated with neuronal plasticity, neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), arginase I and arginase II (AI and II), in the rat EC and PRC using Western blotting. Tissue was collected at 10 hs, 50 hs and 2 weeks post-UVD. In the EC and PRC, nNOS protein expression decreased on the contralateral side at 2 weeks post-UVD but not before. At the same time, eNOS protein expression increased in both regions on the contralateral side. In the EC, AII protein expression increased on the ipsilateral side at 2 weeks post-UVD. In the PRC, AI increased and decreased on the contralateral and ipsilateral sides (respectively) at 2 weeks post-UVD. AII showed a bilateral increase in the PRC at 2 weeks post-UVD. These results demonstrate changes in NOS and arginase protein expression in the PRC and EC following UVD, which are unlikely to be due to the initial severity of the vestibular syndrome because they develop well after vestibular compensation has taken place. Neurochemical changes in these regions of the medial temporal lobe may be implicated in the development of object recognition deficits that contribute to cognitive dysfunction following peripheral vestibular damage.

Effect of low body temperature during unilateral labyrinthectomy on vestibular compensation in the guinea pig.

OBJECTIVES: Vestibular compensation, the recovery that follows unilateral vestibular deafferentation (UVD), is a model for central nervous system plasticity. Recovery from the static symptoms of UVD may involve temperature-dependent processes that modulate the immediate effects of UVD and/or the capability of the central nervous system to undergo adaptive plasticity. In this study we investigated changes in oculomotor and postural vestibular symptoms resulting from low body temperature during UVD. MATERIAL AND METHODS: To study the effect of low temperatures at the time of UVD on vestibular compensation, we compared the rate of compensation and peak values for postural [roll head tilt (RHT) and yaw head tilt (YHT)] and oculomotor [spontaneous nystagmus (SN)] symptoms in three groups of guinea pigs. Animals in Group 1 (n = 6) were maintained at 38 degrees C throughout unilateral labyrinthectomy (UL). Animals in Group 2 (n = 6) were not temperature-controlled and animals in Group 3 (n = 4) were cooled with ice to 25 degrees C throughout UL. RESULTS: Cooled animals showed significantly higher rates of SN upon recovery from anaesthesia and took a significantly longer time to compensate. Cooled animals were also slower to compensate for postural symptoms (RHT and YHT), with 2 animals showing no compensation for RHT 52 h after UL. CONCLUSION: Hypothermia (25 degrees C) during UVD surgery exacerbates postural and oculomotor symptoms following UL and significantly slows recovery.

Activation of the hypothalamic-pituitary-adrenal axis following vestibular deafferentation in pigmented guinea pig.

Twelve male pigmented guinea pigs underwent either a unilateral vestibular deafferentation (UVD) (n=6) or sham operation (n=6). Compared to the pre-operated salivary cortisol concentrations, the UVD operation resulted in a significant increase in night cortisol concentrations (P<0.05) and a significant interaction between the night cortisol concentration and time (P<0.05). There was no significant difference between the pre- and post-UVD morning salivary cortisol concentrations; nor between the pre- and post-sham morning or night salivary cortisol concentrations. This study suggests that the ocular-motor and postural syndrome is causing the activation of the hypothalamic-pituitary-adrenal (HPA) axis.

Interaction between the hypothalamic-pituitary-adrenal axis and behavioural compensation following unilateral vestibular deafferentation.

Vestibular compensation is defined as the process of behavioural recovery that occurs following the loss of sensory input from one or both vestibular labyrinths. The visual and postural instability resulting from the vestibular damage must alter the homeostasis of the subject; however, very little research has been conducted that investigates the interaction between vestibular compensation and the adaptive stress response of the body, i.e. the hypothalamic-pituitary-adrenal (HPA) axis. The aim of this review is to describe and evaluate the experimental evidence indicating a link between vestibular compensation and the body's response to stress, via the HPA axis.