Current Projects

1)         We study how long-term memory is formed, focusing on mechanisms of synaptic plasticity that depend on local protein synthesis. We found that long-term memories form in association with mRNA transport and protein synthesis at particular synapses; that is, the modification of synapses is specific to the content of a memory. We also discovered a mechanism that regulates synaptic protein synthesis, involving microRNAs and the RISC pathway. In this mechanism, a neurotransmitter receptor associated protein called Rapsyn targets a RISC component, called Armitage, for Proteasome-mediated degradation. In addition, we have found that microRNA biogenesis is regulated by neural activity, and will determine whether this form of regulation occurs as memories form. To aid in this analysis, two-photon live imaging of Drosophila undergoing olfactory associative conditioning is being employed. To date, we have observed specific patterns of odor-induced synaptic output from the first order olfactory interneurons known as Projection Neurons, patterns that alter with associative conditioning. This preparation permits the manipulations of gene function and neural activity to be examined for effects on specific biochemical reporters, all during the induction of a memory. Our work has also opened up interesting avenues for translational research, including efforts to treat memory disorders and epilepsy. [See publication, Ashraf et al., 2006; Patents Pending: “Compositions and Methods to Modulate Memory”]


2)         The cellular and biochemical signature of memory is transformed as a memory transits a nascent labile state into a consolidated and stable form. In prior work, we found that the translation of mRNA at synapses, regulated by the RISC pathway, is associated with the induction of stable memory in Drosophila. We next considered whether the regulation of small RNA biogenesis is an additional control point in this pathway. In the odor, shock associative conditioning paradigm, repeated electric foot-shocks serve as the aversive stimulus, acting via the release of dopamine into the adult brain memory center known as the Mushroom Body. Using high-throughput RNA sequence analysis and RT-PCR analysis, we found this release controls the biogenesis of a number of small RNAs, including piRNAs, snoRNAs, tRNA fragments, and miRNAs. A number of the regulated small RNAs were derived from the patterned degradation of mRNA.


Among the induced miRNA loci was let-7C, one of the original miRNA’s identified as heterochronic genes in C. elegans. Let-7 was induced specifically in the Mushroom Body, where its expression suppressed the synthesis of a nuclear BTB protein, Abrupt, in a specific subset of Mushroom Body neurons. Abrupt is known as a developmental regulator of dendritic arbor branching, and controls branch number in a dosage dependent fashion. We found that transgenic manipulation of Abrupt level in Mushroom Body neurons dramatically regulated the number of pre-synaptic sites at axonal termini. This regulation was likewise observed after odor, foot-shock conditioning and was required for memory consolidation. Our observations point to a role for widespread transient synapse assembly as a prologue to consolidation, a phenomenon that might be referred to as synaptic ‘priming’. We suspect that such priming events reflect the transfer of a memory ‘engram’ between neuronal populations, from neurons that hold short-term memory to those that encode it in a stable, consolidated state. Some current work in progress aims to extend these results to the zebrafish.


3)         We uncovered a neural circuit mediating the fly’s directional response to light and gravity. A set of four neurons deep within the brain controls the animal’s directional response to light sensation and gravity. When these neurons are functionally silenced, the animal’s light and gravitational responses are reversed; they walk away from light and in the direction of gravity, the opposite of their normal behaviors. Another site for regulation of phototactic and geotactic responses is the Ellipsoid Body, a component of the central brain structure known as the Central Complex. We also study how visual experience is remembered, particularly with regard to the wavelength of light, as flies make directional choices in a walking maze. In a genetic screen, we identified mutant animals that cannot form a memory of the association of light with aversive heat. We are now characterizing the genes that are required to form this memory. Our plan is to identify the full circuitry controlling these behaviors, from sensory input to motor output. An exciting prospect is to examine how these circuits differ in architecture and function in fruitflies that display distinct light and/or gravity directed behaviors, including other Drosophila species


4)         A number of years ago, we showed that the developing retina controls the timing and size of synaptic target field development in the brain via the axonal transport of two signaling molecules, Hedgehog and Spitz. A third retinal signal called Jelly Belly was recently uncovered by Iris Salecker and colleagues. Hedgehog is partitioned for release at both ends of the photoreceptor neuron. When secreted from the cell body, Hh regulates eye development; when transported along axons and secreted into the brain, Hh controls neuronal development there. The partitioning of Hh for cell body vs. axonal release balances neuronal development between these two fields. We found a conserved amino acid motif located at the Hh C-terminus that targets the protein for axonal release. There is also evidence for an opposing localization signal for cell body release, located at the N-terminus [See publication, Chu et al., 2006]. In ongoing work, we have identified a set of secretory and transport proteins that recognize these signals and control targeted release.  We have also determined, in collaboration with David Robbins (Dartmouth) that some mutant forms of Hedgehog found in human holoprosencephaly are refractory to the Hedgehog auto-cleavage reaction, but retain significant developmental signaling activity in their uncleaved form (Tokhunts et al., 2010, J Biol. Chem.). We are examining the intracellular trafficking and signaling properties of these mutant forms of the protein.


5)         We study the converse problem of retrograde axonal transport, where neurons in the brain inform the photoreceptor neurons, via their synaptic connections, of the spectral sensitivity to which to tune their light reception. A subset of photoreceptor neurons in the fly retina is dedicated to the reception of the spectral information for color vision. These neurons choose to express different light-sensitive pigments (Rhodopsins). We have found that the neurons in the brain, to which the photoreceptors communicate their light reception, transmit a retrograde signal that determines which Rhodopsin genes the photoreceptors express. By coordinating Rhodopsin expression in this way, the neurons in the brain determine the color of light that they are detecting, and therefore can properly encode this color input in the visual field.