A potential role for neuronal connexin 36 in the pathogenesis of amyotrophic lateral sclerosis.

Neuronal gap junctional protein connexin 36 (Cx36) contributes to neuronal death following a range of acute brain insults such as ischemia, traumatic brain injury and epilepsy. Whether Cx36 contributes to neuronal death and pathological outcomes in chronic neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), is not known. We show here that the expression of Cx36 is significantly decreased in lumbar segments of the spinal cord of both human ALS subjects and SOD1 mice as compared to healthy human and wild-type mouse controls, respectively.

Death of Neurons following Injury Requires Conductive Neuronal Gap Junction Channels but Not a Specific Connexin.

Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury).

Neuronal gap junctions play a role in the secondary neuronal death following controlled cortical impact.

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI) and epilepsy. Recent studies in mice showed a critical role for neuronal gap junctions in NMDA receptor-mediated excitotoxicity and ischemia-mediated neuronal death. Here, using controlled cortical impact (CCI) in adult mice, as a model of TBI, and Fluoro-Jade B staining for analysis of neuronal death, we set to determine whether neuronal gap junctions play a role in the CCI-mediated secondary neuronal death.

Neuronal gap junction coupling is regulated by glutamate and plays critical role in cell death during neuronal injury.

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI), and epilepsy. The coupling of neurons by gap junctions (electrical synapses) increases during neuronal injury. We report here that the ischemic increase in neuronal gap junction coupling is regulated by glutamate via group II metabotropic glutamate receptors (mGluRs).

Interplay of chemical neurotransmitters regulates developmental increase in electrical synapses.

Coupling of neurons by electrical synapses (gap junctions) transiently increases in the mammalian CNS during development. We report here that the developmental increase in neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein) are regulated by an interplay between the activity of group II metabotropic glutamate receptors (mGluRs) and GABA(A) receptors. Specifically, using dye coupling, electrotonic coupling, Western blots and small interfering RNA in the rat and mouse hypothalamus and cortex in vivo and in vitro, we demonstrate that activation of group II mGluRs augments, and inactivation prevents, the developmental increase in neuronal gap junction coupling and Cx36 expression.

Neuronal gap junctions are required for NMDA receptor-mediated excitotoxicity: implications in ischemic stroke.

N-methyl-D-aspartate receptors (NMDARs) play an important role in cell survival versus cell death decisions during neuronal development, ischemia, trauma, and epilepsy. Coupling of neurons by electrical synapses (gap junctions) is high or increases in neuronal networks during all these conditions. In the developing CNS, neuronal gap junctions are critical for two different types of NMDAR-dependent cell death.

Use of calcium imaging for analysis of neuronal gap junction coupling.

We recently used Western blots for connexin 36 and neuronal dye coupling with neurobiotin to measure developmental decrease in neuronal gap junction coupling in cell cultures. To ask whether Ca2+ imaging also can be used to measure changes in the amount of neuronal gap junction coupling, we defined a Ca2+ coupling coefficient as the percentage of neurons with bicuculline-induced increases in intracellular Ca2+ that are suppressed by blocking gap junctions. We demonstrate in rat and mouse hypothalamic neuronal cultures that the Ca2+ coupling coefficient decreases during culture development, this decrease is prevented by manipulations that also prevent developmental decrease in neuronal gap junction coupling, and the coefficient is low in cultures lacking connexin 36.

Regulation of cholinergic phenotype in developing neurons.

Specification of neurotransmitter phenotype is critical for neural circuit development and is influenced by intrinsic and extrinsic factors. Recent findings in rat hypothalamus in vitro suggest the role of neurotransmitter glutamate in the regulation of cholinergic phenotype. Here we extended our previous studies on the mechanisms of glutamate-dependent regulation of cholinergic phenotypic properties in hypothalamic neurons.

Non-cholinergic excitation in neurons after a chronic glutamate receptor blockade.

Previous experiments revealed a dramatic increase in excitatory acetylcholine transmission in hypothalamic cultures during a chronic decrease in glutamate activity. Data suggested that in the absence of glutamate excitation, acetylcholine becomes the major excitatory neurotransmitter. However, non-cholinergic excitatory activity was also detected in some neurons.

Calcium-dependent regulation of cholinergic cell phenotype in the hypothalamus in vitro.

Glutamate is a major fast excitatory neurotransmitter in the CNS including the hypothalamus. Our previous experiments in hypothalamic neuronal cultures showed that a long-term decrease in glutamate excitation upregulates ACh excitatory transmission. Data suggested that in the absence of glutamate activity in the hypothalamus in vitro, ACh becomes the major excitatory neurotransmitter and supports the excitation/inhibition balance.