The GABA(A) receptor RDL acts in peptidergic PDF neurons to promote sleep in Drosophila.

Sleep is regulated by a circadian clock that times sleep and wake to specific times of day and a homeostat that drives sleep as a function of prior wakefulness. To analyze the role of the circadian clock, we have used the fruit fly Drosophila. Flies display the core behavioral features of sleep, including relative immobility, elevated arousal thresholds, and homeostatic regulation. We assessed sleep-wake modulation by a core set of circadian pacemaker neurons that express the neuropeptide PDF. We find that disruption of PDF function increases sleep during the late night in light:dark and the first subjective day of constant darkness. Flies deploy genetic and neurotransmitter pathways to regulate sleep that are similar to those of their mammalian counterparts, including GABA. We find that RNA interference-mediated knockdown of the GABA(A) receptor gene, Resistant to dieldrin (Rdl), in PDF neurons reduces sleep, consistent with a role for GABA in inhibiting PDF neuron function. Patch-clamp electrophysiology reveals GABA-activated picrotoxin-sensitive chloride currents on PDF+ neurons. In addition, RDL is detectable most strongly on the large subset of PDF+ pacemaker neurons. These results suggest that GABAergic inhibition of arousal-promoting PDF neurons is an important mode of sleep-wake regulation in vivo.

Differential modulation by nicotine of substantia nigra versus ventral tegmental area dopamine neurons.

Midbrain dopamine (DA) neurons are found in two nuclei, the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). The SNc dopaminergic projections to the dorsal striatum are involved in voluntary movement and habit learning, whereas the VTA projections to the ventral striatum contribute to reward and motivation. Nicotine induces profound DA release from VTA dopamine neurons but substantially less from the SNc. Nicotinic acetylcholine receptor (nAChR) expression differs between these nuclei, but it is unknown whether there are differences in nAChR expression on the afferent projections to these nuclei. Here we have compared the nicotinic modulation of excitatory and inhibitory synaptic inputs to VTA and SNc dopamine neurons. Although nicotine enhances both the excitatory and inhibitory drive to SNc DA cells with response magnitudes similar to those seen in the VTA, the prevalence of these responses in SNc is much lower. We also found that a mixture of nAChR subtypes underlies the synaptic modulation in SNc, further distinguishing this nucleus from the VTA, where alpha7 nAChRs enhance glutamate inputs and non-alpha7 receptors enhance GABA inputs. Finally, we compared the nicotine sensitivity of DA neurons in these two nuclei and found larger response magnitudes in VTA relative to SNc. Thus the observed differences in nicotine-induced DA release from VTA and SNc are likely due to differences in nAChR expression on the afferent inputs as well as on the DA neurons themselves. This may explain why nicotine has a greater effect on behaviors associated with the VTA than the SNc.

The ion channel narrow abdomen is critical for neural output of the Drosophila circadian pacemaker.

Circadian clocks consist of transcriptional feedback loops housed in interdependent pacemaker neurons. Yet little is known about the neuronal output components essential for rhythmic behavior. Drosophila mutants of a putative ion channel, narrow abdomen (na), exhibit poor circadian rhythms and suppressed daylight activity. We find that NA is expressed in pacemaker neurons and induced expression within circadian neurons is sufficient to rescue these mutant phenotypes. Selective na rescue in distinct pacemaker neurons influences rhythmicity and timing of behavior. Oscillations of the clock protein PERIOD are intact in na mutants, indicating an output role. Pore residues are required for robust rescue consistent with NA action as an ion channel. In na mutants, expression of potassium currents and the key neuropeptide PDF are elevated, the latter consistent with reduced release. These data implicate NA and the pacemaker neural network in controlling phase and rhythmicity.

Factors affecting habituation of PC12 cells to ATP.

Extracellular ATP triggers catecholamine secretion from PC12 cells by activating ionotropic purine receptors. Repeated stimulation by ATP leads to habituation of the secretory response. In this paper, we use amperometric detection to monitor the habituation of PC12 cells to multiple stimulations of ATP or its agonist. Cells habituate to 30 microm ATP slower than they do to 300 or 600 microm ATP. Modifying external Mg2+ affects the response of cells to 30 microm ATP, but does not affect habituation, suggesting that habituation does not necessarily correspond to either stimulus intensity or cellular response. Mg2+ affects the initial response of PC12 cells to 2MeSATP in a manner similar to ATP. Increasing external [Mg2+] to 3.0 mm, however, eliminates habituation to 2MeSATP. This habituation can be partially restored by costimulation with 100 microm UTP. Background application of UTP increases habituation to both ATP and 2MeSATP. This suggests that ATP-sensitive metabotropic (P2Y) receptors play a role in the habituation process. Finally, although Ca2+ influx through voltage-operated calcium channels does not appear to contribute to secretion during ATP stimulation, blocking these channels with nicardipine increases habituation. This suggests a role for voltage-operated calcium channels in the habituation process.

Long-lived retrograde fluorescent labeling of corticospinal neurons in the living animal.

For pathophysiological studies, it is advantageous to label specific neuronal populations in living animals. This study aimed to establish a method for stable and long-lasting fluorescent labeling of corticospinal neurons in the living animal. The two fluorescent dyes Fluoro-Red and Fluoro-Green were injected in the cervical spinal cord of anesthetized newborn rats. After a recovery period, treated rats were returned to the mother. After 24 h and 14 days, fixed brain sections revealed wide-spread fluorescence in elongated or pyramidal-shaped cell profiles in a discrete internal cortical layer, consistent with layer V pyramidal cells. Labeled neurons displayed spontaneous synaptic activity using the slice patch clamp method. These results suggest that these dyes are effective tools for pathophysiological and slice patch clamp studies focused on specific neuron groups.

Short- and long-term modulation of synaptic inputs to brain reward areas by nicotine.

Dopamine signaling in brain reward areas is a key element in the development of drug abuse and dependence. Recent anatomical and electrophysiological research has begun to elucidate both complexity and specificity in synaptic connections between ventral tegmental neurons and their inputs. Specifically, the activity of dopamine neurons in the ventral tegmental area relies on the combination of both excitatory and inhibitory inputs. Controlling endogenous neurotransmission to dopamine neurons is one mechanism by which drugs of abuse affect both transient and long-term changes in synaptic activity. Here, we review recent findings concerning glutamatergic, GABAergic, and cholinergic inputs to dopamine neurons, and their roles in the reinforcement associated with drug abuse. Importantly, several studies support that a single drug exposure can lead to changes in synaptic strength that are associated with learning and memory. Ultimately, these cellular changes could underlie the long-lasting effects of drugs. Furthermore, nicotinic acetylcholine receptors in the ventral tegmental area emerge as a possible common target for the behavioral and cellular actions not only of nicotine, but also of several other drugs of abuse. Finally, we explore age-related differences in nicotine sensitivity in order to understand both human epidemiological data, and laboratory animal behavioral findings that suggest adolescents are more susceptible to developing nicotine dependence.

Synaptic mechanisms underlie nicotine-induced excitability of brain reward areas.

A single nicotine exposure increases dopamine levels in the mesolimbic reward system for hours, but nicotine concentrations experienced by smokers desensitize nAChRs on dopamine neurons in seconds to minutes. Here, we show that persistent modulation of both GABAergic and glutamatergic synaptic transmission by nicotine can contribute to the sustained increase in dopamine neuron excitability. Nicotine enhances GABAergic transmission transiently, which is followed by a persistent depression of these inhibitory inputs due to nAChR desensitization. Simultaneously, nicotine enhances glutamatergic transmission through nAChRs that desensitize less than those on GABA neurons. The net effect is a shift toward excitation of the dopamine reward system. These results suggest that spatial and temporal differences in nicotinic receptor activity on both excitatory and inhibitory neurons in reward areas coordinate to reinforce nicotine self-administration.