McCabe, Brian D.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. The a7 subtype of nAChRs is involved
in neurological pathologies such as Parkinson’s disease, Alzheimer’s disease, addiction, epilepsy and autism spectrum
disorders. The Drosophila melanogaster a7 (Da7) has the closest sequence homology to the vertebrate a7 subunit and it can
form homopentameric receptors just as the vertebrate counterpart. The Da7 subunits are essential for the function of the
Giant Fiber circuit, which mediates the escape response of the fly. To further characterize the receptor function, we
generated different missense mutations in the Da7 nAChR’s ligand binding domain. We characterized the effects of
targeted expression of two UAS-constructs carrying a single mutation, D197A and Y195T, as well as a UAS-construct carrying
a triple D77T, L117Q, I196P mutation in a Da7 null mutant and in a wild type background. Expression of the triple mutation
was able to restore the function of the circuit in Da7 null mutants and had no disruptive effects when expressed in wild
type. In contrast, both single mutations severely disrupted the synaptic transmission of Da7-dependent but not
glutamatergic or gap junction dependent synapses in wild type background, and did not or only partially rescued the
synaptic defects of the null mutant. These observations are consistent with the formation of hybrid receptors, consisting of
D197A or Y195T subunits and wild type Da7 subunits, in which the binding of acetylcholine or acetylcholine-induced
conformational changes of the Da7 receptor are altered and causes inhibition of cholinergic responses. Thus targeted
expression of D197A or Y195T can be used to selectively disrupt synaptic transmission of Da7-dependent synapses in
neuronal circuits. Hence, these constructs can be used as tools to study learning and memory or addiction associated
behaviors by allowing the manipulation of neuronal processing in the circuits without affecting other cellular signaling.

While the mammalian brain functions within a very narrow range of oxygen concentrations
and temperatures, the fruit fly, Drosophila melanogaster, has employed strategies to deal
with a much wider range of acute environmental stressors. The foraging (for) gene encodes
the cGMP-dependent protein kinase (PKG), has been shown to regulate thermotolerance
in many stress-adapted species, including Drosophila, and could be a potential therapeutic
target in the treatment of hyperthermia in mammals. Whereas previous thermotolerance
studies have looked at the effects of PKG variation on Drosophila behavior or excitatory
postsynaptic potentials at the neuromuscular junction (NMJ), little is known about PKG
effects on presynaptic mechanisms. In this study, we characterize presynaptic calcium
([Ca^2+]i) dynamics at the Drosophila larval NMJ to determine the effects of high temperature
stress on synaptic transmission. We investigated the neuroprotective role of PKG modulation
both genetically using RNA interference (RNAi), and pharmacologically, to
determine if and how PKG affects presynaptic [Ca^2+]i dynamics during hyperthermia. We
found that PKG activity modulates presynaptic neuronal Ca^2+ responses during acute
hyperthermia, where PKG activation makes neurons more sensitive to temperatureinduced
failure of Ca^2+ flux and PKG inhibition confers thermotolerance and maintains normal
Ca^2+ dynamics under the same conditions. Targeted motoneuronal knockdown of PKG
using RNAi demonstrated that decreased PKG expression was sufficient to confer thermoprotection.
These results demonstrate that the PKG pathway regulates presynaptic motoneuronal
Ca^2+ signaling to influence thermotolerance of presynaptic function during acute
hyperthermia.