HI-induced bax translocation to the mitochondria, ER and nucleus correlates with the activation of subcellular cell death signaling cascades

Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603

phological defects suggesting that these lines contain mutations in genes that are specifically required for the tangential migration of FBM neurons. We are continuing to identify new mutants and map these mutations to chromosomes. We expect that this screen will aid us to elucidate the molecular and genetic pathways underlying the development of branchiomotor neurons. Keywords: Zebrafish; Facial motor neuron; Neuronal migration; Forward genetic screen

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the biochemical estimation of the enzymatic activity and protein concentration of SDH in LGB and SC (P < 0.05) in the treatment groups compared to the control groups. Reduced staining intensity and biochemical quantification of SDH suggests that ethanol impairs the metabolic process via Kreb’s cycle in the neurons of these visual relay centers investigated. Keywords: Alcohol; Lateral geniculate body; Superior colliculus; Vision

doi:10.1016/j.ijdevneu.2006.09.172 doi:10.1016/j.ijdevneu.2006.09.173

[P111]

[P112] Chronic ethanol administration impaired activity of succinic dehydrogenase in the lateral geniculate body and superior colliculus of adult wistar rats F.A. Fakoya 1,∗ , P.B. Fakunle 2 , P.D. Shallie 3 1 Obafemi Awolowo University, Nigeria; 2 Ladoke Akintola Uni-

versity of Technology, Nigeria; Nigeria

3 Olabisi

Onabanjo University,

Ethanol has been established to impair vision particularly at the periphery (retina), but the level of impact on neuronal metabolic activity of the visual pathway have not been investigated, hence the effects of chronic administration of ethanol on the activity of succinic dehydrogenase (SDH) was investigated on the intracranial visual relay centres—lateral geniculate body (LGB) and superior colliculus (SC) of the adult wistar rat. Forty adult wistar rats (average weight 200 g) were randomly distributed into four groups T1, T2, C1 and C2 (n = 10). The animals were fed with standard mouse chow and provided water ad libitum. The animals in group T1 and T2 were given 25% ethanol in 2% sucrose solution as their drinking water, while C1 animals received distilled water and C2 animals received 2% sucrose solution as controls. All the groups were exposed for a period of 6 weeks except T2 animals that were allowed an extra 2-week ethanol withdrawal period. The animals were sacrificed by cervical dislocation, the whole brain removed, weighed and frozen. Frozen brains (n − 5) were trimmed to the regions of LGB and SC by Paxinos stereotaxic coordinate method and cryostat sections obtained at 10 ␮m thickness. Sections were histochemically stained for the enzyme SDH according to the method of (Nachlas et al., 1957), as well as with nissl stain for neurodifferentiation and counting. The remaining brains (n − 5) were dissected at 4 ◦ C to obtain the LGB and SC, pooled and homogenized for biochemical estimation of the SDH activity and protein concentration, respectively. Chronic administration of ethanol resulted in a 13% and 16% loss of neurons in the LBG and SC, respectively (P < 0.01). However, no abnormal morphology of the neuron was observed. The brains from the treatment group weighed less than the controls but this difference was not statistically significant. The histochemical findings showed a decreased staining intensity of SDH in both LGB and SC of treatment sections compared to the controls, which was supported by the mean valves obtained from

HI-induced bax translocation to the mitochondria, ER and nucleus correlates with the activation of subcellular cell death signaling cascades M. Gill ∗ , R. Perez-Polo The University of Texas- Medical Branch, USA Hypoxic-ischemic (HI) insult produces a characteristic necrotic core surrounded by an apoptotic penumbra. Recent reports correlate increased activation of ER cell death signaling cascades with increased necrosis, and increased activation of the intrinsic mitochondrial cell death pathway with increased apoptosis. In addition, current research proposes that Bax, a known protagonist in the intrinsic pathway, has alternative functions at the ER interface. We hypothesize that HI insult increases Bax expression at the mitochondria, ER and nucleus, and that this increased Bax expression correlates with increased activation of respective organelle cell death signaling cascades. Utilizing a permanent left common carotid electrocoagulation followed by a 90 min hypoxic interval (7.8% O2 ) in P7 Wistar rat pups, we extracted the ipsilateral cortex at 0.5, 1, 2, 3, 6, 12, 24, 48 and 72 h after HI for differential ultracentrifugationmediated enrichment of organelles for Western blot analysis and DNA-histone cell death ELISA. In addition, we prepared tissue for confocal immunohistochemical analysis at 1, 3, 6 and 24 h after HI. We observed that HI insult increased cell death at all time points after HI. Furthermore, HI significantly increased mitochondrial, ER and nuclear Bax expression. Correlating with increased Bax expression, HI increased cytosolic cleaved caspase 3 expression and decreased mitochondrial AIF expression. HI, also, significantly increased ER cleaved caspase 12 expression. In addition, HI increased nuclear phospho-p53 (Ser15) expression. Confocal immunohistochemical analysis confirmed the increase in nuclear Bax expression illustrating a peri- and intranuclear staining pattern. Thus, HI increased Bax expression at the mitochondria, ER and nucleus, and this increased Bax expression correlated with increased activation of respective organelle cell death signalling cascades, suggesting a mediating role for Bax in the integration of HI-induced neuropathological signaling. Further analysis of Bax actions at each organelle interface may provide insights into

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Poster abstracts / Int. J. Devl Neuroscience 24 (2006) 495–603

the operant mechanisms by which Bax engages each respective cell death signaling cascade. Acknowledgements Research supported in part by PO1 HD 039833 and NIEHS Environmental Toxicology Training Grant T32-07254. M.B.G. Keywords: Bax; Translocation; Cell death doi:10.1016/j.ijdevneu.2006.09.174 [P113] High anxiety/aggressive mice exhibit serotonergic developmental deficits that persist into adulthood D.L. Nehrenberg 1,∗ , R.E. Peterson 1 , M. Styner 1 , H.-G. Kim 2 , W.C. Wetsel 2 , J.M. Lauder 1 1 University

of North Carolina at Chapel Hill, USA; University, USA

2 Duke

Mouse models of human affective disorders are consistently associated with deficient serotonergic function, as manifested by reduced serotonin (5-HT) immunoreactive (IR) neurons and 5-HT contents. Adult ICR mice selectively bred for highaggression (NC900) reproduce common behavioral, pharmacological and neurological traits of human anxiety compared to mice selectively bred for low-aggression (NC100). Here, we examined whether NC900 mice also reproduce what is considered to be a primary etiologic feature of affective disorders, alterations in serotonin development. First, we evaluated whether embryonic day 13.5 (E13.5) NC900 mice exhibited decreased numbers of 5-HT IR neurons and reduced 5-HT contents by HPLC-EC, relative to NC100 mice and unselected ICR mice, and determined whether these deficits persisted into adulthood. Second, we determined whether our 5-HT neuronal measures were negatively correlated with adult anxiety-like behaviors and aggression. E13.5 NC900 animals exhibited dramatic reductions in the number of 5-HT IR neurons compared to NC100 mice, and these deficits persisted into adulthood (Fig. 1). NC900 mice also show reduced 5-HT contents in ventral striatum and amygdala, primary brain sites of motivation and emotion. Moreover, NC900 reliably expressed more anxiety-like and aggressive behaviors than NC100 animals. Taken together, these results, obtained from a forward, behavioral genetic mouse model, corroborate results obtained from a reverse genetic model of anxiety and aggression (Hendricks et al., 2003). Our findings provide further support for the notion that deficient serotonergic development is a primary pathogenic feature of affective disorders, like anxiety and aggression.

Fig. 1. (A) E13.5 NC900 mice express deficits in number of 5-HT IR cells relative to NC100 mice, (B) which persist into adulthood.

Acknowledgements NIH grants T32 ES007126 and MRDDRC P30 HD031108. Keywords: Serotonin; Anxiety; Aggression; GABAA receptor doi:10.1016/j.ijdevneu.2006.09.175 [P114] High resolution imaging of facial motor neuron migration in zebrafish P.K. Grant 2,∗ , C.B. Moens 1 1 HHMI,

Fred Hutchinson Cancer Research Center, USA; of Washington, USA

2 University

In a developing brain, the spatial patterning required for appropriate specification of neurons is not necessarily that required for the formation of proper connectivity. This means that certain populations of neurons must migrate to allow the environment in which the neuron finally resides to be different from that in which it was specified. The facial motor neurons (FMNs) of cranial nerve VII in the vertebrate hindbrain provide an opportunity to study tangential migration, migrating posteriorly from their birthplace in rhombomere 4 (r4) to reside in rhombomere 6 (r6). While a number of factors have been shown to be required for this migration to occur, little is known about the actual cellular behavior of the facial motor neurons as they migrate and how they interact with other cells in their migratory path. By utilizing the Isl1 enhancer region we can drive expression of various fluorescent proteins in the cranial motor neurons and combine that with complementary reporters in surrounding cells allowing for four-dimensional imaging of both migrating