Background

The fruit fly (Drosophila melanogaster) has been used to make key discoveries in a variety of fundamental areas in neuroscience including learning and memory (Bolduc et al., 2008; Cervantes-Sandoval et al., 2016), synapse formation and regulation (Genç et al., 2017), and circadian rhythms (Allada et al., 1998; Guo et al., 2016). Mutants identified through both forward and reverse genetic screens have also provided useful models of human neurological disorders including Fragile X syndrome, Parkinson’s Disease, Huntington Disease and epilepsy disorders (Pallos et al., 2008; Parker et al., 2011; Liu et al., 2012; Sears and Broadie, 2017). Much of what we have learned in this research comes from electrophysiological recordings and calcium imaging at the neuromuscular junction (NMJ), from neurons in dissociated primary culture, or from the central nervous system of embryos and larvae. Though these methods have been instrumental in our understanding, to elucidate the underlying cellular mechanisms of neurological processes in adult animals, it is important to have electrophysiological access to individual neurons of the adult brain.

Recordings from neurons in the adult CNS were made possible by development of two complementary systems in the mid-2000s. One involves exposing and desheathing a small area of brain making it possible to obtain intracellular recordings from neurons in a live, behaving adult fly (Wilson et al., 2004; Hige et al., 2015; Nagel and Wilson, 2016). This preparation is best suited to recording from populations of neurons on the dorsal surface of the brain. The second preparation involves removing the whole brain from the adult head capsule and placing it in a recording chamber (Gu and O’Dowd, 2006 and 2007). This provides access to neurons in the entire brain and allows for easy environmental manipulations. Although the process is invasive and may cause damage to the brain, intact neurons and functional circuits can be persevered and maintained for up to one hour after skillful dissection. Labs have used whole brain dissection and whole-cell recordings to characterize the electrical properties of circadian neurons (Sheeba et al., 2008b), uncover the electrical cellular mechanisms responsible for sleep and arousal (Sheeba et al., 2008a), discover a new light-sensing pathway in the brain (Ni et al., 2017), determine the mechanism of action for a common pesticide (Qiao et al., 2014), find a memory suppressor miRNA that regulates an autism susceptibility gene (Guven-Ozkan et al., 2016), and describe synaptic dysfunction in a model of Parkinson’s Disease (Sun et al., 2016).

Our lab uses this protocol extensively to study the cellular mechanisms of genetic epilepsy associated with mutations in SCN1A, a gene that encodes NaV1.1 sodium channels that are highly expressed in inhibitory, GABAergic neurons in the human brain. Using homologous recombination, and more recently CRISPR/Cas9 mediated gene editing, we have introduced specific SCN1A missense mutations into the same location in the Drosophila sodium channel gene, para. We have shown that all of the mutations causing febrile seizure phenotypes in humans that we have examined (K1270T, S1231R, R1648H/C), also result in heat-induced seizure phenotypes in the adult fly (Sun et al., 2012; Schutte et al., 2014 and 2016). To evaluate how specific mutations alter sodium currents and neuronal activity, we perform electrophysiological analyses of sodium currents in knock-in flies carrying SCN1A mutations, focused primarily on GABAergic, local neurons (LNs) in the antennal lobe. Whole-cell recordings from the cell bodies of LNs can be used to evaluate sodium currents and firing properties in mutant compared to wild-type neurons. The ability to rapidly exchange extracellular recording solutions in the ex vivo preparation allows fast and reversible elevation of the temperature to assess constitutive and temperature-dependent changes in sodium currents and firing properties in knock-in mutant compared to wild-type neurons. Fast perfusion also facilitates evaluation of the acute effects of potential anti-convulsant drugs on sodium currents and firing properties. Here we present our updated protocol for ex vivo whole-cell recordings in adult Drosophila brains, including fly dissection and preparation, data acquisition, and analysis.