Brief Change in Dopamine Activity during Consolidation Impairs Long-Term Memory via Sleep Disruption.

Sleep disturbances are associated with poor long-term memory (LTM) formation, yet the underlying cell types and neural circuits involved have not been fully decoded. Dopamine neurons (DANs) are involved in memory processing at multiple stages. Here, we show that brief activation of protocerebral anterior medial DANs (PAM-DANs) or inhibition of a pair of dorsal posterior medial (DPM) neurons during the first few hours of memory consolidation impairs 24 h LTM. Interestingly, sleep deprivation elevates the neural activity of PAM-DANs and DPM neurons, and brief thermos-activation of PAM-DANs or inactivation of DPM neurons results in sleep loss and fragmentation. Pharmacological rescue of sleep after this manipulation restores LTM. A specific subset of PAM-DANs, PAM-α1 that synapse onto DPM neurons specify the microcircuit that links sleep and memory. PAM-DANs, including PAM-α1, form functional synapses with DPM neurons mainly via Dop1R1 receptor to inhibit DPM. Our data suggest that the post-training activity of PAM(-α1)-DPM microcircuit, especially during memory consolidation, plays an essential role in maintaining the sleep necessary for LTM consolidation, providing a new cellular and circuit basis for the complex relationship between sleep and memory.

Subtype-Specific Roles of Ellipsoid Body Ring Neurons in Sleep Regulation in Drosophila.

The ellipsoid body (EB) is a major structure of the central complex of the Drosophila melanogaster brain. Twenty-two subtypes of EB ring neurons have been identified based on anatomic and morphologic characteristics by light-level microscopy and EM connectomics. A few studies have associated ring neurons with the regulation of sleep homeostasis and structure. However, cell type-specific and population interactions in the regulation of sleep remain unclear. Using an unbiased thermogenetic screen of EB drivers using female flies, we found the following: (1) multiple ring neurons are involved in the modulation of amount of sleep and structure in a synergistic manner; (2) analysis of data for ΔP(doze)/ΔP(wake) using a mixed Gaussian model detected 5 clusters of GAL4 drivers which had similar effects on sleep pressure and/or depth: lines driving arousal contained R4m neurons, whereas lines that increased sleep pressure had R3m cells; (3) a GLM analysis correlating ring cell subtype and activity-dependent changes in sleep parameters across all lines identified several cell types significantly associated with specific sleep effects: R3p was daytime sleep-promoting, and R4m was nighttime wake-promoting; and (4) R3d cells present in 5HT7-GAL4 and in GAL4 lines, which exclusively affect sleep structure, were found to contribute to fragmentation of sleep during both day and night. Thus, multiple subtypes of ring neurons distinctively control sleep amount and/or structure. The unique highly interconnected structure of the EB suggests a local-network model worth future investigation; understanding EB subtype interactions may provide insight how sleep circuits in general are structured.SIGNIFICANCE STATEMENT How multiple brain regions, with many cell types, can coherently regulate sleep remains unclear, but identification of cell type-specific roles can generate opportunities for understanding the principles of integration and cooperation. The ellipsoid body (EB) of the fly brain exhibits a high level of connectivity and functional heterogeneity yet is able to tune multiple behaviors in real-time, including sleep. Leveraging the powerful genetic tools available in Drosophila and recent progress in the characterization of the morphology and connectivity of EB ring neurons, we identify several EB subtypes specifically associated with distinct aspects of sleep. Our findings will aid in revealing the rules of coding and integration in the brain.

V3 Interneurons Are Active and Recruit Spinal Motor Neurons during In Vivo Fictive Swimming in Larval Zebrafish.

Survival for vertebrate animals is dependent on the ability to successfully find food, locate a mate, and avoid predation. Each of these behaviors requires motor control, which is set by a combination of kinematic properties. For example, the frequency and amplitude of motor output combine in a multiplicative manner to determine features of locomotion such as distance traveled, speed, force (thrust), and vigor. Although there is a good understanding of how different populations of excitatory spinal interneurons establish locomotor frequency, there is a less thorough mechanistic understanding for how locomotor amplitude is established. Recent evidence indicates that locomotor amplitude is regulated in part by a subset of functionally and morphologically distinct V2a excitatory spinal interneurons (Type II, nonbursting) in larval and adult zebrafish. Here, we provide direct evidence that most V3 interneurons (V3-INs), which are a developmentally and genetically defined population of ventromedial glutamatergic spinal neurons, are active during fictive swimming. We also show that elimination of the spinal V3-IN population reduces the proportion of active motor neurons (MNs) during fictive swimming but does not alter the range of locomotor frequencies produced. These data are consistent with V3-INs providing excitatory drive to spinal MNs during swimming in larval zebrafish and may contribute to the production of locomotor amplitude independently of locomotor frequency.

Rest Is Required to Learn an Appetitively-Reinforced Operant Task in Drosophila.

Maladaptive operant conditioning contributes to development of neuropsychiatric disorders. Candidate genes have been identified that contribute to this maladaptive plasticity, but the neural basis of operant conditioning in genetic model organisms remains poorly understood. The fruit fly Drosophila melanogaster is a versatile genetic model organism that readily forms operant associations with punishment stimuli. However, operant conditioning with a food reward has not been demonstrated in flies, limiting the types of neural circuits that can be studied. Here we present the first sucrose-reinforced operant conditioning paradigm for flies. In the paradigm, flies walk along a Y-shaped track with reward locations at the terminus of each hallway. When flies turn in the reinforced direction at the center of the track, they receive a sucrose reward at the end of the hallway. Only flies that rest early in training learn the reward contingency normally. Flies rewarded independently of their behavior do not form a learned association but have the same amount of rest as trained flies, showing that rest is not driven by learning. Optogenetically-induced sleep does not promote learning, indicating that sleep itself is not sufficient for learning the operant task. We validated the sensitivity of this assay to detect the effect of genetic manipulations by testing the classic learning mutant dunce. Dunce flies are learning-impaired in the Y-Track task, indicating a likely role for cAMP in the operant coincidence detector. This novel training paradigm will provide valuable insight into the molecular mechanisms of disease and the link between sleep and learning.

Covert sleep-related biological processes are revealed by probabilistic analysis in Drosophila.

Sleep pressure and sleep depth are key regulators of wake and sleep. Current methods of measuring these parameters in Drosophila melanogaster have low temporal resolution and/or require disrupting sleep. Here we report analysis tools for high-resolution, noninvasive measurement of sleep pressure and depth from movement data. Probability of initiating activity, P(Wake), measures sleep depth while probability of ceasing activity, P(Doze), measures sleep pressure. In vivo and computational analyses show that P(Wake) and P(Doze) are largely independent and control the amount of total sleep. We also develop a Hidden Markov Model that allows visualization of distinct sleep/wake substates. These hidden states have a predictable relationship with P(Doze) and P(Wake), suggesting that the methods capture the same behaviors. Importantly, we demonstrate that both the Doze/Wake probabilities and the sleep/wake substates are tied to specific biological processes. These metrics provide greater mechanistic insight into behavior than measuring the amount of sleep alone.

A Serotonin-Modulated Circuit Controls Sleep Architecture to Regulate Cognitive Function Independent of Total Sleep in Drosophila.

Both the structure and the amount of sleep are important for brain function. Entry into deep, restorative stages of sleep is time dependent; short sleep bouts selectively eliminate these states. Fragmentation-induced cognitive dysfunction is a feature of many common human sleep pathologies. Whether sleep structure is normally regulated independent of the amount of sleep is unknown. Here, we show that in Drosophila melanogaster, activation of a subset of serotonergic neurons fragments sleep without major changes in the total amount of sleep, dramatically reducing long episodes that may correspond to deep sleep states. Disruption of sleep structure results in learning deficits that can be rescued by pharmacologically or genetically consolidating sleep. We identify two reciprocally connected sets of ellipsoid body neurons that form the heart of a serotonin-modulated circuit that controls sleep architecture. Taken together, these findings define a circuit essential for controlling the structure of sleep independent of its amount.

Intraspinal serotonergic signaling suppresses locomotor activity in larval zebrafish.

Serotonin (5HT) is a modulator of many vital processes in the spinal cord (SC), such as production of locomotion. In the larval zebrafish, intraspinal serotonergic neurons (ISNs) are a source of spinal 5HT that, despite the availability of numerous genetic and optical tools, has not yet been directly shown to affect the spinal locomotor network. In order to better understand the functions of ISNs, we used a combination of strategies to investigate ISN development, morphology, and function. ISNs were optically isolated from one another by photoconverting Kaede fluorescent protein in individual cells, permitting morphometric analysis as they developed in vivo. ISN neurite lengths and projection distances exhibited the greatest amount of change between 3 and 4 days post-fertilization (dpf) and appeared to stabilize by 5 dpf. Overall ISN innervation patterns were similar between cells and between SC regions. ISNs possessed rostrally-extending neurites resembling dendrites and a caudally-extending neurite resembling an axon, which terminated with an enlarged growth cone-like structure. Interestingly, these enlargements remained even after neurite extension had ceased. Functionally, application of exogenous 5HT reduced spinally-produced motor nerve bursting. A selective 5HT reuptake inhibitor and ISN activation with channelrhodopsin-2 each produced similar effects to 5HT, indicating that spinally-intrinsic 5HT originating from the ISNs has an inhibitory effect on the spinal locomotor network. Taken together this suggests that the ISNs are morphologically mature by 5 dpf and supports their involvement in modulating the activity of the spinal locomotor network. © 2018 Wiley Periodicals, Inc. Develop Neurobiol, 2018.

Regulation of Eag by Ca2+/calmodulin controls presynaptic excitability in Drosophila.

Drosophila ether-à-go-go ( eag) is the founding member of a large family of voltage-gated K+ channels, the KCNH family, which includes Kv10, 11, and 12. Concurrent binding of calcium/calmodulin (Ca2+/CaM) to NH2- and COOH-terminal sites inhibits mammalian EAG1 channels at submicromolar Ca2+ concentrations, likely by causing pore constriction. Although the Drosophila EAG channel was believed to be Ca2+-insensitive (Schönherr R, Löber K, Heinemann SH. EMBO J 19: 3263-3271, 2000.), both the NH2- and COOH-terminal sites are conserved. In this study we show that Drosophila EAG is inhibited by high Ca2+ concentrations that are only present at plasma membrane Ca2+ channel microdomains. To test the role of this regulation in vivo, we engineered mutations that block CaM-binding to the major COOH-terminal site of the endogenous eag locus, disrupting Ca2+-dependent inhibition. eag CaMBD mutants have reduced evoked release from larval motor neuron presynaptic terminals and show decreased Ca2+ influx in stimulated adult projection neuron presynaptic terminals, consistent with an increase in K+ conductance. These results are predicted by a conductance-based multicompartment model of the presynaptic terminal in which some fraction of EAG is localized to the Ca2+ channel microdomains that control neurotransmitter release. The reduction of release in the larval neuromuscular junction drives a compensatory increase in motor neuron somatic excitability. This misregulation of synaptic and somatic excitability has consequences for systems-level processes and leads to defects in associative memory formation in adults. NEW & NOTEWORTHY Regulation of excitability is critical to tuning the nervous system for complex behaviors. We demonstrate in this article that the EAG family of voltage-gated K+ channels exhibit conserved gating by Ca2+/CaM. Disruption of this inhibition in Drosophila results in decreased evoked neurotransmitter release due to truncated Ca2+ influx in presynaptic terminals. In adults, disrupted Ca2+ dynamics cripples memory formation. These data demonstrate that the biophysical details of channels have important implications for cell function and behavior.

Intraspinal serotonergic neurons consist of two, temporally distinct populations in developing zebrafish.

Zebrafish intraspinal serotonergic neuron (ISN) morphology and distribution have been examined in detail at different ages; however, some aspects of the development of these cells remain unclear. Although antibodies to serotonin (5-HT) have detected ISNs in the ventral spinal cord of embryos, larvae, and adults, the only tryptophan hydroxylase (tph) transcript that has been described in the spinal cord is tph1a. Paradoxically, spinal tph1a is only expressed transiently in embryos, which brings the source of 5-HT in the ISNs of larvae and adults into question. Because the pet1 and tph2 promoters drive transgene expression in the spinal cord, we hypothesized that tph2 is expressed in spinal cords of zebrafish larvae. We confirmed this hypothesis through in situ hybridization. Next, we used 5-HT antibody labeling and transgenic markers of tph2-expressing neurons to identify a transient population of ISNs in embryos that was distinct from ISNs that appeared later in development. The existence of separate ISN populations may not have been recognized previously due to their shared location in the ventral spinal cord. Finally, we used transgenic markers and immunohistochemical labeling to identify the transient ISN population as GABAergic Kolmer-Agduhr double-prime (KA″) neurons. Altogether, this study revealed a novel developmental paradigm in which KA″ neurons are transiently serotonergic before the appearance of a stable population of tph2-expressing ISNs.

Coordination of fictive motor activity in the larval zebrafish is generated by non-segmental mechanisms.

The cellular and network basis for most vertebrate locomotor central pattern generators (CPGs) is incompletely characterized, but organizational models based on known CPG architectures have been proposed. Segmental models propose that each spinal segment contains a circuit that controls local coordination and sends longer projections to coordinate activity between segments. Unsegmented/continuous models propose that patterned motor output is driven by gradients of neurons and synapses that do not have segmental boundaries. We tested these ideas in the larval zebrafish, an animal that swims in discrete episodes, each of which is composed of coordinated motor bursts that progress rostrocaudally and alternate from side to side. We perturbed the spinal cord using spinal transections or strychnine application and measured the effect on fictive motor output. Spinal transections eliminated episode structure, and reduced both rostrocaudal and side-to-side coordination. Preparations with fewer intact segments were more severely affected, and preparations consisting of midbody and caudal segments were more severely affected than those consisting of rostral segments. In reduced preparations with the same number of intact spinal segments, side-to-side coordination was more severely disrupted than rostrocaudal coordination. Reducing glycine receptor signaling with strychnine reversibly disrupted both rostrocaudal and side-to-side coordination in spinalized larvae without disrupting episodic structure. Both spinal transection and strychnine decreased the stability of the motor rhythm, but this effect was not causal in reducing coordination. These results are inconsistent with a segmented model of the spinal cord and are better explained by a continuous model in which motor neuron coordination is controlled by segment-spanning microcircuits.

Episodic swimming in the larval zebrafish is generated by a spatially distributed spinal network with modular functional organization.

Despite the diverse methods vertebrates use for locomotion, there is evidence that components of the locomotor central pattern generator (CPG) are conserved across species. When zebrafish begin swimming early in development, they perform short episodes of activity separated by periods of inactivity. Within these episodes, the trunk flexes with side-to-side alternation and the traveling body wave progresses rostrocaudally. To characterize the distribution of the swimming CPG along the rostrocaudal axis, we performed transections of the larval zebrafish spinal cord and induced fictive swimming using N-methyl-d-aspartate (NMDA). In both intact and spinalized larvae, bursting is found throughout the rostrocaudal extent of the spinal cord, and the properties of fictive swimming observed were dependent on the concentration of NMDA. We isolated series of contiguous spinal segments by performing multiple spinal transections on the same larvae. Although series from all regions of the spinal cord have the capacity to produce bursts, the capacity to produce organized episodes of fictive swimming has a rostral bias: in the rostral spinal cord, only 12 contiguous body segments are necessary, whereas 23 contiguous body segments are necessary in the caudal spinal cord. Shorter series of segments were often active but produced either continuous rhythmic bursting or sporadic, nonrhythmic bursting. Both episodic and continuous bursting alternated between the left and right sides of the body and showed rostrocaudal progression, demonstrating the functional dissociation of the circuits responsible for episodic structure and fine burst timing. These findings parallel results in mammalian locomotion, and we propose a hierarchical model of the larval zebrafish swimming CPG.

Elevated triglycerides correlate with progression of diabetic neuropathy.

OBJECTIVE: To evaluate mechanisms underlying diabetic neuropathy progression using indexes of sural nerve morphometry obtained from two identical randomized, placebo-controlled clinical trials. RESEARCH DESIGN AND METHODS: Sural nerve myelinated fiber density (MFD), nerve conduction velocities (NCVs), vibration perception thresholds, clinical symptom scores, and a visual analog scale for pain were analyzed in participants with diabetic neuropathy. A loss of > or =500 fibers/mm(2) in sural nerve MFD over 52 weeks was defined as progressing diabetic neuropathy, and a MFD loss of < or =100 fibers/mm(2) during the same time interval as nonprogressing diabetic neuropathy. The progressing and nonprogressing cohorts were matched for baseline characteristics using an O'Brien rank-sum and baseline MFD. RESULTS: At 52 weeks, the progressing cohort demonstrated a 25% decrease (P < 0.0001) from baseline in MFD, while the nonprogressing cohort remained unchanged. MFD was not affected by active drug treatment (P = 0.87), diabetes duration (P = 0.48), age (P = 0.11), or BMI (P = 0.30). Among all variables tested, elevated triglycerides and decreased peroneal motor NCV at baseline significantly correlated with loss of MFD at 52 weeks (P = 0.04). CONCLUSIONS: In this cohort of participants with mild to moderate diabetic neuropathy, elevated triglycerides correlated with MFD loss independent of disease duration, age, diabetes control, or other variables. These data support the evolving concept that hyperlipidemia is instrumental in the progression of diabetic neuropathy.

Rosiglitazone treatment reduces diabetic neuropathy in streptozotocin-treated DBA/2J mice.

Diabetic neuropathy (DN) is a common complication of diabetes. Currently, there is no drug treatment to prevent or slow the development of DN. Rosiglitazone (Rosi) is a potent insulin sensitizer and may also slow the development of DN by a mechanism independent of its effect on hyperglycemia. A two by two design was used to test the effect of Rosi treatment on the development of DN. Streptozotocin-induced diabetic DBA/2J mice were treated with Rosi. DN and oxidative stress were quantified, and gene expression was profiled using the Affymetrix Mouse Genome 430 2.0 microarray platform. An informatics approach identified key regulatory elements activated by Rosi. Diabetic DBA/2J mice developed severe hyperglycemia, DN, and elevated oxidative stress. Rosi treatment did not affect hyperglycemia but did reduce oxidative stress and prevented the development of thermal hypoalgesia. Two novel transcription factor binding modules were identified that may control genes correlated to changes in DN after Rosi treatment: SP1F_ZBPF and EGRF_EGRF. These targets may be useful in designing drugs with the same efficacy as Rosi in treating DN but with fewer undesirable effects.

Mouse models of diabetic neuropathy.

Diabetic neuropathy (DN) is a debilitating complication of type 1 and type 2 diabetes. Rodent models of DN do not fully replicate the pathology observed in human patients. We examined DN in streptozotocin (STZ)-induced [B6] and spontaneous type 1 diabetes [B6Ins2(Akita)] and spontaneous type 2 diabetes [B6-db/db, BKS-db/db]. Despite persistent hyperglycemia, the STZ-treated B6 and B6Ins2(Akita) mice were resistant to the development of DN. In contrast, DN developed in both type 2 diabetes models: the B6-db/db and BKS-db/db mice. The persistence of hyperglycemia and development of DN in the B6-db/db mice required an increased fat diet while the BKS-db/db mice developed severe DN and remained hyperglycemic on standard mouse chow. Our data support the hypothesis that genetic background and diet influence the development of DN and should be considered when developing new models of DN.