Neuroscience
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Using Drosophila mutants and pharmacological blockers, we provide the first evidence that distinct types of K(+) channels differentially influence sub-cellular Ca(2+) regulation and growth cone morphology during neuronal development. Fura-2-based imaging revealed in cultured embryonic neurons that the loss of either voltage-gated, inactivating Shaker channels or Ca(2+)-gated Slowpoke BK channels led to robust spontaneous Ca(2+) transients that preferentially occurred within the growth cone. In contrast, loss of voltage-gated, non-inactivating Shab channels did not show such a disparity and sometimes produced soma-specific Ca(2+) transients. ⋯ Loss of Shaker currents increased the size of lamellipodia and the number of filopodia, structures associated with the actin cytoskeleton. Interestingly, loss of Slowpoke currents strongly influenced tubulin regulation, enhancing the number of microtubule loop structures per growth cone. Together, our findings support the idea that individual K(+) channel subunits differentially regulate spontaneous sub-cellular Ca(2+) fluctuations in growing neurons that may influence activity-dependent growth cone formation.
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Comparative Study
Acute hypoxia and programmed cell death in developing CNS: Differential vulnerability of chick optic tectum layers.
The chick optic tectum displays an alternating pattern of cellular and plexiform layers and at embryonic day (ED) 12 there are mainly four cellular layers: transient cell compartment 3 (TCC3), compartment "h-i-j"(C"h-i-j"), stratum griseum centrale (SGC) and subventricular zone (SvZ). In the present work we characterized the programmed cell death (PCD) of these layers and their vulnerability to acute hypoxia at ED12, and also identified the main cellular type involved in hypoxic cell death. The colocalization of three independent markers of cell degeneration: pyknotic nuclei by Hoechst staining, fragmented DNA by TdT-mediated dUTP nick-end labeling (TUNEL), and presence of active caspase-3 by immunofluorescence, was analyzed in embryos that developed in normoxic conditions (control embryos) and embryos that were subjected to hypoxia (8% O(2)/92% N(2)) for 60 min (hypoxic embryos), followed by 0-12 h of normoxic recovery. ⋯ In addition, the significant colocalization between the neuron specific nuclear protein (NeuN) and TUNEL signal showed that hypoxia affected primarily neurons. In conclusion, our findings demonstrate that in the chick optic tectum at ED12, PCD is layer dependent and that acute hypoxia causes a transient increase in neuronal death in a delayed fashion, which is also layer dependent. The morphological features of the neuronal death process at the light microscope level resembled apoptosis.
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Osmoprotective genes are tonicity-activated genes involved in cellular osmoadaptation to hypertonicity and considered to be regulated by a specific transcription factor called tonicity-responsive enhancer-binding protein (TonEBP). In the brain we had previously established that TonEBP was expressed and tonicity-induced in neurons only. Here we have compared in various brain regions of rats subjected to systemic hypertonicity, the cellular expression of TonEBP through immunocytochemistry and the cellular expression of osmoprotective genes, namely aldose reductase (AR), sodium-dependent myo-inositol transporter (SMIT), betaine/GABA transporter (BGT1) and taurine transporter (TauT), by in situ hybridization using non-radioactive digoxigenin-labeled riboprobes. ⋯ The present work reveals large discrepancies between the cellular distribution of the tonicity-induced expression of osmoprotective genes and that of their regulatory transactivator TonEBP. Depending on the cell subsets and the osmoprotective genes, TonEBP may appear insufficient or conversely unnecessary for the tonicity-induced activation of an osmoprotective gene. Altogether our results show that brain cells, even from the same class, activate distinct osmoprotective genes through distinct activation processes to adapt to hypertonicity.
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Activation of D1-like (D1, D5) or D2-like (D1, D3, D4) dopamine receptors in the nucleus accumbens shell is sufficient to reinstate cocaine-seeking behavior in rats. The goal of these experiments was to assess whether cooperative activation of D1-like and D2-like dopamine receptors in the accumbens shell is required to promote cocaine reinstatement. Rats were initially trained to self-administer cocaine (0.25 mg, i.v.) using a fixed-ratio schedule of reinforcement for approximately 21 days. ⋯ Similarly, administration of the selective D1/5 dopamine receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH-23390) (1.0 microg) into the nucleus accumbens shell prior to quinpirole (3.0 microg) blocked reinstatement of drug-seeking behavior elicited by this D2/3 dopamine receptor agonist. Moreover, intra-accumbal shell co-administration of subthreshold doses of quinpirole (1.5 microg) and SKF-81297 (0.1 microg) promoted cocaine-seeking behavior. Collectively, these results indicate that cooperative activation of D1-like and D2-like dopamine receptors in the nucleus accumbens shell is necessary to reinstate cocaine seeking in rats.
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Comparative Study
The intra-arterial injection of microglia protects hippocampal CA1 neurons against global ischemia-induced functional deficits in rats.
In the present study, we have attempted to elucidate the effects of the intra-arterial injection of microglia on the global ischemia-induced functional and morphological deficits of hippocampal CA1 neurons. When PKH26-labeled immortalized microglial cells, GMIR1, were injected into the subclavian artery, these exogenous microglia were found to accumulate in the hippocampus at 24 h after ischemia. In hippocampal slices prepared from medium-injected rats subjected to ischemia 48 h earlier, synaptic dysfunctions including a significant reduction of synaptic responses and a marked reduction of long-term potentiation (LTP) of the CA3-CA1 Schaffer collateral synapses were observed. ⋯ Furthermore, the arterial-injected microglia prevented the ischemia-induced decline of the brain-derived neurotrophic factor (BDNF) levels in CA1 neurons. These observations strongly suggest that the arterial-injection of microglia protected CA1 neurons against the ischemia-induced neuronal degeneration. The restoration of the ischemia-induced synaptic deficits and the resultant reduction of the BDNF levels in CA1 neurons, possibly by the release of diffusible factor(s), might thus contribute to the protective effect of the arterial-injection of microglia against ischemia-induced neuronal degeneration.