The Effect of Optogenetically Activating Glia on Neuronal Function

Glia, or glial cells, are considered a vital component of the nervous system, serving as an electrical insulator and a protective barrier from the interstitial (extracellular) media. Certain glial cells (i.e., astrocytes, microglia, and oligodendrocytes) within the CNS have been shown to directly affect neural functions, but these properties are challenging to study due to the difficulty involved with selectively-activating specific glia. To overcome this hurdle, we selectively expressed light-sensitive ion channels (i.e., channel rhodopsin, ChR2-XXL) in glia of larvae and adult Drosophila melanogaster. Upon activation of ChR2, both adults and larvae showed a rapid contracture of body wall muscles with the animal remaining in contracture even after the light was turned off. During ChR2-XXL activation, electrophysiological recordings of evoked excitatory junction potentials within body wall muscles of the larvae confirmed a train of motor nerve activity. Additionally, when segmental nerves were transected from the CNS and exposed to light, there were no noted differences in quantal or evoked responses. This suggests that there is not enough expression of ChR2-XXL to influence the segmental axons to detect in our paradigm. Activation of the glia within the CNS is sufficient to excite the motor neurons.

Investigations into modifications of neuromuscular physiology by axonal transport disruptions in Drosophila SOD1 mutants

Cu/Zn superoxide dismutase (SOD1) is a cytoplasmic antioxidant enzyme, which, when mutant in humans, is linked to familial cases of the motor neurodegenerative disease amyotrophic lateral sclerosis (ALS). The Drosophila SOD1 gene (Sod) shares a highly conserved sequence with the human homolog, and this study includes examinations of the established hypomorphic n108 allele (Sodn108), alongside a knock-in construct of the G85R allele found in human ALS patients (SodG85R). In addition to previously documented decreased adult lifespan and attenuated motor function, we show that Sod mutant Drosophila can display significant mortality during larval and pupal development. Immunostaining of neuronal membrane at neuromuscular synapses in Sod mutant larvae revealed presynaptic terminals of abnormal morphology, with incompletely segregated and enlarged synaptic boutons along the motor terminal branches, in which vital staining indicated mitochondrial aggregation. We demonstrate strong genetic interactions between SodG85R and the axon transport-linked Pk mutants PkPk and PkSple in larval NMJ morphology and neuromuscular transmission. Intracellular recordings of larval excitatory junction potentials (EJPs) demonstrate enhanced EJP size in the double-mutant of PkPk and SodG85R. To examine synaptic terminal excitability, maintained by Ca2+ channel action and independent of Na+ channel function, we used the NaV blocker TTX, along with the KV1 blocker 4-aminopyridine (4-AP) and the commonly used broad-spectrum K+ channel blocker tetraethylammonium (TEA). We were able to induce prolonged “plateau-like” EJPs, which were further extended in Pk mutants and Pk;Sod double-mutants. These observations were corroborated with focal EJP recording from individual boutons. Altogether, this study highlights alterations in synaptic morphology and function at a developmental stage prior to neurodegeneration and death of Sod mutant organisms, along with a potential role of axonal transport in the maintenance of neuronal health.

Action of octopamine and tyramine on muscles of Drosophila melanogaster larvae

Octopamine (OA) and tyramine (TA) play important roles in homeostatic mechanisms, behavior, and modulation of neuromuscular junctions in arthropods. However, direct actions of these amines on muscle force production that are distinct from effects at the neuromuscular synapse have not been well studied. We utilize the technical benefits of the Drosophila larval preparation to distinguish the effects of OA and TA on the neuromuscular synapse from their effects on contractility of muscle cells. In contrast to the slight and often insignificant effects of TA, the action of OA was profound across all metrics assessed. We demonstrate that exogenous OA application decreases the input resistance of larval muscle fibers, increases the amplitude of excitatory junction potentials (EJPs), augments contraction force and duration, and at higher concentrations (10−5 and 10−4 M) affects muscle cells 12 and 13 more than muscle cells 6 and 7. Similarly, OA increases the force of synaptically driven contractions in a cell-specific manner. Moreover, such augmentation of contractile force persisted during direct muscle depolarization concurrent with synaptic block. OA elicited an even more profound effect on basal tonus. Application of 10−5 M OA increased synaptically driven contractions by ∼1.1 mN but gave rise to a 28-mN increase in basal tonus in the absence of synaptic activation. Augmentation of basal tonus exceeded any physiological stimulation paradigm and can potentially be explained by changes in intramuscular protein mechanics. Thus we provide evidence for independent but complementary effects of OA on chemical synapses and muscle contractility.

Loss of responsiveness of circular smooth muscle cells from the guinea pig ileum is associated with changes in gap junction coupling

Gap junction coupling and neuromuscular transmission to smooth muscle were studied in the first 4 h after preparations were set up in vitro. Intracellular recordings were made from smooth muscle cells of guinea pig ileum. Fast inhibitory junction potentials (IJPs) were small (1.3 ± 1.0 mV) in the first 30 min but increased significantly over the first 120 min to 15.8 ± 0.9 mV ( n = 12, P < 0.001). Comparable increases in slow IJPs and excitatory junction potentials were also observed. During the same period, resting membrane potential depolarized from −58.8 ± 1.4 to −47.2 ± 0.4 mV ( n = 12, P < 0.001). Input resistance, estimated by intracellular current injection, decreased in parallel ( P < 0.05), and dye coupling, measured by intracellular injection of carboxyfluorescein, increased ( P < 0.001). Input resistance was higher and dye coupling was less in longitudinal than circular smooth muscle cells. Gap junction blockers [carbenoxolone (100 μM), 18β-glycyrrhetinic acid (10 μM), and 2-aminoethoxydiphenyl borate (50 μM)] hyperpolarized coupled circular smooth muscle cells, reduced the amplitude of fast and slow IJPs and excitatory junction potentials, increased input resistance, and reduced dye coupling. Local application of ATP (10 mM) mimicked IJPs and showed comparable increases in amplitude over the first 120 min; carbenoxolone and 2-aminoethoxydiphenyl borate significantly reduced ATP-evoked hyperpolarizations in coupled cells. In contrast, synaptic transmission between myenteric neurons was not suppressed during the first 30 min. Gap junction coupling between circular smooth muscle cells in isolated preparations was initially disrupted but recovered over the next 120 min to a steady level. This was associated with potent effects on neuromuscular transmission and responses to exogenous ATP.

Heat Shock–Mediated Thermoprotection of Larval Locomotion Compromised by Ubiquitous Overexpression of Hsp70 in Drosophila melanogaster

Maintaining the competence of locomotor circuitry under stressful conditions can benefit organisms by enabling locomotion to more tolerable microhabitats. We show that prior heat shock protects locomotion and the locomotor central pattern generator of larval Drosophila against subsequent hyperthermic stress. We combined molecular genetic, electrophysiological, and behavioral techniques to investigate heat shock–mediated thermoprotection. Prior heat shock increased the distance traveled by larvae during hyperthermia before failure. The frequency of the rhythm of peristaltic locomotor contractions and the velocity of locomotion were both less thermosensitive after heat shock and were less susceptible to failure at high temperatures. Rhythmic coordinated motor patterns, recorded intracellularly as excitatory junction potentials in body wall muscles of dissected preparations, were centrally generated because patterns could still be generated in the absence of sensory feedback (sensory function disrupted with shibire). Prior heat shock protected central circuit operation during hyperthermic stress by increasing the temperature at which it failed. Overexpression of Hsp70 after a heat shock using transgenic flies ( traII) did not enhance thermoprotection, as expected, but had deleterious effects on parameters of behavior.