Biology subjects

O'Kane, C. J.

Publications and source records attributed to O'Kane, C. J..

6 recordsLinked to original sources

bioRxiv2022

Mushroom body output neurons MBONa1/a2 define an odor intensity channel that regulates behavioral odor discrimination learning in larval Drosophila

The sensitivity of animals to sensory input must be regulated to ensure that signals are detected and also discriminable. However, how circuits regulate the dynamic range of sensitivity to sensory stimuli is not well understood. A given odor is represented in the insect mushroom bodies (MBs) by sparse combinatorial coding by Kenyon cells (KCs), forming an odor quality representation. To address how intensity of sensory stimuli is processed at the level of the MB input region, the calyx, we characterized a set of novel mushroom body output neurons that respond only to high odor concentrations. We show that a pair of MB calyx output neurons, MBONa1/2, are postsynaptic in the MB calyx, where they receive extensive synaptic inputs from KC dendrites, the inhibitory feedback neuron APL, and octopaminergic sVUM1 neurons, but relatively few inputs from projection neurons. This pattern is broadly consistent in the third instar larva as well as in the first instar connectome. MBONa1/a2 presynaptic terminals innervate a region immediately surrounding the MB medial lobe output region in the ipsilateral and contralateral brain hemispheres. By monitoring calcium activity using jRCamP1b, we find that MBONa1/a2 responses are odor-concentration dependent, responding only to ethyl acetate (EA) concentrations higher than a 200-fold dilution, in contrast to MB neurons which are relatively concentration-invariant and respond to EA dilutions as low as 10-4. Optogenetic activation of the calyx-innervating sVUM1 modulatory neurons originating in the SEZ (Subesophageal zone), did not show a detectable effect on MBONa1/a2 odor responses. Optogenetic activation of MBONa1/a2 using CsChrimson impaired odor discrimination learning compared to controls. We propose that MBONa1/a2 form an output channel of the calyx, summing convergent sensory and modulatory input, firing only to high odor concentration, and might affect the activity of downstream MB targets.

neuroscience↗

bioRxiv2021

Spastic paraplegia protein ortholog, reticulon-like 1, governs presynaptic ER organization and Ca2+ dynamics

Neuronal endoplasmic reticulum (ER) appears continuous throughout the cell. Its shape and continuity are influenced by ER-shaping proteins, mutations in which can cause distal axon degeneration in Hereditary Spastic Paraplegia (HSP). We therefore asked how loss of Rtnl1, a Drosophila ortholog of the human HSP gene RTN2 (SPG12), which encodes an ER-shaping protein, affects ER organization and the function of presynaptic terminals. Loss of Rtnl1 depleted ER membrane markers at Drosophila presynaptic motor terminals, and appeared to deplete narrow tubular ER while leaving cisternae largely unaffected, thus suggesting little change in resting Ca2+ storage capacity. Nevertheless, these changes were accompanied by major reductions in activity-evoked Ca2+ fluxes in the cytosol, ER lumen, and mitochondria, as well as reduced evoked and spontaneous neurotransmission. We found that reduced STIM-mediated ER-plasma membrane contacts underlie presynaptic Ca2+ defects in Rtnl1 mutants. Our results show the importance of ER architecture in presynaptic physiology and function which are therefore potential factors in the pathology of HSP.

neuroscience↗

bioRxiv2020

Distant homologies and domain conservation of the Hereditary Spastic Paraplegia protein SPG11/ALS5/spatacsin

Loss-of-function mutations in SPG11 protein (spatacsin) are a common cause of autosomal recessive hereditary spastic paraplegia with thin corpus callosum. To identify regions of the protein that may have functions that are disrupted in disease, we carried out bioinformatic analyses of its conserved regions. An N-terminal region of around 650 amino-acid residues, present in SPG11 across a wide range of metazoan animals, was missing from many insect lineages. Evolutionary loss of this domain correlated with loss of its binding partner, the AP-5 adaptor complex, suggesting that its main function is interaction with AP-5 in intracellular trafficking, and that the remainder of SPG11 carries out AP-5-independent functions. At the C-terminus of SPG11, a spatacsin_C domain showed sequence similarity and predicted structural homology to the Vps16_C domain of the HOPS complex protein Vps16. It localized to acidic compartments, consistent with a role in endolysosomal or autolysosomal transport, like Vps16. Mass spectrometry analysis of binding partners of this domain identified membrane trafficking proteins, some SM proteins, and several aminoacyl-tRNA synthetases. Since mutations affecting SPG11 or aminoacyl-tRNA synthetases can both cause Charcot-Marie-Tooth neuropathy (CMT) type 2, we suggest autolysosomal trafficking as a target process in CMT type 2.

neuroscience↗

bioRxiv2020

Endoplasmic reticulum (ER) lumenal indicators in Drosophila reveal effects of HSP-related mutations on ER calcium dynamics

Genes for ER-shaping proteins are among the most commonly mutated in Hereditary Spastic Paraplegia (HSP). Mutation of these genes in model organisms can lead to disruption of the ER network. To investigate how the physiological roles of the ER might be affected by such disruption, we developed tools to interrogate its Ca2+ signaling function. We generated GAL4-driven Ca2+ sensors targeted to the ER lumen, to record ER Ca2+ fluxes in identified Drosophila neurons. Using GAL4 lines specific for Type Ib or Type Is larval motor neurons, we compared the responses of different lumenal indicators to electrical stimulation, in axons and presynaptic terminals. The most effective sensor, ER-GCaMP6-210, had a Ca2+ affinity close to the expected ER lumenal concentration. Repetitive nerve stimulation generally showed a transient increase of lumenal Ca2+ in both the axon and presynaptic terminals. Mutants lacking neuronal reticulon and REEP proteins, homologs of human HSP proteins, showed a larger ER lumenal evoked response compared to wild type; we propose mechanisms by which this phenotype could lead to neuronal dysfunction or degeneration. Our lines are useful additions to a Drosophila Ca2+ imaging toolkit, to explore the physiological roles of ER, and its pathophysiological roles in HSP and in axon degeneration more broadly.

neuroscience↗

bioRxiv2018

GAL4 drivers specific for Type Ib and Type Is motor neurons in Drosophila

The Drosophila larval neuromuscular system is extensively used by researchers to study neuronal cell biology, and Drosophila glutamatergic motor neurons (MNs) have become a major model system. There are two main Types of glutamatergic MNs, Ib and Is, with different structural and physiological properties at synaptic level at the neuromuscular junction. To generate genetic tools to identify and manipulate MNs of each Type, we screened for GAL4 driver lines for this purpose. Here we describe GAL4 drivers specific for examples of neurons within each Type, Ib or Is. These drivers showed high expression levels and were expressed in only few MNs, making them amenable tools for specific studies of both axonal and synapse biology in identified Type I MNs.

neuroscience↗

bioRxiv2018

Octopaminergic neurons have multiple targets in Drosophila larval mushroom body calyx and regulate behavioral odor discrimination

Discrimination of sensory signals is essential for an organism to form and retrieve memories of relevance in a given behavioural context. Sensory representations are modified dynamically by changes in behavioral state, facilitating context-dependent selection of behavior, through signals carried by noradrenergic input in mammals, or octopamine (OA) in insects. To understand the circuit mechanisms of this signaling, we characterized the function of two OA neurons, sVUM1 neurons, that originate in the subesophageal zone (SEZ) and target the input region of the memory center, the mushroom body (MB) calyx, in larval Drosophila. We find that sVUM1 neurons target multiple neurons, including olfactory projection neurons (PNs), the inhibitory neuron APL, and a pair of extrinsic output neurons, but relatively few mushroom body intrinsic neurons, Kenyon cells. PN terminals carried the OA receptor Oamb, a Drosophila 1-adrenergic receptor ortholog. Using an odor discrimination learning paradigm, we showed that optogenetic activation of OA neurons compromised discrimination of similar odors but not learning ability. Our results suggest that sVUM1 neurons modify odor representations via multiple extrinsic inputs at the sensory input area to the MB olfactory learning circuit.

neuroscience↗