Circadian clocks are endogenous timekeeping mechanisms, which give the sense of time-of-day to most organisms. To help the organisms to adapt to daily fluctuations in the environment, circadian clocks are reset by various environmental cues. Light is one of the cardinal environmental cues that synchronize circadian clocks. In a standard 12:12 light-dark condition, Drosophila exhibits bimodal activity pattern in the anticipation of lights-on and -off. The morning peak of activity is generated by Pigment Dispersing Factor (PDF) positive small ventro-lateral neurons (sLNvs) called the M-oscillators, while the evening peak of activity is generated by the dorsolateral neurons (LNds) and the 5th sLNv together referred to as the E-oscillators. Since the Drosophila circadian clock is extremely sensitive to light, a brief light exposure can robustly shift the phase of circadian behavior. The model for this resetting posits that circadian photoreception is cell-autonomous: the photoreceptor CRYPTOCHROME (CRY) senses light, binds to TIMELESS (TIM) and promotes its degradation via JETLAG (JET). However, it was more recently proposed that interactions between circadian neurons are also required for phase resetting. The goal of my thesis was to map the neuronal circuitry controlling circadian photoreception in Drosophila. In the first half of my dissertation (Chapter II), using a novel severe jetset mutant and JET RNAi, we identified M- and E-oscillators as critical light sensing neurons. We also found that JET functions cell-autonomously to promote TIM degradation in M- and E-oscillators, and non-autonomously in E-oscillators when expressed in M-oscillators. However, JET expression was required in both groups of neurons to phase-shift locomotor rhythms in response to light input. Thus M- and E-oscillators cooperate to shift circadian behavior in response to photic cues. In chapter III, unexpectedly, we found that light can delay or advance circadian behavior even when the M- or E-oscillators are genetically ablated or incapacitated suggesting that behavioral phase shifts in response to light are largely a consequence of cell autonomous light detection by CRY and governed by the molecular properties of the pacemaker. Nevertheless, neural interactions are integral in modulating light responses. The M-oscillator neurotransmitter, PDF was important in coordinating M- and E-oscillators for circadian behavioral response to light input. Moreover, we uncover a potential role for a subset of Dorsal neurons in control of phase advances specifically. Hence, neural modulation of cell autonomous light detection contributes to plasticity of circadian behavior and facilitates its adaptation to environmental inputs.

The mammalian spinal cord contains the neuronal circuitry necessary to generate rhythmic locomotor activity in the absence of inputs from the higher brain centre or sensory system. This circuitry is regulated by local neuromodulatory inputs, which can adjust the strength and timing of locomotor output. The free radical gas nitric oxide has been shown to act as an important neuromodulator of spinal circuits, which control locomotion in other vertebrate models such as the tadpole and lamprey. Despite this, the involvement of the NO-mediated soluble guanylate cyclase/cyclic guanosine monophosphate secondary messenger-signalling pathway (NO/sGC/cGMP) in mammalian locomotion has largely been under-investigated. The NADPH diaphorase histochemical reaction was used to identify sources of NO in the lumbar spinal cord. The largest population NADPH diaphorase reactive neurons were located in the dorsal horn, followed by the laminae of the ventral horn, particularly around the central canal (lamina X) and lamina VII. NADPH diaphorase reactive neurons were found along a rostrocaudal gradient between lumbar segments L1 to L5. These results show that that discrete neuronal sources of NO are present in the developing mouse spinal cord, and that these cells increase in number during the developmental period postnatal day P1 – P12. NADPH diaphorase was subsequently used to identify NADPH diaphorase reactive neurons at P12 in the mouse model of ALS using the SODG93A transgenic mouse. Physiological recordings of ventral root output were made to assess the contribution of NO to the regulation induced rhythmic fictive locomotion in the in vitro isolated spinal cord preparation. Exogenous NO inhibits central pattern generator (CPG) output while facilitating and inhibiting motor neuron output at low and high concentrations respectively. Removal of endogenous NO increases CPG output while decreasing motor neuron output and these effects are mediated by cGMP. These data suggest that an endogenous tone of NO is involved in the regulation of fictive locomotion and that this involves the NO/sGC/cGMP pathway. Intracellular recordings from presumed motor neurons and a heterogeneous, unidentified sample of interneurons shows that NO modulates the intrinsic properties of spinal neurons. These data suggest that the net effect of NO appears to be a reduction in motor neuron excitability.