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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. Abstract The 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 Ih 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 to the superficial layers as well as the deep layers. These findings are fundamentally relevant to understanding the role of the EC in the formation and consolidation of episodic memory.

The study of aggression in flies has a long history (Jacobs, 1960, 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, Hoffmann, 1987). Only recently, however, have modern techniques been used to explore this behavior and establish the fly as an accepted model system to study molecular mechanisms underlying aggression. Drosophila exhibit complex multi-component aggressive behaviors that are easily discernable and scored (Chen et al., 2002, Nilsen et al., 2004). These behaviors, along with many others, are often very similar to those observed in mammals (Nichols, 2006). It is known that male flies will fight with each other, but not with females, to establish social dominance (Yurkovic et al., 2006). Interestingly, sexually dimorphic aggressive behavioral patterns have been shown to be mediated by the fruitless gene (Vrontou et al., 2006, Chan and Kravitz, 2007). Recently, the neurotransmitter octopamine also has been found to mediate aspects of aggression in the fly (Hoyer et al., 2008, Zhou et al., 2008). Genetic studies with fly strains bred for hyper- and hypoaggressiveness, combined with microarray analysis, has delineated some of the molecular factors underlying aggression (Dierick and Greenspan, 2006). Although several differentially expressed genes have been identified, only one, for a cytochrome P450 (Cyp6a20), was determined to affect aggression directly. Somewhat surprisingly, serotonin levels in these strains did not differ. Serotonin has been strongly implicated in mediating aggressive behaviors in other animals, ranging from the more evolutionarily related crustacean, to birds, and mammals (Kravitz, 2000, Sperry et al., 2003, Popova, 2006). In the lobster, infusion of serotonin into the hemolymph elicits a socially dominant posture (Livingstone et al., 1980), as well as encouraging subordinate engagement of dominants (Huber et al., 1997). Attempts to alter serotonin levels in crayfish using biosynthetic precursors and inhibitors also have been found to modulate aggressive and social behaviors (Panksepp and Huber, 2002). The effects of serotonin in crustaceans are likely mediated by receptors similar to mammalian 5-HT1A and 5-HT2 receptors, although they are more dissimilar to their mammalian counterparts with regard to ligand binding profiles than the corresponding Drosophila receptors (Spitzer et al., 2008). In mammals, many aspects of the serotonin system including biosynthesis, metabolism, and receptor activation, primarily of 5-HT1A, 5-HT1B, and 5-HT2 receptors, have been demonstrated to modulate aggressive behaviors (Popova, 2006). In the fly, the effect of manipulating serotonin levels on aggression has been studied by Baier and coworkers (Baier et al., 2002), who examined the potential role of serotonin by feeding flies a metabolic precursor to serotonin, 5-HTP, and a biosynthesis inhibitor, pCPA, to raise and lower endogenous levels, respectively. Neither of these treatments, however, was found to alter aggression in their assays. These unexpected results should not be surprising, however, because two of the three families of serotonin receptors in Drosophila are functional orthologs of the mammalian 5-HT1A and 5-HT2 receptors (there is also a 5-HT7 receptor ortholog). In mammalian systems these two receptor subtypes often act to oppose one another functionally. Moreover, in previous crayfish and lobster studies, it was found that the rate of manipulation of serotonin levels (i.e. rapidly or slowly) was more important for modulation of aggressive behaviors than absolute levels (Ma et al., 1992, Panksepp and Huber, 2002, Panksepp et al., 2003), and it may be that a different treatment protocol would have uncovered a role for serotonin. Nevertheless, in the flies bred for hyperaggressivness by Dierick and Greenspan, mentioned above, follow-up studies manipulating 5-HT levels demonstrated that serotonin does modulate aspects of aggression in these flies, implicating serotonin in this behavior (Dierick and Greenspan, 2007). In flies, serotonin has been demonstrated to modulate sleep, circadian behaviors, and sensory processing (Nichols et al., 2002, Yuan et al., 2006, Nichols, 2007). In both flies and mammals the effects of serotonin are primarily mediated through interactions with a number of G-protein coupled receptor proteins to initiate various effector pathways (Nichols and Nichols, 2008). Significantly, the orthologous serotonin receptor families present in Drosophila are the 5-HT2, 5-HT1A-like, and 5-HT7 receptors (Witz et al., 1990, Saudou et al., 1992, Colas et al., 1995). The 5-HT1A-like receptors include the 5-HT1ADro and 5-HT1BDro (formerly the 5-HTdro2A and 5-HTdro2B) receptors, which are believed to be duplicated genes and homologues to the mammalian 5-HT1A receptor (Saudou et al., 1992). Of all the serotonin receptors, the 5-HT1A, 5-HT1B, and 5-HT2, receptors have been the most strongly implicated in mammalian and human behaviors. Here, we have used pharmacological methods to probe individually 5-HT2 and 5-HT1A-like receptor circuitries for their roles in modulating aggression in Drosophila, and show that 5-HT2- and 5-HT1A-like receptors have differential effects on aggressive behaviors in the fly

SummaryWe 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.

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-HT 1A -like and 5-HT 2 , and 5-HT 2 and 5-HT 7 receptor circuits in mediating short-term learning and memory. Examination of long-term memory suggests that 5-HT 1A -like receptors are necessary for consolidation and important for recall, 5-HT 2 receptors are important for consolidation and recall, and 5-HT 7 receptors are involved in all three phases. Importantly, we have validated our pharmacological results with genetic experiments and showed that hypomorph strains for 5-HT 2 Dro and 5-HT 1B Dro receptors, as well as knockdown of 5-HT 7 Dro 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.