KCNQ Channels Show Conserved Ethanol Block and Function in Ethanol Behaviour

In humans, KCNQ2/3 channels form an M-current that regulates neuronal excitability, with mutations in these channels causing benign neonatal familial convulsions. The M-current is important in mechanisms of neural plasticity underlying associative memory and in the response to ethanol, with KCNQ controlling the release of dopamine after ethanol exposure. We show that dKCNQ is broadly expressed in the nervous system, with targeted reduction in neuronal KCNQ increasing neural excitability and KCNQ overexpression decreasing excitability and calcium signalling, consistent with KCNQ regulating the resting membrane potential and neural release as in mammalian neurons. We show that the single KCNQ channel in Drosophila (dKCNQ) has similar electrophysiological properties to neuronal KCNQ2/3, including conserved acute sensitivity to ethanol block, with the fly channel (IC₅₀ = 19.8 mM) being more sensitive than its mammalian ortholog (IC₅₀ = 42.1 mM). This suggests that the role of KCNQ in alcohol behaviour can be determined for the first time by using Drosophila. We present evidence that loss of KCNQ function in Drosophila increased sensitivity and tolerance to the sedative effects of ethanol. Acute activation of dopaminergic neurons by heat-activated TRP channel or KCNQ-RNAi expression produced ethanol hypersensitivity, suggesting that both act via a common mechanism involving membrane depolarisation and increased dopamine signalling leading to ethanol sedation.     

The Role of Dopamine in Drosophila Larval Classical Olfactory Conditioning

Learning and memory is not an attribute of higher animals. Even Drosophila larvae are able to form and recall an association of a given odor with an aversive or appetitive gustatory reinforcer. As the Drosophila larva has turned into a particularly simple model for studying odor processing, a detailed neuronal and functional map of the olfactory pathway is available up to the third order neurons in the mushroom bodies. At this point, a convergence of olfactory processing and gustatory reinforcement is suggested to underlie associative memory formation. The dopaminergic system was shown to be involved in mammalian and insect olfactory conditioning. To analyze the anatomy and function of the larval dopaminergic system, we first characterize dopaminergic neurons immunohistochemically up to the single cell level and subsequent test for the effects of distortions in the dopamine system upon aversive (odor-salt) as well as appetitive (odor-sugar) associative learning. Single cell analysis suggests that dopaminergic neurons do not directly connect gustatory input in the larval suboesophageal ganglion to olfactory information in the mushroom bodies. However, a number of dopaminergic neurons innervate different regions of the brain, including protocerebra, mushroom bodies and suboesophageal ganglion. We found that dopamine receptors are highly enriched in the mushroom bodies and that aversive and appetitive olfactory learning is strongly impaired in dopamine receptor mutants. Genetically interfering with dopaminergic signaling supports this finding, although our data do not exclude on naïve odor and sugar preferences of the larvae. Our data suggest that dopaminergic neurons provide input to different brain regions including protocerebra, suboesophageal ganglion and mushroom bodies by more than one route. We therefore propose that different types of dopaminergic neurons might be involved in different types of signaling necessary for aversive and appetitive olfactory memory formation respectively, or for the retrieval of these memory traces. Future studies of the dopaminergic system need to take into account such cellular dissociations in function in order to be meaningful.     

A carboxy-terminal domain determines the subunit specificity of KCNQ K+ channel assembly

Mutations in KCNQ K⁺ channel genes underlie several human pathologies. KCNQ α-subunits form either homotetramers or hetero-oligomers with a restricted subset of other KCNQ α-subunits or with KCNE β-subunits. KCNQ1 assembles with KCNE β-subunits but not with other KCNQ α-subunits. By contrast, KCNQ3 interacts with KCNQ2, KCNQ4 and KCNQ5. Using a chimaeric strategy, we show that a cytoplasmic carboxy-terminal subunit interaction domain (sid) suffices to transfer assembly properties between KCNQ3 and KCNQ1. A chimaera (KCNQ1-sid_Q3) carrying the si domain of KCNQ3 within the KCNQ1 backbone interacted with KCNQ2, KCNQ3 and KCNQ4 but not with KCNQ1. This interaction was shown by enhancement of KCNQ2 currents, testing for dominant-negative effects of pore mutants, determining its effects on surface expression and co-immunoprecipitation experiments. Conversely, a KCNQ3-sid_Q1 chimaera no longer affects KCNQ2 but interacts with KCNQ1. We conclude that the si domain suffices to determine the subunit specificity of KCNQ channel assembly.     

Effects of KCNQ2 Gene Truncation on M-Type Kv7 Potassium Currents

The KCNQ2 gene product, Kv7.2, is a subunit of the M-channel, a low-threshold voltage-gated K⁺ channel that regulates mammalian and human neuronal excitability. Spontaneous mutations one of the KCNQ2 genes cause disorders of neural excitability such as Benign Familial Neonatal Seizures. However there appear to be no reports in which both human KCNQ2 genes are mutated. We therefore asked what happens to M-channel function when both KCNQ2 genes are disrupted. We addressed this using sympathetic neurons isolated from mice in which the KCNQ2 gene was truncated at a position corresponding to the second transmembrane domain of the Kv7.2 protein. Since homozygote KCNQ2−/− mice die postnatally, experiments were largely restricted to neurons from late embryos. Quantitative PCR revealed an absence of KCNQ2 mRNA in ganglia from KCNQ2−/− embryos but 100–120% increase of KCNQ3 and KCNQ5 mRNAs; KCNQ2+/− ganglia showed ∼30% less KCNQ2 mRNA than wild-type (+/+) ganglia but 40–50% more KCNQ3 and KCNQ5 mRNA. Neurons from KCNQ2−/− embryos showed a complete absence of M-current, even after applying the Kv7 channel enhancer, retigabine. Neurons from heterozygote KCNQ2+/− embryos had ∼60% reduced M-current. In contrast, M-currents in neurons from adult KCNQ2+/− mice were no smaller than those in neurons from wild-type mice. Measurements of tetraethylammonium block did not indicate an increased expression of Kv7.5-containing subunits, implying a compensatory increase in Kv7.2 expression from the remaining KCNQ2 gene. We conclude that mouse embryonic M-channels have an absolute requirement for Kv7.2 subunits for functionality, that the reduced M-channel activity in heterozygote KCNQ2+/− mouse embryos results primarily from a gene-dosage effect, and that there is a compensatory increase in Kv7.2 expression in adult mice.     

Models of heterogeneous dopamine signaling in an insect learning and memory center

The Drosophila mushroom body exhibits dopamine dependent synaptic plasticity that underlies the acquisition of associative memories. Recordings of dopamine neurons in this system have identified signals related to external reinforcement such as reward and punishment. However, other factors including locomotion, novelty, reward expectation, and internal state have also recently been shown to modulate dopamine neurons. This heterogeneity is at odds with typical modeling approaches in which these neurons are assumed to encode a global, scalar error signal. How is dopamine dependent plasticity coordinated in the presence of such heterogeneity? We develop a modeling approach that infers a pattern of dopamine activity sufficient to solve defined behavioral tasks, given architectural constraints informed by knowledge of mushroom body circuitry. Model dopamine neurons exhibit diverse tuning to task parameters while nonetheless producing coherent learned behaviors. Notably, reward prediction error emerges as a mode of population activity distributed across these neurons. Our results provide a mechanistic framework that accounts for the heterogeneity of dopamine activity during learning and behavior.     

Increased dopaminergic signaling impairs aversive olfactory memory retention in Drosophila

Dopamine is necessary for the aversive olfactory associative memory formation in Drosophila, but its effect on other stages of memory is not known. Herein, we studied the effect of enhanced dopaminergic signaling on aversive olfactory memory retention in flies. We used l-3,4-dihydroxyphenylalanine (l-DOPA) to elevate dopamine levels: l-DOPA-treated flies exhibited a normal learning performance, but a decrease in 1-h memory. Dopamine transporter (DAT) mutant flies or flies treated with the DAT inhibitor desipramine exhibited poor memory retention. Flies subjected to heat stress after training exhibited a decrease in memory. Memory was restored by blocking dopaminergic neuronal output during heat stress, suggesting that dopamine is involved in heat stress-induced memory impairment in flies. Taken together, our findings suggest that increased dopaminergic signaling impairs aversive olfactory memory retention in flies.     

Amyloid-β Peptide Exacerbates the Memory Deficit Caused by Amyloid Precursor Protein Loss-of-Function in Drosophila

The amyloid precursor protein (APP) plays a central role in Alzheimer’s disease (AD). APP can undergo two exclusive proteolytic pathways: cleavage by the α-secretase initiates the non-amyloidogenic pathway while cleavage by the β-secretase initiates the amyloidogenic pathway that leads, after a second cleavage by the γ-secretase, to amyloid-β (Aβ) peptides that can form toxic extracellular deposits, a hallmark of AD. The initial events leading to AD are still unknown. Importantly, aside from Aβ toxicity whose molecular mechanisms remain elusive, several studies have shown that APP plays a positive role in memory, raising the possibility that APP loss-of-function may participate to AD. We previously showed that APPL, the Drosophila APP ortholog, is required for associative memory in young flies. In the present report, we provide the first analysis of the amyloidogenic pathway’s influence on memory in the adult. We show that transient overexpression of the β-secretase in the mushroom bodies, the center for olfactory memory, did not alter memory. In sharp contrast, β-secretase overexpression affected memory when associated with APPL partial loss-of-function. Interestingly, similar results were observed with Drosophila Aβ peptide. Because Aβ overexpression impaired memory only when combined to APPL partial loss-of-function, the data suggest that Aβ affects memory through the APPL pathway. Thus, memory is altered by two connected mechanisms—APPL loss-of-function and amyloid peptide toxicity—revealing in Drosophila a functional interaction between APPL and amyloid peptide.     

The hangover gene negatively regulates bouton addition at the Drosophila neuromuscular junction

The synaptic growth of neurons during the development and adult life of an animal is a very dynamic and highly regulated process. During larval development in Drosophila new boutons and branches are added at the glutamatergic neuromuscular junction (NMJ) until a balance between neuronal activity and morphological structures is reached. Analysis of several Drosophila mutants suggest that bouton number and size might be regulated by separate signaling processes [Budnik, V., 1996. Synapse maturation and structural plasticity at Drosophila neuromuscular junctions. Curr. Opin. Neurobiol. 6, 858-867.]. Here we show a new role for Hangover as a negative regulator of bouton number at the NMJ. The hangover gene (hang) encodes a nuclear zinc finger protein. It has a function in neuronal plasticity mediating ethanol tolerance, a behavior that develops upon previous experience with ethanol. hang^AE10 mutants have more boutons and an extended synaptic span. Moreover, Hang expression in the motoneuron is required for the regulation of bouton number and the overall length of muscle innervation. However, the increase in bouton number does not correlate with a change in synaptic transmission, suggesting a mechanism independent from neuronal activity leads to the surplus of synaptic boutons. In contrast, we find that expression levels of the cell adhesion molecule Fasciclin II (FASII) are reduced in the hang mutant. This finding suggests that the increase in bouton number in hang mutants is caused by a reduction in FASII expression, thus, linking the regulation of nuclear gene expression with the addition of boutons at the NMJ regulated by cell adhesion molecules.     

A Single Pair of Neurons Modulates Egg-Laying Decisions in Drosophila

Animals have to judge environmental cues and choose the most suitable option for them from many different options. Female fruit flies selecting an optimum site to deposit their eggs is a biologically important reproductive behavior. When given the direct choice between ovipositing their eggs in a sucrose-containing medium or a caffeine-containing medium, female flies prefer the latter. However, the neural circuits and molecules that regulate this decision-making processes during egg-laying site selection remain poorly understood. In the present study, we found that amnesiac (amn) mutant flies show significant defects in egg-laying decisions, and such defects can be reversed by expressing the wild-type amn transgene in two dorsal paired medial (DPM) neurons in the brain. Silencing neuronal activity with an inward rectifier potassium channel (Kir2.1) in DPM neurons also impairs egg-laying decisions. Finally, the activity in mushroom body αβ neurons is required for the egg-laying behavior, suggesting a possible “DPM-αβ neurons” brain circuit modulating egg-laying decisions. Our results highlight the brain circuits and molecular mechanisms of egg-laying decisions in Drosophila.     

Apelin-13 Enhances Arcuate POMC Neuron Activity via Inhibiting M-Current

The hypothalamus is a key element of the neural circuits that control energy homeostasis. Specific neuronal populations within the hypothalamus are sensitive to a variety of homeostatic indicators such as circulating nutrient levels and hormones that signal circulating glucose and body fat content. Central injection of apelin secreted by adipose tissues regulates feeding and glucose homeostasis. However, the precise neuronal populations and cellular mechanisms involved in these physiological processes remain unclear. Here we examine the electrophysiological impact of apelin-13 on proopiomelanocortin (POMC) neuron activity. Approximately half of POMC neurons examined respond to apelin-13. Apelin-13 causes a dose-dependent depolarization. This effect is abolished by the apelin (APJ) receptor antagonist. POMC neurons from animals pre-treated with pertussis toxin still respond to apelin, whereas the Gβγ signaling inhibitor gallein blocks apelin-mediated depolarization. In addition, the effect of apelin is inhibited by the phospholipase C and protein kinase inhibitors. Furthermore, single-cell qPCR analysis shows that POMC neurons express the APJ receptor, PLC-β isoforms, and KCNQ subunits (2, 3 and 5) which contribute to M-type current. Apelin-13 inhibits M-current that is blocked by the KCNQ channel inhibitor. Therefore, our present data indicate that apelin activates APJ receptors, and the resultant dissociation of the Gαq heterotrimer triggers a Gβγ-dependent activation of PLC-β signaling that inhibits M-current.     

Immunohistochemical analysis of a novel dehydroepiandrosterone sulfotransferase-like protein in Drosophila neural circuits

Sulfotransferase (ST)-catalyzed sulfation plays an important role in various neuronal functions such as homeostasis of catecholamine neurotransmitters and hormones. Drosophila is a popular model for the study of memory and behavioral manifestations because it is able to mimic the intricate neuroregulation and recognition in humans. However, there has been no evidence indicating that cytosolic ST(s) is(are) present in Drosophila. The aim of this study is to investigate whether or not cytosolic ST(s) is(are) expressed in the Drosophila nervous system. Immunoblot analysis demonstrated the presence of dehydroepiandrosterone (DHEA) ST-like protein in Drosophila brain and a sensitive fluorometric assay revealed its sulfating activity toward DHEA. Immunohistochemical staining demonstrated this DHEA ST-like protein to be abundant in specific neurons as well as in several bundles of nerve fibers in Drosophila. Clarification of a possible link between ST and a neurotransmitter-mediated effect may eventually aid in designing approaches for alleviating neuronal disorders in humans.     

amnesiac regulates sleep onset and maintenance in Drosophila melanogaster

The adenylate cyclase/cAMP signaling pathway and adult mushroom bodies (MBs) have been shown to play an important role in sleep regulation in Drosophila. The amnesiac (amn) gene, encodes a neuropeptide that is homologous with vertebrate pituitary adenylate cyclase-activating peptide (PACAP), is expressed in dorsal paired medial (DPM) neurons and is required for the middle-term memory (MTM) in flies. However, the role of amn on regulation of sleep is as yet unknown. Here we provide evidence that amn plays a major role on sleep maintenance and onset in Drosophila. Flies with the amnesiac allele, loss-of-function amn^X8 mutation, showed a fragmented sleep pattern and short sleep latency. Moreover, homeostatic regulation was disrupted in amn^X8 mutants after sleep deprivation. Sleep maintenance was also influenced by disruption of neurotransmission in DPM neurons with increased sleep bout number and decreased sleep bout length. Furthermore, age-related sleep fragmentation and initiation were inhibited in amn^X8 mutant flies. These data suggest that amn is required in initiation and maintenance of sleep.     

Regulation of the voltage-gated K(+) channels KCNQ2/3 and KCNQ3/5 by ubiquitination. Novel role for Nedd4-2

The muscarine-sensitive K(+) current (M-current) stabilizes the resting membrane potential in neurons, thus limiting neuronal excitability. The M-current is mediated by heteromeric channels consisting of KCNQ3 subunits in association with either KCNQ2 or KCNQ5 subunits. The role of KCNQ2/3/5 in the regulation of neuronal excitability is well established; however, little is known about the mechanisms that regulate the cell surface expression of these channels. Ubiquitination by the Nedd4/Nedd4-2 ubiquitin ligases is known to regulate a number of membrane ion channels and transporters. In this study, we investigated whether Nedd4/Nedd4-2 could regulate KCNQ2/3/5 channels. We found that the amplitude of the K(+) currents mediated by KCNQ2/3 and KCNQ3/5 were reduced by Nedd4-2 (but not Nedd4) in a Xenopus oocyte expression system. Deletion experiments showed that the C-terminal region of the KCNQ3 subunit is required for the Nedd4-2-mediated regulation of the heteromeric channels. Glutathione S-transferase fusion pulldowns and co-immunoprecipitations demonstrated a direct interaction between KCNQ2/3 and Nedd4-2. Furthermore, Nedd4-2 could ubiquitinate KCNQ2/3 in transfected cells. Taken together, these data suggest that Nedd4-2 is potentially an important regulator of M-current activity in the nervous system.     

Drosophila Neurotrophins Reveal a Common Mechanism for Nervous System Formation

Neurotrophic interactions occur in Drosophila, but to date, no neurotrophic factor had been found. Neurotrophins are the main vertebrate secreted signalling molecules that link nervous system structure and function: they regulate neuronal survival, targeting, synaptic plasticity, memory and cognition. We have identified a neurotrophic factor in flies, Drosophila Neurotrophin (DNT1), structurally related to all known neurotrophins and highly conserved in insects. By investigating with genetics the consequences of removing DNT1 or adding it in excess, we show that DNT1 maintains neuronal survival, as more neurons die in DNT1 mutants and expression of DNT1 rescues naturally occurring cell death, and it enables targeting by motor neurons. We show that Spätzle and a further fly neurotrophin superfamily member, DNT2, also have neurotrophic functions in flies. Our findings imply that most likely a neurotrophin was present in the common ancestor of all bilateral organisms, giving rise to invertebrate and vertebrate neurotrophins through gene or whole-genome duplications. This work provides a missing link between aspects of neuronal function in flies and vertebrates, and it opens the opportunity to use Drosophila to investigate further aspects of neurotrophin function and to model related diseases.     

NGF inhibits M/KCNQ currents and selectively alters neuronal excitability in subsets of sympathetic neurons depending on their M/KCNQ current background

M/KCNQ currents play a critical role in the determination of neuronal excitability. Many neurotransmitters and peptides modulate M/KCNQ current and neuronal excitability through their G protein-coupled receptors. Nerve growth factor (NGF) activates its receptor, a member of receptor tyrosine kinase (RTK) superfamily, and crucially modulates neuronal cell survival, proliferation, and differentiation. In this study, we studied the effect of NGF on the neuronal (rat superior cervical ganglion, SCG) M/KCNQ currents and excitability. As reported before, subpopulation SCG neurons with distinct firing properties could be classified into tonic, phasic-1, and phasic-2 neurons. NGF inhibited M/KCNQ currents by similar proportion in all three classes of SCG neurons but increased the excitability only significantly in tonic SCG neurons. The effect of NGF on excitability correlated with a smaller M-current density in tonic neurons. The present study indicates that NGF is an M/KCNQ channel modulator and the characteristic modulation of the neuronal excitability by NGF may have important physiological implications.     

Spike-Timing Theory of Working Memory

Working memory (WM) is the part of the brain''s memory system that provides temporary storage and manipulation of information necessary for cognition. Although WM has limited capacity at any given time, it has vast memory content in the sense that it acts on the brain''s nearly infinite repertoire of lifetime long-term memories. Using simulations, we show that large memory content and WM functionality emerge spontaneously if we take the spike-timing nature of neuronal processing into account. Here, memories are represented by extensively overlapping groups of neurons that exhibit stereotypical time-locked spatiotemporal spike-timing patterns, called polychronous patterns; and synapses forming such polychronous neuronal groups (PNGs) are subject to associative synaptic plasticity in the form of both long-term and short-term spike-timing dependent plasticity. While long-term potentiation is essential in PNG formation, we show how short-term plasticity can temporarily strengthen the synapses of selected PNGs and lead to an increase in the spontaneous reactivation rate of these PNGs. This increased reactivation rate, consistent with in vivo recordings during WM tasks, results in high interspike interval variability and irregular, yet systematically changing, elevated firing rate profiles within the neurons of the selected PNGs. Additionally, our theory explains the relationship between such slowly changing firing rates and precisely timed spikes, and it reveals a novel relationship between WM and the perception of time on the order of seconds.     

Additive Expression of Consolidated Memory through Drosophila Mushroom Body Subsets

Associative olfactory memory in Drosophila has two components called labile anesthesia-sensitive memory and consolidated anesthesia-resistant memory (ARM). Mushroom body (MB) is a brain region critical for the olfactory memory and comprised of 2000 neurons that can be classified into αβ, α′β′, and γ neurons. Previously we demonstrated that two parallel pathways mediated ARM consolidation: the serotonergic dorsal paired medial (DPM)–αβ neurons and the octopaminergic anterior paired lateral (APL)–α′β′ neurons. This finding prompted us to ask how this composite ARM is retrieved. Here, we showed that blocking the output of αβ neurons and that of α′β′ neurons each impaired ARM retrieval, and blocking both simultaneously had an additive effect. Knockdown of radish and octβ2R in αβ and α′β′ neurons, respectively, impaired ARM. A combinatorial assay of radish mutant background rsh¹ and neurotransmission blockade confirmed that ARM retrieved from α′β′ neuron output is independent of radish. We identified MBON-β2β′2a and MBON-β′2mp as the MB output neurons downstream of αβ and α′β′ neurons, respectively, whose glutamatergic transmissions also additively contribute to ARM retrieval. Finally, we showed that α′β′ neurons could be functionally subdivided into α′β′m neurons required for ARM retrieval, and α′β′ap neurons required for ARM consolidation. Our work demonstrated that two parallel neural pathways mediating ARM consolidation in Drosophila MB additively contribute to ARM expression during retrieval.