Lipophorin receptors regulate mushroom body development and complex behaviors in Drosophila.

BACKGROUND: Drosophila melanogaster lipophorin receptors (LpRs), LpR1 and LpR2, are members of the LDLR family known to mediate lipid uptake in a range of organisms from Drosophila to humans. The vertebrate orthologs of LpRs, ApoER2 and VLDL-R, function as receptors of a glycoprotein involved in development of the central nervous system, Reelin, which is not present in flies. ApoER2 and VLDL-R are associated with the development and function of the hippocampus and cerebral cortex, important association areas in the mammalian brain, as well as with neurodevelopmental and neurodegenerative disorders linked to those regions. It is currently unknown whether LpRs play similar roles in the Drosophila brain. RESULTS: We report that LpR-deficient flies exhibit impaired olfactory memory and sleep patterns, which seem to reflect anatomical defects found in a critical brain association area, the mushroom bodies (MB). Moreover, cultured MB neurons respond to mammalian Reelin by increasing the complexity of their neurite arborization. This effect depends on LpRs and Dab, the Drosophila ortholog of the Reelin signaling adaptor protein Dab1. In vitro, two of the long isoforms of LpRs allow the internalization of Reelin, suggesting that Drosophila LpRs interact with human Reelin to induce downstream cellular events. CONCLUSIONS: These findings demonstrate that LpRs contribute to MB development and function, supporting the existence of a LpR-dependent signaling in Drosophila, and advance our understanding of the molecular factors functioning in neural systems to generate complex behaviors in this model. Our results further emphasize the importance of Drosophila as a model to investigate the alterations in specific genes contributing to neural disorders.

An Early Disturbance in Serotonergic Neurotransmission Contributes to the Onset of Parkinsonian Phenotypes in Drosophila melanogaster.

Parkinson's disease (PD) is a neurodegenerative disease characterized by motor symptoms and dopaminergic cell loss. A pre-symptomatic phase characterized by non-motor symptoms precedes the onset of motor alterations. Two recent PET studies in human carriers of mutations associated with familial PD demonstrate an early serotonergic commitment-alteration in SERT binding-before any dopaminergic or motor dysfunction, that is, at putative PD pre-symptomatic stages. These findings support the hypothesis that early alterations in the serotonergic system could contribute to the progression of PD, an idea difficult to be tested in humans. Here, we study some components of the serotonergic system during the pre-symptomatic phase in a well-characterized Drosophila PD model, Pink1B9 mutant flies. We detected lower brain serotonin content in Pink1B9 flies, accompanied by reduced activity of SERT before the onset of motor dysfunctions. We also explored the consequences of a brief early manipulation of the serotonergic system in the development of motor symptoms later in aged animals. Feeding young Pink1B9 flies with fluoxetine, a SERT blocker, prevents the loss of dopaminergic neurons and ameliorates motor impairment observed in aged mutant flies. Surprisingly, the same pharmacological manipulation in young control flies results in aged animals exhibiting a PD-like phenotype. Our findings support that an early dysfunction in the serotonergic system precedes and contributes to the onset of the Parkinsonian phenotype in Drosophila.

Assessing olfactory, memory, social and circadian phenotypes associated with schizophrenia in a genetic model based on Rim.

Schizophrenia shows high heritability and several of the genes associated with this disorder are involved in calcium (Ca2+) signalling and synaptic function. One of these is the Rab-3 interacting molecule-1 (RIM1), which has recently been associated with schizophrenia by Genome Wide Association Studies (GWAS). However, its contribution to the pathophysiology of this disorder remains unexplored. In this work, we use Drosophila mutants of the orthologue of RIM1, Rim, to model some aspects of the classical and non-classical symptoms of schizophrenia. Rim mutants showed several behavioural features relevant to schizophrenia including social distancing and altered olfactory processing. These defects were accompanied by reduced evoked Ca2+ influx and structural changes in the presynaptic terminals sent by the primary olfactory neurons to higher processing centres. In contrast, expression of Rim-RNAi in the mushroom bodies (MBs), the main memory centre in flies, spared learning and memory suggesting a differential role of Rim in different synapses. Circadian deficits have been reported in schizophrenia. We observed circadian locomotor activity deficits in Rim mutants, revealing a role of Rim in the pacemaker ventral lateral clock neurons (LNvs). These changes were accompanied by impaired day/night remodelling of dorsal terminal synapses from a subpopulation of LNvs and impaired day/night release of the circadian neuropeptide pigment dispersing factor (PDF) from these terminals. Lastly, treatment with the commonly used antipsychotic haloperidol rescued Rim locomotor deficits to wildtype. This work characterises the role of Rim in synaptic functions underlying behaviours disrupted in schizophrenia.

The Drosophila ortholog of the schizophrenia-associated CACNA1A and CACNA1B voltage-gated calcium channels regulate memory, sleep and circadian rhythms.

Schizophrenia exhibits up to 80% heritability. A number of genome wide association studies (GWAS) have repeatedly shown common variants in voltage-gated calcium (Cav) channel genes CACNA1C, CACNA1I and CACNA1G have a major contribution to the risk of the disease. More recently, studies using whole exome sequencing have also found that CACNA1B (Cav2.2 N-type) deletions and rare disruptive variants in CACNA1A (Cav2.1 P/Q-type) are associated with schizophrenia. The negative symptoms of schizophrenia include behavioural defects such as impaired memory, sleep and circadian rhythms. It is not known how variants in schizophrenia-associated genes contribute to cognitive and behavioural symptoms, thus hampering the development of treatment for schizophrenia symptoms. In order to address this knowledge gap, we studied behavioural phenotypes in a number of loss of function mutants for the Drosophila ortholog of the Cav2 gene family called cacophony (cac). cac mutants showed several behavioural features including decreased night-time sleep and hyperactivity similar to those reported in human patients. The change in timing of sleep-wake cycles suggested disrupted circadian rhythms, with the loss of night-time sleep being caused by loss of cac just in the circadian clock neurons. These animals also showed a reduction in rhythmic circadian behaviour a phenotype that also could be mapped to the central clock. Furthermore, reduction of cac just in the clock resulted in a lengthening of the 24 h period. In order to understand how loss of Cav2 function may lead to cognitive deficits and underlying cellular pathophysiology we targeted loss of function of cac to the memory centre of the fly, called the mushroom bodies (MB). This manipulation was sufficient to cause reduction in both short- and intermediate-term associative memory. Memory impairment was accompanied by a decrease in Ca2+ transients in response to a depolarizing stimulus, imaged in the MB presynaptic terminals. This work shows loss of cac Cav2 channel function alone causes a number of cognitive and behavioural deficits and underlying reduced neuronal Ca2+ transients, establishing Drosophila as a high-throughput in vivo genetic model to study the Cav channel pathophysiology related to schizophrenia.

Study of the release of endogenous amines in Drosophila brain in vivo in response to stimuli linked to aversive olfactory conditioning.

A highly challenging question in neuroscience is to understand how aminergic neural circuits contribute to the planning and execution of behaviors, including the generation of olfactory memories. In this regard, electrophysiological techniques like Local Field Potential or imaging methods have been used to answer questions relevant to cell and circuit physiology in different animal models, such as the fly Drosophila melanogaster. However, these techniques do not provide information on the neurochemical identity of the circuits of interest. Different approaches including fast scan cyclic voltammetry, allow researchers to identify and quantify in a timely fashion the release of endogenous neuroactive molecules, but have been only used in in vitro Drosophila brain preparations. Here, we report a procedure to record for the first time the release of endogenous amines -dopamine, serotonin and octopamine- in adult fly brain in vivo, by fast scan cyclic voltammetry. As a proof of principle, we carried out recordings in the calyx region of the Mushroom Bodies, the brain area mainly associated to the generation of olfactory memories in flies. By using principal component regression in normalized training sets for in vivo recordings, we detect an increase in octopamine and serotonin levels in response to electric shock and olfactory cues respectively. This new approach allows the study of dynamic changes in amine neurotransmission that underlie complex behaviors in Drosophila and shed new light on the contribution of these amines to olfactory processing in this animal model.

Dop1R1, a type 1 dopaminergic receptor expressed in Mushroom Bodies, modulates Drosophila larval locomotion.

As in vertebrates, dopaminergic neural systems are key regulators of motor programs in insects, including the fly Drosophila melanogaster. Dopaminergic systems innervate the Mushroom Bodies (MB), an important association area in the insect brain primarily associated to olfactory learning and memory, but that has been also implicated with the execution of motor programs. The main objectives of this work is to assess the idea that dopaminergic systems contribute to the execution of motor programs in Drosophila larvae, and then, to evaluate the contribution of specific dopaminergic receptors expressed in MB to these programs. Our results show that animals bearing a mutation in the dopamine transporter show reduced locomotion, while mutants for the dopaminergic biosynthetic enzymes or the dopamine receptor Dop1R1 exhibit increased locomotion. Pan-neuronal expression of an RNAi for the Dop1R1 confirmed these results. Further studies show that animals expressing the RNAi for Dop1R1 in the entire MB neuronal population or only in the MB γ-lobe forming neurons, exhibit an increased motor output, as well. Interestingly, our results also suggest that other dopaminergic receptors do not contribute to larval motor behavior. Thus, our data support the proposition that CNS dopamine systems innervating MB neurons modulate larval locomotion and that Dop1R1 mediates this effect.

Characterization of a presymptomatic stage in a Drosophila Parkinson's disease model: Unveiling dopaminergic compensatory mechanisms.

Parkinson's disease (PD) is a degenerative disorder characterized by several motor symptoms including shaking, rigidity, slow movement and difficult walking, which has been associated to the death of nigro-striatal dopaminergic neurons. >90% of PD patients also present olfactory dysfunction. Although the molecular mechanisms responsible for this disease are not clear, hereditary PD is linked to mutations in specific genes, including the PTEN-induced putative kinase 1 (PINK1). In this work we provide for the first time a thorough temporal description of the behavioral effects induced by a mutation in the PINK1 gene in adult Drosophila, a previously described animal model for PD. Our data suggests that the motor deficits associated to PD are fully revealed only by the third week of age. However, olfactory dysfunction is detected as early as the first week of age. We also provide immunofluorescence and neurochemical data that let us propose for the first time the idea that compensatory changes occur in this Drosophila model for PD. These compensatory changes are associated to specific components of the dopaminergic system: the biosynthetic enzymes, Tyrosine hydroxylase and Dopa decarboxylase, and the Dopamine transporter, a plasma membrane protein involved in maintaining dopamine extracellular levels at physiologically relevant levels. Thus, our behavioral, immunofluorescence and neurochemical data help define for the first time presymptomatic and symptomatic phases in this PD animal model, and that compensatory changes occur in the dopaminergic neurons in the presymptomatic stage.

Characterization of a Novel Drosophila SERT Mutant: Insights on the Contribution of the Serotonin Neural System to Behaviors.

A better comprehension on how different molecular components of the serotonergic system contribute to the adequate regulation of behaviors in animals is essential in the interpretation on how they are involved in neuropsychiatric and pathological disorders. It is possible to study these components in "simpler" animal models including the fly Drosophila melanogaster, given that most of the components of the serotonergic system are conserved between vertebrates and invertebrates. Here we decided to advance our understanding on how the serotonin plasma membrane transporter (SERT) contributes to serotonergic neurotransmission and behaviors in Drosophila. In doing this, we characterized for the first time a mutant for Drosophila SERT (dSERT) and additionally used a highly selective serotonin-releasing drug, 4-methylthioamphetamine (4-MTA), whose mechanism of action involves the SERT protein. Our results show that dSERT mutant animals exhibit an increased survival rate in stress conditions, increased basal motor behavior, and decreased levels in an anxiety-related parameter, centrophobism. We also show that 4-MTA increases the negative chemotaxis toward a strong aversive odorant, benzaldehyde. Our neurochemical data suggest that this effect is mediated by dSERT and depends on the 4-MTA-increased release of serotonin in the fly brain. Our in silico data support the idea that these effects are explained by specific interactions between 4-MTA and dSERT. In sum, our neurochemical, in silico, and behavioral analyses demonstrate the critical importance of the serotonergic system and particularly dSERT functioning in modulating several behaviors in Drosophila.