Nicotine protects rat hypoglossal motoneurons from excitotoxic death via downregulation of connexin 36

Abstract

Motoneuron disease including amyotrophic lateral sclerosis may be due, at an early stage, to deficit in the extracellular clearance of the excitatory transmitter glutamate. A model of glutamate-mediated excitotoxic cell death based on pharmacological inhibition of its uptake was used to investigate how activation of neuronal nicotinic receptors by nicotine may protect motoneurons. Hypoglossal motoneurons (HMs) in neonatal rat brainstem slices were exposed to the glutamate uptake blocker DL-threo-β-benzyloxyaspartate (TBOA) that evoked large Ca2+ transients time locked among nearby HMs, whose number fell by about 30% 4 h later. As nicotine or the gap junction blocker carbenoxolone suppressed bursting, we studied connexin 36 (Cx36), which constitutes gap junctions in neurons and found it largely expressed by HMs. Cx36 was downregulated when nicotine or carbenoxolone was co-applied with TBOA. Expression of Cx36 was preferentially observed in cytosolic rather than membrane fractions after nicotine and TBOA, suggesting protein redistribution with no change in synthesis. Nicotine raised the expression of heat shock protein 70 (Hsp70), a protective factor that binds the apoptotic-inducing factor (AIF) whose nuclear translocation is a cause of cell death. TBOA increased intracellular AIF, an effect blocked by nicotine. These results indicate that activation of neuronal nicotinic receptors is an early tool for protecting motoneurons from excitotoxicity and that this process is carried out via the combined decrease in Cx36 activity, overexpression of Hsp70 and fall in AIF translocation. Thus, retarding or inhibiting HM death may be experimentally achieved by targeting one of these processes leading to motoneuron death.

Nicotine‐mediated neuroprotection of rat spinal networks against excitotoxicity

“…Although former preclinical studies have indicated that nicotine can protect neurons against insults like excitotoxicity or oxygen/glucose deprivation (Gahring, Meyer, & Rogers, ; More & Dong, ), the present investigation was prompted by the recent observation of nicotine protection against excitotoxicity applied to rat brainstem motoneurons (Corsini et al., , ). There was, however, no report of similar effects on the rat spinal cord, an issue that was explored using an in vitro model of spinal cord injury fully validated in our laboratory.…”

“…In accordance with previous protocols for Ca 2+ imaging (Corsini, Tortora, Rauti, & Nistri, ; Fabbro, Skorinkin, Grandolfo, Nistri, & Giniatullin, ; Sharifullina & Nistri, ), organotypic spinal slices grown in vitro for 3 weeks were incubated with the fluorescent calcium dye Fluo 3‐AM (4 μM, Molecular Probes, Invitrogen, Carlsbad, CA, USA) for 1 hr at room temperature in Dulbecco's modified Eagle's medium. The fluorescent dye was washed out with the same medium containing strychnine (1 μM) and bicuculline (20 μM) for 30 min.…”

Functional up‐regulation of the M‐current by retigabine contrasts hyperexcitability and excitotoxicity on rat hypoglossal motoneurons

“…Immunohistochemistry was performed on 450 μm thick slices as described previously (Corsini et al . ). After stabilization, slices were either immediately fixed in 4% paraformaldehyde for 4 h or were incubated (4 h) in either recording Krebs solution (in m m : 130 NaCl, 3 KCl, 1.5 NaH 2 PO 4 , 1.5 CaCl 2 , 1 MgCl 2 , 25 NaHCO 3 and 12 glucose; pH 7.4; 300–320 mOsm) alone as the sham condition or supplemented with selected drugs and fixed afterwards.…”

“…), Ca 2+ overload (Sharifullina & Nistri, ; Corsini et al . ), oxidative stress, mitochondrial damage (Tortora et al . ) and an unfolded protein response (Corsini et al .…”

“…), nicotine (Corsini et al. , ) and propofol (Ghezzi et al . ), which, although acting on different molecular targets, share the ability to reduce HM hyperexcitability and inhibit bursting.…”