Kathleen K. Siwicki

Pharmacological Rescue of Synaptic Plasticity, Courtship Behavior, and Mushroom Body Defects in a Drosophila Model of Fragile X Syndrome

Fragile X syndrome is a leading heritable cause of mental retardation that results from the loss of FMR1 gene function. A Drosophila model for Fragile X syndrome, based on the loss of dfmr1 activity, exhibits phenotypes that bear similarity to Fragile X-related symptoms. Herein, we demonstrate that treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium can rescue courtship and mushroom body defects observed in these flies. Furthermore, we demonstrate that dfmr1 mutants display cognitive deficits in experience-dependent modification of courtship behavior, and treatment with mGluR antagonists or lithium restores these memory defects. These findings implicate enhanced mGluR signaling as the underlying cause of the cognitive, as well as some of the behavioral and neuronal, phenotypes observed in the Drosophila Fragile X model. They also raise the possibility that compounds having similar effects on metabotropic glutamate receptors may ameliorate cognitive and behavioral defects observed in Fragile X patients.

Circadian fluctuations of period protein immunoreactivity in the CNS and the visual system of Drosophila

When the protein encoded by the period (per) gene, which influences circadian rhythms in Drosophila melanogaster, was labeled with an anti- per antibody in adult flies sectioned at different times of day, regular fluctuations in the intensity of immunoreactivity were observed in cells of the visual system and central brain. These fluctuations persisted in constant darkness. Time courses of the changing levels of staining were altered in the per-short mutant: in light/dark cycles, the phase was earlier than in wild-type, and in constant darkness the period was shorter. In a per-long mutant and in behaviorally subnormal germline transformants (involving transduced per DNA), staining intensities were much fainter than in wild-type. Factors involved in initiating or maintaining the per protein cycling were investigated by examining the immunoreactivity in visual system mutants and by exposing wild-type flies to altered light/dark regimes. These genetic and environmental manipulations affected the expression of the per protein in ways that usually parallelled their effects on circadian behaviors.

Antibodies to the period gene product of drosophila reveal diverse tissue distribution and rhythmic changes in the visual system

Polyclonal antibodies were prepared against the period gene product, which influences biological rhythms in D. melanogaster, by using small synthetic peptides from the per sequence as immunogens. The peptide that elicited the best antibody reagent was a small domain near the site of the pers (short period) mutation. Specific immunohistochemical staining was detected in a variety of tissue types: the embryonic CNS; a few cell bodies in the central brain of pupae; these and other cells in the central brain of adults, as well as imaginal cells in the eyes, optic lobes, and the gut. The intensity of per-specific staining in the visual system was found to oscillate, defining a free-running circadian rhythm with a peak in the middle of the night.

Drosophila Lacking dfmr1 Activity Show Defects in Circadian Output and Fail to Maintain Courtship Interest

Fragile X mental retardation is a prominent genetic disorder caused by the lack of the FMR1 gene product, a known RNA binding protein. Specific physiologic pathways regulated by FMR1 function have yet to be identified. Adult dfmr1 (also called dfxr) mutant flies display arrhythmic circadian activity and have erratic patterns of locomotor activity, whereas overexpression of dFMR1 leads to a lengthened period. dfmr1 mutant males also display reduced courtship activity which appears to result from their inability to maintain courtship interest. Molecular analysis fails to reveal any defects in the expression of clock components; however, the CREB output is affected. Morphological analysis of neurons required for normal circadian behavior reveals subtle abnormalities, suggesting that defects in axonal pathfinding or synapse formation may cause the observed behavioral defects.

Mushroom Body Ablation Impairs Short-Term Memory and Long-Term Memory of Courtship Conditioning in Drosophila melanogaster

We have evaluated the role of the Drosophila mushroom bodies (MBs) in courtship conditioning, in which experience with mated females causes males to reduce their courtship toward virgins (Siegel and Hall, 1979). Whereas previous studies indicated that MB ablation abolished learning in an olfactory conditioning paradigm (deBelle and Heisenberg, 1994), MB-ablated males were able to learn in the courtship paradigm. They resumed courting at naive levels within 30 min after training, however, while the courtship of control males remained depressed 1 hr after training. We also describe a novel courtship conditioning paradigm that established long-term memory, lasting 9 days. In MB-ablated males, memory dissipated completely within 1 day. Our results indicate that the MBs are not required for learning and immediate recall of courtship conditioning but are required for consolidation of short-term and long-term associative memories.

Neuroanatomical studies of period gene expression in the hawkmoth, Manduca sexta.

In the nervous system of the hawkmoth, Manduca sexta, cells expressing the period (per)gene were mapped by in situ hybridization and immunocytochemical methods. Digoxigenin‐labeled riboprobes were transcribed from a 1‐kb M. sexta per cDNA. Monoclonal anti‐PER antibodies were raised to peptide antigens translated from both M. sexta and Drosophila melanogaster per cDNAs. These reagents revealed a widespread distribution of per gene products in M. sexta eyes, optic lobes, brains, and retrocerebral complexes. Labeling for per mRNA was prominent in photoreceptors and in glial cells throughout the brain, and in a cluster of 100–200 neurons adjacent to the accessory medulla of the optic lobes. Daily rhythms of per mRNA levels were detected only in glial cells. PER‐like immunoreactivity was observed in nuclei of most neurons and glial cells and in many photoreceptor nuclei. Four neurosecretory cells in the pars lateralis of each brain hemisphere exhibited both nuclear and cytoplasmic staining with anti‐PER antibodies. These cells were positively identified as Ia1 neurosecretory cells that express corazonin immunoreactivity. Anti‐corazonin labeled their projections in the brain and their neurohemal endings in the corpora cardiaca and corpora allata. Four pairs of PER‐expressing neurosecretory cells previously described in the silkmoth, Anthereae pernyi, are likely to be homologous to these PER/corazonin‐expressing Ia1 cells of M. sexta. Other findings, such as widespread nuclear localization of M. sexta PER and rhythmic expression in glial cells, are reminiscient of the period gene of D. melanogaster, suggesting that some functions of per may be conserved in this lepidopteran species. J. Comp. Neurol. 447:366–380, 2002.

Fruitless, doublesex and the genetics of social behavior in Drosophila melanogaster

Two genes coding for transcription factors, fruitless and doublesex, have been suggested to play important roles in the regulation of sexually dimorphic patterns of social behavior in Drosophila melanogaster. The generalization that fruitless specified the development of the nervous system and doublesex specified non-neural tissues culminated with claims that fruitless was both necessary and sufficient to establish sex-specific patterns of behavior. Several recent articles refute this notion, however, demonstrating that at a minimum, both fruitless and doublesex are involved in establishing sexually dimorphic features of neural circuitry and behavior in fruit flies.

Associative learning and memory in Drosophila: beyond olfactory conditioning

The associative learning abilities of the fruit fly, Drosophila melanogaster, have been demonstrated in both classical and operant conditioning paradigms. Efforts to identify the neural pathways and cellular mechanisms of learning have focused largely on olfactory classical conditioning. Results derived from various genetic and molecular manipulations provide considerable evidence that this form of associative learning depends critically on neural activity and cAMP signaling in brain neuropil structures called mushroom bodies. Three other behavioral learning paradigms in Drosophila serve as the main subject of this review. These are (1) visual and motor learning of flies tethered in a flight simulator, (2) a form of spatial learning that is independent of visual and olfactory cues, and (3) experience-dependent changes in male courtship behavior. The present evidence suggests that at least some of these modes of learning are independent of mushroom bodies. Applying targeted genetic manipulations to these behavioral paradigms should allow for a more comprehensive understanding of neural mechanisms responsible for diverse forms of associative learning and memory.

A Drosophila DEG/ENaC subunit functions specifically in gustatory neurons required for male courtship behavior.

Detection of specific female pheromones stimulates courtship behavior in Drosophila melanogaster males, but the chemosensory molecules, cells, and mechanisms involved remain poorly understood. Here we show that ppk25, a DEG/ENaC ion channel subunit required for normal male response to females, is expressed at highest levels in a single sexually dimorphic gustatory neuron of most taste hairs on legs and wings, but not in neurons that detect courtship-inhibiting pheromones or food. Synaptic inactivation of ppk25-expressing neurons, or knockdown of ppk25 expression in all gustatory neurons, significantly impairs male response to females, whereas gustatory expression of ppk25 rescues the courtship behavior of ppk25 mutant males. Remarkably, the only other detectable albeit significantly weaker expression of ppk25 occurs in olfactory neurons implicated in modulation of courtship behavior. However, expression of ppk25 in olfactory neurons is not required for male courtship under our experimental conditions. These data show that ppk25 functions specifically in peripheral taste neurons involved in activation of courtship behavior, an unexpected function for this type of channel. Furthermore, our work identifies a small subset of gustatory neurons with an essential role in activation of male courtship behavior, most likely in response to female pheromones.

Proctolin in the lobster nervous system

The pentapeptide proctolin (Arg-Tyr-Leu-Pro-Thr) is present in high concentrations in neurosecretory organs of the lobster, Homarus americanus. The central nervous system contains ca. 1400 proctolin-immunoreactive neurons, which appear to serve a variety of different functions. Some of these neurons have been specifically identified and analyzed biochemically to determine which classical neurotransmitters coexist with the peptide. These include: serotonin-proctolin cell pairs in the fifth thoracic and first abdominal ganglia; a large dopamine-proctolin neuron in the circumesophageal ganglion; and cholinergic-proctolin sensory neurons which innervate a mechanoreceptor in the scaphognathite. With these identified neurons we have begun to investigate the physiological actions of proctolin, the interactions between cotransmitters, and the development of multiple transmitter phenotypes in individual neurons.