• The Kv2.2 voltage-gated potassium channel: From the gene to its potential physiological role in arousal

      Hermanstyne, Tracey; Misonou, Hiroaki; Mong, Jessica Aurora (2012)
      Voltage-gated potassium (Kv) channels are involved in various physiological processes such as repolarization of neuronal and cardiac action potentials, calcium signaling, cellular proliferation, migration and behavioral rhythms. These channels exhibit distinct biophysical and biochemical characteristics primarily through the formation of heteromeric tetramers into a functional channel. Mammalian Kv2 delayed rectifier channels are, however, the unique exception in this subfamily because previous studies have shown these isotypes are localized in distinct domains of neuronal membranes and lack the capability to form heteromeric channels. In this dissertation, we report a novel form of rat Kv2.2, which has not been previously recognized. Our data indicate that this novel form of Kv2.2 is indeed the predominant form expressed in the brain and is co-localized in the same neuronal membrane domains with Kv2.1. In addition, co-immunoprecipitation and electrophysiology experiments showed that Kv2.1 and Kv2.2 are capable of forming heteromeric channels. However, through specific immunostaining, we found that Kv2.2 is expressed in a specific set of neurons, which have negligible levels of Kv2.1, suggesting a unique physiological role of Kv2.2. These neurons are located in the magnocellular peroptic area (MCPO) and the horizontal limb of diagonal band of Broca (HDB) of the basal forebrain (BF). It has been shown that the MCPO/HDB are implicated in the regulation of cortical activity and the sleep-wake cycle. Using specific immunolabeling and knock-in mice in which GFP is expressed in GABAergic neurons, we found that Kv2.2 is abundantly expressed in a sub-population of GABAergic neurons in this region which establishes Kv2.2 as a molecular target to study the role of this specific sub-population of BF GABAergic neurons. In conclusion we hypothesize, that the Kv2.2-GABAergic neurons has a functional role in the regulation of the sleep-wake cycle. Using c-FOS immunolabeling and polysomnographic recordings of Kv2.2 knockout mice, we found that Kv2.2-GABAergic neurons are preferentially active during the wake state and that the knockout mice exhibit an increase wakefulness phenotype, respectively. Taken all together, these results challenges the present dogma of Kv2 channels as well as reveal a significant aspect of BF GABAergic neurons in the promotion of wakefulness.
    • Nerve gas-induced seizures: Role of basal forebrain cholinergic projections in rapid neuronal and glial activation in the brain

      Zimmer, Lee Alexander; Shipley, Michael T. (1999)
      Soman, an, irreversible inhibitor of cholinesterase, causes intense convulsions, neuropathology and, ultimately, death. We used the immediate early gene product, Fos, as a marker for neuronal activity to pinpoint the earliest brain regions involved in the initiation and maintenance of soman-induced convulsions. The rapid induction of Fos in the piriform, cortex (PC) and the pontine nucleus locus coeruleus (LC), taken together with previous anatomical, electrophysiological, and neurochemical studies, suggests that prolonged, excessive exposure to synaptically released acetylcholine (ACh) and norepinephrine triggers seizures in PC and subsequently in other cortical and subcortical structures. By 24 hr following soman injection, there is marked neuropathology in the PC. Using immunocytochemical markers specific for astrocytes (GFAP) and glia (OX-42), we aimed to determine if soman-induced seizures also cause selective, rapid activation of astrocytes and microglia in the PC and other brain regions. The results demonstrate that: (i) there is a rapid increase (45--60 min) in GFAP staining in astrocytes of the piriform restricted to the same layers in which neurons express Fos; (ii) between 1 and 8 hr, ramified microglia in PC and hippocampus alter their morphology to resemble active then reactive microglia. These results suggest that intensely active neurons provide local signals triggering reactive changes in neighboring glia. To investigate the role of ACh in soman-induced seizures, cholinergic neurons in the nucleus of the diagonal band (NDB) were lesioned unilaterally with 192 IgG-saporin. NDB lesions inhibited the rapid activation of Fos in PC and inhibited changes in GFAP staining induced by staining. Electrical stimulation electrodes were implanted unilaterally in the NDB to focally activate projections to PC. Stimulation of NDB induced Fos and GFAP staining in PC identical to those following soman. Fos and GFAP staining elicited by NDB stimulation was blocked by scopolamine. These results suggest that ACh release from NDB terminals in PC triggers the initiation of seizures and gliosis following soman administration via a muscarinic receptor mechanism. Next, we examined the role of microglia during CNS injury using an in vitro slice paradigm that eliminated blood-borne monocytes and other serum factors before injury. Our findings indicate that resting microglia transform into macrophage-like cells following brain injury in the absence of monocytes and other serum factors. Finally, the effects of stimulation of the NDB on the spontaneous discharge of neurons and evoked field potentials in PC were investigated in anesthetized rats. The results of this study suggest that activation of cholinergic inputs to PC increases the excitability of pyramidal cells, probably by a disinhibitory mechanism involving muscarinic receptor activation.