Inhibition of RhoA reduces propofol-mediated growth cone collapse, axonal transport impairment, loss of synaptic connectivity, and behavioural deficits.

BACKGROUND: Exposure of the developing brain to propofol results in cognitive deficits. Recent data suggest that inhibition of neuronal apoptosis does not prevent cognitive defects, suggesting mechanisms other than neuronal apoptosis play a role in anaesthetic neurotoxicity. Proper neuronal growth during development is dependent upon growth cone morphology and axonal transport. Propofol modulates actin dynamics in developing neurones, causes RhoA-dependent depolymerisation of actin, and reduces dendritic spines and synapses. We hypothesised that RhoA inhibition prevents synaptic loss and subsequent cognitive deficits. The present study tested whether RhoA inhibition with the botulinum toxin C3 (TAT-C3) prevents propofol-induced synapse and neurite loss, and preserves cognitive function. METHODS: RhoA activation, growth cone morphology, and axonal transport were measured in neonatal rat neurones (5-7 days in vitro) exposed to propofol. Synapse counts (electron microscopy), dendritic arborisation (Golgi-Cox), and network connectivity were measured in mice (age 28 days) previously exposed to propofol at postnatal day 5-7. Memory was assessed in adult mice (age 3 months) previously exposed to propofol at postnatal day 5-7. RESULTS: Propofol increased RhoA activation, collapsed growth cones, and impaired retrograde axonal transport of quantum dot-labelled brain-derived neurotrophic factor, all of which were prevented with TAT-C3. Adult mice previously treated with propofol had decreased numbers of total hippocampal synapses and presynaptic vesicles, reduced hippocampal dendritic arborisation, and infrapyramidal mossy fibres. These mice also exhibited decreased hippocampal-dependent contextual fear memory recall. All anatomical and behavioural changes were prevented with TAT-C3 pre-treatment. CONCLUSION: Inhibition of RhoA prevents propofol-mediated hippocampal neurotoxicity and associated cognitive deficits.

A synthetic small-molecule Isoxazole-9 protects against methamphetamine relapse.

Adult neurogenesis in the dentate gyrus (DG) is strongly influenced by drug-taking behavior and may have a role in the etiology of drug-seeking behavior. However, mechanistic studies on the relationship of neurogenesis on drug seeking are limited. Outbred Wistar rats experienced extended access methamphetamine self-administration and individual differences in drug taking defined animals with higher preferred and lower preferred levels of drug intake. Forced abstinence from higher preferred levels of drug taking enhanced neurogenesis and neuronal activation of granule cell neurons (GCNs) in the DG and produced compulsive-like drug reinstatement. Systemic treatment with the drug Isoxazole-9 (a synthetic small molecule known to modulate neurogenesis in the adult rodent brain) during abstinence blocked compulsive-like context-driven methamphetamine reinstatement. Isoxazole-9 modulated neurogenesis, neuronal activation and structural plasticity of GCNs, and expression of synaptic proteins associated with learning and memory in the DG. These findings identify a subset of newly born GCNs within the DG that could directly contribute to drug-seeking behavior. Taken together, these results support a direct role for the importance of adult neurogenesis during abstinence in compulsive-like drug reinstatement.

Developmental effects of wheel running on hippocampal glutamate receptor expression in young and mature adult rats.

Recent evidence suggests that the behavioral benefits associated with voluntary wheel running in rodents may be due to modulation of glutamatergic transmission in the hippocampus, a brain region implicated in learning and memory. However, the expression of the glutamatergic ionotropic N-methyl-d-aspartate receptor (GluN) in the hippocampus in response to chronic sustained voluntary wheel running has not yet been investigated. Further, the developmental effects during young and mature adulthood on wheel running output and GluN expression in hippocampal subregions has not been determined, and therefore is the main focus of this investigation. Eight-week-old and 16-week-old male Wistar rats were housed in home cages with free access to running wheels and running output was monitored for 4weeks. Wheel access was terminated and tissues from the dorsal and ventral hippocampi were processed for Western blot analysis of GluN subunit expression. Young adult runners demonstrated an escalation in running output but this behavior was not evident in mature adult runners. In parallel, young adult runners demonstrated a significant increase in total GluN (1 and 2A) subunit expression in the dorsal hippocampus (DH), and an opposing effect in the ventral hippocampus (VH) compared to age-matched sedentary controls; these changes in total protein expression were not associated with significant alterations in the phosphorylation of the GluN subunits. In contrast, mature adult runners demonstrated a reduction in total GluN2A expression in the DH, without producing alterations in the VH compared to age-matched sedentary controls. In conclusion, differential running activity-mediated modulation of GluN subunit expression in the hippocampal subregions was revealed to be associated with developmental effects on running activity, which may contribute to altered hippocampal synaptic activity and behavioral outcomes in young and mature adult subjects.

Protracted abstinence from chronic ethanol exposure alters the structure of neurons and expression of oligodendrocytes and myelin in the medial prefrontal cortex.

In rodents, chronic intermittent ethanol vapor exposure (CIE) produces alcohol dependence, alters the structure and activity of pyramidal neurons and decreases the number of oligodendroglial progenitors in the medial prefrontal cortex (mPFC). In this study, adult Wistar rats were exposed to seven weeks of CIE and were withdrawn from CIE for 21 days (protracted abstinence; PA). Tissue enriched in the mPFC was processed for Western blot analysis and Golgi-Cox staining to investigate the long-lasting effects of CIE on the structure of mPFC neurons and the levels of myelin-associated proteins. PA increased dendritic arborization within apical dendrites of pyramidal neurons. These changes occurred concurrently with hypophosphorylation of the N-methyl-d-aspartate (NMDA) receptor 2B (NR2B) at Tyr-1472. PA increased myelin basic protein (MBP) levels which occurred concurrently with hypophosphorylation of the premyelinating oligodendrocyte bHLH transcription factor Olig2 in the mPFC. Given that PA is associated with increased sensitivity to stress and hypothalamic-pituitary-adrenal (HPA) axis dysregulation, and stress alters oligodendrocyte expression as a function of glucocorticoid receptor (GR) activation, the levels of total GR and phosphorylated GR were also evaluated. PA produced hypophosphorylation of the GR at Ser-232 without affecting expression of total protein. These findings demonstrate persistent and compensatory effects of ethanol in the mPFC long after cessation of CIE, including enhanced myelin production and impaired GR function. Collectively, these results suggest a novel relationship between oligodendrocytes and GR in the mPFC, in which stress may alter frontal cortex function in alcohol dependent subjects by promoting hypermyelination, thereby altering the cellular composition and white matter structure in the mPFC.

Methamphetamine differentially affects BDNF and cell death factors in anatomically defined regions of the hippocampus.

Methamphetamine exposure reduces hippocampal long-term potentiation (LTP) and neurogenesis and these alterations partially contribute to hippocampal maladaptive plasticity. The potential mechanisms underlying methamphetamine-induced maladaptive plasticity were identified in the present study. Expression of brain-derived neurotrophic factor (BDNF; a regulator of LTP and neurogenesis), and its receptor tropomyosin-related kinase B (TrkB) were studied in the dorsal and ventral hippocampal tissue lysates in rats that intravenously self-administered methamphetamine in a limited access (1h/day) or extended access (6h/day) paradigm for 17days post baseline sessions. Extended access methamphetamine enhanced expression of BDNF with significant effects observed in the dorsal and ventral hippocampus. Methamphetamine-induced enhancements in BDNF expression were not associated with TrkB receptor activation as indicated by phospho (p)-TrkB-706 levels. Conversely, methamphetamine produced hypophosphorylation of N-methyl-d-aspartate (NMDA) receptor subunit 2B (GluN2B) at Tyr-1472 in the ventral hippocampus, indicating reduced receptor activation. In addition, methamphetamine enhanced expression of anti-apoptotic protein Bcl-2 and reduced pro-apoptotic protein Bax levels in the ventral hippocampus, suggesting a mechanism for reducing cell death. Analysis of Akt, a pro-survival kinase that suppresses apoptotic pathways and pAkt at Ser-473 demonstrated that extended access methamphetamine reduces Akt expression in the ventral hippocampus. These data reveal that alterations in Bcl-2 and Bax levels by methamphetamine were not associated with enhanced Akt expression. Given that hippocampal function and neurogenesis vary in a subregion-specific fashion, where dorsal hippocampus regulates spatial processing and has higher levels of neurogenesis, whereas ventral hippocampus regulates anxiety-related behaviors, these data suggest that methamphetamine self-administration initiates distinct allostatic changes in hippocampal subregions that may contribute to the altered synaptic activity in the hippocampus, which may underlie enhanced negative affective symptoms and perpetuation of the addiction cycle.

Time course of morphine's effects on adult hippocampal subgranular zone reveals preferential inhibition of cells in S phase of the cell cycle and a subpopulation of immature neurons.

Opiates, such as morphine, decrease neurogenesis in the adult hippocampal subgranular zone (SGZ), raising the possibility that decreased neurogenesis contributes to opiate-induced cognitive deficits. However, there is an incomplete understanding of how alterations in cell cycle progression and progenitor maturation contribute to this decrease. The present study examined how morphine regulates progenitor cell cycle, cell death and immature SGZ neurons (experiment 1) as well as the progression of SGZ progenitors through key stages of maturation (experiment 2). In experiment 1, mice received sham or morphine pellets (s.c., 0 and 48 h) and bromodeoxyuridine (BrdU) 2 h prior to sacrifice (24, 72 or 96 h). Morphine decreased both the number of S phase and total cycling cells, as there were fewer cells immunoreactive (IR) for the S phase marker BrdU and the cell cycle marker Ki67. The percentage of Ki67-IR cells that were BrdU-IR was decreased after 24 but not 96 h of morphine, suggesting a disproportionate effect on S phase cells relative to all cycling cells at this time point. Cell death (activated caspase-3 counts) was increased after 24 but not 96 h. In experiment 2, nestin-green fluorescent protein (GFP) mice given BrdU 1 day prior to morphine or sham surgery (0 and 48 h, sacrifice 96 h) had fewer Ki67-IR cells, but no change in BrdU-IR cell number, suggesting that this population of BrdU-IR cells was less sensitive to morphine. Interestingly, examination of key stages of progenitor cell maturation revealed that morphine increased the percent of BrdU-IR cells that were type 2b and decreased the percent that were immature neurons. These data suggest that chronic morphine decreases SGZ neurogenesis by inhibiting dividing cells, particularly those in S phase, and progenitor cell progression to a more mature neuronal stage.

Determination of key aspects of precursor cell proliferation, cell cycle length and kinetics in the adult mouse subgranular zone.

Neurogenesis studies on the adult mouse hippocampal subgranular zone (SGZ) typically report increases or decreases in proliferation. However, key information is lacking about these proliferating SGZ precursors, from the fundamental--what dose of bromodeoxyuridine (BrdU) is appropriate for labeling all S phase cells?--to the detailed--what are the kinetics of BrdU-labeled cells and their progeny? To address these questions, adult C57BL/6J mice were injected with BrdU and BrdU-immunoreactive (IR) cells were quantified. Initial experiments with a range of BrdU doses (25-500 mg/kg) suggested that 150 mg/kg labels all actively dividing precursors in the mouse SGZ. Experiments using a saturating dose of BrdU suggested BrdU bioavailability is less than 15 min, notably shorter than in the developing mouse brain. We next explored precursor division and maturation by tracking the number of BrdU-IR cells and colabeling of BrdU with other cell cycle proteins from 15 min to 30 days after BrdU. We found that BrdU and the Gap2 and mitosis (G2/M) phase protein pHisH3 maximally colocalized 8 h after BrdU, indicating that the mouse SGZ precursor cell cycle length is 14 h. In addition, triple labeling with BrdU and proliferating cell nuclear antigen (PCNA) and Ki-67 showed that BrdU-IR precursors and/or their progeny express these endogenous cell cycle proteins up to 4 days after BrdU injection. However, the proportion of BrdU/Ki-67-IR cells declined at a greater rate than the proportion of BrdU/PCNA-IR cells. This suggests that PCNA protein is detectable long after cell cycle exit, and that reliance on PCNA may overestimate the length of time a cell remains in the cell cycle. These findings will be critical for future studies examining the regulation of SGZ precursor kinetics in adult mice, and hopefully will encourage the field to move beyond counting BrdU-IR cells to a more mechanistic analysis of adult neurogenesis.

Orphanin FQ/nociceptin and naloxone benzoylhydrazone activate distinct receptors in BE(2)-C human neuroblastoma cells.

kappa(3) opioid receptors have a unique binding and analgesic profile, as originally defined by naloxone benzoylhydrazone (NalBzoH). Although antisense studies demonstrated the close relationship between kappa(3) opioid and Orphan opioid receptor-like receptor (ORL1) and implied they were generated from the same gene, these studies also revealed differences in the sensitivity profiles of NalBzoH and orphanin FQ/nociceptin (OFQ/N), indicating that they were not identical. To help define the relationship between kappa(3) and ORL1 receptors, we utilized BE(2)-C human neuroblastoma cells that natively express functional ORL1 and kappa(3) opioid receptors. (125)I-[Tyr(14)]OFQ/N binds to a single population of receptors in BE(2)-C cells. Competition binding and adenylyl cyclase studies clearly illustrated marked selectivity differences between the ORL1 and the kappa(3) sites. Furthermore, antisense DNA targeting ORL1 blocked the inhibition of cAMP by OFQ/N, but not by NalBzoH. Thus, the receptor mechanisms mediating the activity of OFQ/N and NalBzoH in BE(2)-C cells are distinct.

beta-Funaltrexamine inactivates ORL1 receptors in BE(2)-C human neuroblastoma cells.

The potential interactions of natively expressed mu-opioid and opioid receptor-like (ORL1) receptors were studied by exposing intact BE(2)-C cells to agonists or antagonists for 1 h. Pretreatment with the mu-opioid receptor agonist, [D-Ala(2), N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), or the ORL1 receptor agonist, orphanin FQ/nociceptin desensitized both mu-opioid and ORL1 receptor responses. beta-Funaltrexamine (beta-FNA) pretreatment also blocked both mu-opioid and ORL1 receptor responses, but only mu-opioid receptor binding was reduced. Moreover, beta-FNA (1 microM) failed to inhibit specific ORL1 receptor binding.