We have established that sensitivity to volatile anesthetics is strongly altered by mutations in two ion channel genes, narrow abdomen (na) and the ryanodine receptor (Ryr). The na gene encodes an unusual homolog of voltage-gated sodium and calcium channels that is expressed broadly in the central nervous system of all metazoans, and whose human ortholog, NALCN, has been implicated in susceptibility to bipolar disorder. In addition to their effects on general anesthesia, na mutations affect the arousal state of flies in the absence of anesthetics. Free roaming mutant flies have a tendency to periodically (every 3-10 seconds) lose postural control and enter a quasi-sleep state from which they spontaneously recover. The period of this abrupt shift in postural control correlates with a rhythmic electrical signal that can be recorded from the eyes of mutant flies, suggesting that this behavior reflects a rhythmic change in nervous system excitability. As will be described in detail below, mutations in na also interfere with social aggregation. The physiological role of NA remains mysterious, to a large degree because of the limited information available regarding accessory proteins that regulate its function. Previous collaborations identified two proteins, UNC79 and UNC80, which are required for NA expression and which modulate the function of the mammalian NALCN. In an on-going collaboration with Dr. Hong-Sheng Li's laboratory, we have combined isotopic labeling techniques with mass-spectrographic protein identification, to identify additional proteins that physically interact with the NA channel. The best characterized of these is called Lesh on the basis of mutants leg shaking behavior. Mutations in lesh, like those in unc-79 and unc-80, block expression of NA protein and produce phenotypes identical to na mutants, including periodic loss of postural control and electrical oscillations in the visual system. However, unlike the other known accessory subunits, Lesh protein expression is not reduced in na mutants, and lesh overexpression results in elevated levels of NA protein. Therefore, Lesh appears to be a critical regulator of NA expression, and is therefore likely to be a key factor in the regulation of excitability and anesthetic sensitivity. As mentioned above, na mutations affect the tendency of flies to aggregate when placed in an arena that limits movement to two dimensions, a phenomenon called local enhancement. As reported previously, this tendency is independent of the sex or social experience of the flies, requires visual input, and is blocked by very low concentrations of volatile anesthetics. na mutations significantly reduce the tendency of flies to cluster, as measured by their social space index. Replacing na function in neurons that express the neurotransmitter acetylcholine returns their behavior to normal. Interestingly, knocking down na function in neurons of the mushroom bodies, complex neuropils involved in multimodal integration and learning, significantly reduces social space index. Therefore, this behavior requires function of the NA ion channel in a region that mediates complex neural processing. We previously reported that sensitivity to halothane strongly correlates with gene dosage of the ryanodine receptor (Ryr), an ion channel that mediates release of Ca2+ from internal stores. We showed that this effect is mediated by the nervous system, is selective for halothane, and that halothane activates Ca2+ release through Ryr in cultured cells and in central neurons. During the course of the project we identified a collection of point mutants in the Ryr gene, some of which cause truncations, which are predicted to eliminate function and which reduce sensitivity to halothane. Others cause substitutions of highly conserved amino acids and enhance the responsiveness of flies to halothane. To further understand the basis of the anesthetic hypersensitivity of the point mutants, we examined Ca2+ flux in identified neurons of wild-type and mutant Drosophila. Both resistant and hypersensitive mutants showed significantly reduced responses to caffeine, a known Ryr agonist. However, when challenged with halothane, neurons in hypersensitive mutants showed a significantly enhanced response. This suggests that the mutation selectively alters the response of the Ryr channel, enhancing the effect of some agonists but not others. On the basis of whole-cell patch clamp recordings, the primary electrophysiological effect of halothane is a strong hyperpolarization, indicating that internal Ca2+ released by Ryr activates an inhibitory conductance. Therefore, behavioral genetic experiments indicate that Ryr is intimately involved in anesthesia, while imaging and electrophysiology suggest a model in which halothane activates Ryr, causing release of Ca2+ into the cytoplasm, resulting in neuronal inhibition. During the past year, we also completed a collaborative project regarding a gene associated with autism, a fly homolog of an adhesion molecule called Neuroligin (NLG). Drosophila neuroligin-1 (nlg1) has been shown to be required for the homeostatic growth and function of a prototypical synapse, the larval neuromuscular junction. We found that overexpression of NLG1 in the muscle perturbs responsiveness of glutamate receptors (GluRs) and increases the size of the synaptic terminal, but inhibits glutamate release from the nerve terminal. Overexpression-driven terminal growth and inhibition of release do not occur in the absence of Neurexin1 (NRX1), the presumed presynaptic partner of NLG1, indicating that signaling from the muscle to the neuron through the NLG1- NRX1 complex is required. Disruption of GluR function by NLG1 overexpression is insensitive to NRX1 function, indicating that the phenotype is autonomous to the muscle. Manipulations that cause abnormal long-term strengthening of the synapse, such as hyperactivity and upregulation of phosphoinositide-3-kinase, downregulate NLG1 expression. These results suggest NLGs are positioned to mediate transynaptic signaling in response to pathways that regulate long-term synaptic change.
