Aggressive behavior is widespread throughout the animal kingdom, and is a complex social behavior influenced by both genetics and environment. Animals typically fight over resources that include food, territory, and sexual partners. Of all the neurotransmitters, serotonin has been the most implicated in modulating aggressive behaviors in mammalian systems. In the fruit fly, Drosophila melanogaster, the involvement of serotonin itself in aggressive behaviors has been recently established, however, the underlying mechanisms have largely remained elusive. Here we describe the influence of different serotonin receptor subtypes on aggressive behaviors in Drosophila. Drosophila express homologs of three mammalian serotonin receptors: the 5-HT 1A , 5-HT 2 , and 5-HT 7 receptors. Significantly, these receptors mediate important behaviors in mammalian systems ranging from feeding, aggression, and sleep, to cognition. To examine the role of the 5-HT 2 Dro receptor, we utilized the selective 5-HT 2 receptor agonist (R)-1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI), and the 5-HT 2 receptor antagonist, ketanserin. To examine the role of 5-HT 1A -like receptors we used the 5-HT 1A receptor agonist 8-hydroxy-2-dipropylaminotetralin hydrobromide (8-OH-DPAT), and the 5-HT 1A receptor antagonist WAY100635. We find that activation of 5-HT 2 receptors with (R)-DOI appears to decrease overall aggression, whereas activation of 5-HT 1A -like receptors with 8-OH-DPAT increases overall aggression. Furthermore, the different serotonin receptor circuitries appear to mediate different aspects of aggression: 5-HT 2 receptor manipulation primarily alters lunging and boxing, whereas 5-HT 1A -like receptor manipulation primarily affects wing threats and fencing. Elucidating the effects of serotonergic systems on aggression in the fly is a significant advancement not only in establishing the fly as a system to study aggression, but as a system relevant to elucidating molecular mechanisms underlying aggression in mammals, including humans. KeywordsAggression; neuropharmacology; 5-HT; DOI; 8-OH-DPAT The study of aggression in flies has a long history (Jacobs, 1960(Jacobs, , 1978; Drosophila exhibits aggressive behavior in the acquisition of food, territory, and mates, and these behaviors can differ among fly strains (Jacobs, 1960, Dow and Schilcher, 1975, Jacobs, 1978 Corresponding Author: Charles D. Nichols, Ph.D., Dept. of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112, (504) 568-2957, (504) 568-2361 (fax), cnich1@lsuhsc.edu. Section Editor: Dr. Geoffrey M. Schoenbaum Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process e...

The 5-HT7 receptor remains one of the less well characterized serotonin receptors. Although it has been demonstrated to be involved in the regulation of mood, sleep, and circadian rhythms, as well as relaxation of vascular smooth muscles in mammals, the precise mechanisms underlying these functions remain largely unknown. The fruit fly, Drosophila melanogaster, is an attractive model organism to study neuropharmacological, molecular, and behavioral processes that are largely conserved with mammals. Drosophila express a homolog of the mammalian 5-HT7 receptor, as well as homologs for the mammalian 5-HT1A, and 5-HT2, receptors. Each fly receptor couples to the same effector pathway as their mammalian counterpart and have been demonstrated to mediate similar behavioral responses. Here, we report on the expression and function of the 5-HT7Dro receptor in Drosophila. In the larval central nervous system, expression is detected postsynaptically in discreet cells and neuronal circuits. In the adult brain there is strong expression in all large-field R neurons that innervate the ellipsoid body, as well as in a small group of cells that cluster with the PDF-positive LNvs neurons that mediate circadian activity. Following both pharmacological and genetic approaches, we have found that 5-HT7Dro activity is essential for normal courtship and mating behaviors in the fly, where it appears to mediate levels of interest in both males and females. This is the first reported evidence of direct involvement of a particular serotonin receptor subtype in courtship and mating in the fly.

Learning and memory in the fruit fly, Drosophila melanogaster, is a complex behavior with many parallels to mammalian learning and memory. Although many neurotransmitters including acetylcholine, dopamine, glutamate, and GABA have previously been demonstrated to be involved in aversive olfactory learning and memory, the role of serotonin has not been well defined. Here, we present the first evidence of the involvement of individual serotonin receptors in olfactory learning and memory in the fly. We initially followed a pharmacological approach, utilizing serotonin receptor agonists and antagonists to demonstrate that all serotonin receptor families present in the fly are necessary for short term learning and memory. Isobolographic analysis utilizing combinations of drugs revealed functional interactions are occurring between 5-HT1A-like and 5-HT2, and 5-HT2 and 5-HT7 receptor circuits in mediating short term learning and memory. Examination of long term memory suggest that 5-HT1A-like receptors are necessary for consolidation and important for recall, 5-HT2 receptors are important for consolidation and recall, and 5-HT7 receptors are involved in all three phases. Importantly, we have validated our pharmacological results with genetic experiments, and show that hypomorph strains for 5-HT2Dro and 5-HT1BDro receptors, as well as knockdown of 5-HT7Dro mRNA significantly impair performance in short term memory. Our data highlight the importance of the serotonin system and individual serotonin receptors to influence olfactory learning and memory in the fly, and position the fly as a model system to study the role of serotonin in cognitive processes relevant to mammalian CNS function.

SUMMARY We have translated a powerful genetic tool, designer receptors exclusively activated by designer drugs (DREADDs), from mammalian systems to Drosophila melanogaster to selectively, rapidly, reversibly, and dose-dependently control behaviors and physiological processes in the fly. DREADDs are muscarinic acetylcholine G protein-coupled receptors evolved for loss of affinity to acetylcholine and for the ability to be fully activated by an otherwise biologically inert chemical, clozapine-N-oxide. We demonstrate its ability to control a variety of behaviors and processes in larvae and adults, including heart rate, sensory processing, diurnal behavior, learning and memory, and courtship. The advantages of this particular technology include the dose-responsive control of behaviors, the lack of a need for specialized equipment, and the capacity to remotely control signaling in essentially all neuronal and nonneuronal fly tissues.

Key points• Layer V principal neurons of the entorhinal cortex receive the hippocampal output on their proximal and basal dendrites and send their axons to cortical areas, playing a fundamental role in memory processing.• The apical dendrites of these neurons are rich in spines and extend to the entorhinal superficial layers, in the proximity of axons from cortical neurons, which could make synapses onto these spines.• We stimulated afferent fibres in the superficial layers and recorded depolarizing responses in entorhinal layer V neurons, indicating that they receive excitatory inputs onto their distal dendrites.• The responses were completely blocked by glutamatergic receptor antagonists; stimulation of distal afferents could initiate dendritic spikes, which propagated to the soma to generate an action potential.• These results show that the distal dendrites of entorhinal layer V neurons have access to information that could affect the integration of the input from the hippocampus. AbstractThe entorhinal cortex (EC) has a fundamental function in transferring information between the hippocampus and the neocortex. EC layer V principal neurons are the main recipients of the hippocampal output and send processed information to the neocortex, likely playing an important role in memory processing and consolidation. Most of these neurons have apical dendrites that extend to the superficial layers and are rich in spines, which could be the targets of excitatory inputs from fibres innervating that region. We have used electrical stimulation of afferent fibres coupled with whole-cell patch-clamp somatic recordings to study the features of distal excitatory inputs and compare them with those of proximal ones. The amplitude of putative unitary excitatory responses was ∼1.5 times larger for distal compared with proximal inputs. The responses were purely glutamatergic, as they were abolished by a combination of AMPA and NMDA glutamatergic receptor antagonists. Blockade of I h by 4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride (ZD7288) increased temporal summation; the increase was comparable for proximal and distal inputs. Proximal inputs initiated a somatic spike more reliably than distal ones; in some instances, somatic action potentials triggered by distal stimulation were preceded by dendritic spikes that fully propagated to the soma. Altogether, our results show that medial layer V entorhinal neurons receive excitatory synapses at distal dendritic locations, which gives them access to information encoded by inputs V. Medinilla and O. Johnson contributed equally to this work.

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Serotonin is a neurotransmitter involved in many behaviors in mammals including regulation of mood, appetite, sleep, memory, aggression, and sexual behavior. In the fly, serotonin has also been demonstrated to be involved in many of these same complex behaviors. The effects of serotonin are primarily mediated through interactions with G‐protein coupled receptors to initiate multiple signal transduction pathways. Drosophila expresses orthologs of four mammalian 5‐HT receptor subtypes: 5‐HT1ADro, 5‐HT1BDro, 5‐HT2Dro, and 5‐HT7Dro. Here, we present data demonstrating involvement of all three serotonin receptor families in olfactory learning and memory in the fly.We probed the involvement of the 5‐HT1A‐like, 5‐HT2, and 5‐HT7 receptors in short‐term memory in the fly. The 5‐HT1A receptor agonist U92106A and the 5‐HT1A receptor antagonist WAY100635 reduced performance indices in a dose‐dependent manner. The 5‐HT2 receptor agonist DOI, and the 5‐HT2 receptor antagonist ketanserin also abated performance indices in a dose‐dependent manner (IC50 value for DOI = 0.21 mM). A putative 5‐HT7 receptor antagonist SB 269970, also diminished performance levels. Since both agonists and antagonists for a given receptor inhibit performance, a homeostasis likely exists at each receptor, and perturbation of homeostasis disrupts this behavior.Research support: NIMH grants 5R21MH078454‐02 and 3R21MH078454‐01A1S1

Serotonin (5‐HT) influences behaviors that include circadian, sleep, appetite, aggression, locomotion, perception, and sexual behavior. The effects of 5‐HT are mediated through G‐protein coupled receptors, which initiate multiple effector pathways. Disregulation of 5‐HT signaling in humans has been implicated in neuropsychiatric disorders including depression, anxiety disorders, anorexia and schizophrenia. The fruit fly, Drosophila melanogaster, expresses orthologs of three mammalian 5‐HT receptor subtypes: 5‐HT1A, 5‐HT2, and 5‐HT7. The 5‐HT7Dro receptor is orthologous to the mammalian 5‐HT7 receptor and activates adenylate cyclase through Gαs. We show that in fly 3rd instar larvae brain, 5‐HT7Dro expression is observed in discreet populations of neurons in the hemispheres as well as in neurons of the ventral ganglia. In adult brains, 5‐HT7Dro is highly expressed in neurons that innervate the ellipsoid body, a region believed to be involved in higher order behaviors. 5‐HT7Dro neurons also cluster with PDF‐positive circadian pacemaker neurons. Pharmacological studies using the 5‐HT1A/7 receptor agonist 8‐OH‐DPAT with the 5‐HT1A receptor antagonist WAY100635 eliminated circadian activity. These results suggest that 5‐HT7Dro may modulate circadian activity in the fly. Further studies to determine the role of 5‐HT7Dro in additional behaviors are ongoing. Supported by NIMH 1R21MH078454