ABSTRACT
Vertebrate synapsins are abundant synaptic vesicle phosphoproteins that have been proposed to fine-regulate neurotransmitter release by phosphorylation-dependent control of synaptic vesicle motility. However, the consequences of a total lack of all synapsin isoforms due to a knock-out of all three mouse synapsin genes have not yet been investigated. In Drosophila a single synapsin gene encodes several isoforms and is expressed in most synaptic terminals. Thus the targeted deletion of the synapsin gene of Drosophila eliminates the possibility of functional knock-out complementation by other isoforms. Unexpectedly, synapsin null mutant flies show no obvious defects in brain morphology, and no striking qualitative changes in behaviour are observed. Ultrastructural analysis of an identified 'model' synapse of the larval nerve muscle preparation revealed no difference between wild-type and mutant, and spontaneous or evoked excitatory junction potentials at this synapse were normal up to a stimulus frequency of 5 Hz. However, when several behavioural responses were analysed quantitatively, specific differences between mutant and wild-type flies are noted. Adult locomotor activity, optomotor responses at high pattern velocities, wing beat frequency, and visual pattern preference are modified. Synapsin mutant flies show faster habituation of an olfactory jump response, enhanced ethanol tolerance, and significant defects in learning and memory as measured using three different paradigms. Larval behavioural defects are described in a separate paper. We conclude that Drosophila synapsins play a significant role in nervous system function, which is subtle at the cellular level but manifests itself in complex behaviour.
Subject(s)
Behavior, Animal/physiology , Drosophila/physiology , Synapsins/deficiency , Action Potentials/drug effects , Action Potentials/genetics , Action Potentials/physiology , Animals , Animals, Genetically Modified , Behavior, Animal/drug effects , Blotting, Western/methods , Central Nervous System Depressants/pharmacology , Cloning, Molecular/methods , Conditioning, Operant/physiology , DNA Mutational Analysis , Drosophila/genetics , Electric Stimulation/methods , Ethanol/pharmacology , Excitatory Postsynaptic Potentials/genetics , Immunohistochemistry/methods , Immunosorbent Techniques , Membrane Potentials/genetics , Membrane Potentials/physiology , Microscopy, Electron , Motor Activity/drug effects , Motor Activity/physiology , Mutagenesis/physiology , Neuromuscular Junction/genetics , Neuromuscular Junction/metabolism , Neuromuscular Junction/physiology , Psychomotor Performance/physiology , Sexual Behavior/drug effects , Sexual Behavior/physiology , Synapses/metabolism , Synapses/ultrastructure , Synapsins/genetics , Synapsins/physiology , Synaptic Vesicles/genetics , Synaptic Vesicles/metabolism , Synaptic Vesicles/ultrastructure , Time Factors , Tissue Distribution , Visual Perception/genetics , Visual Perception/physiology , Walking/physiology , Wings, Animal/physiologyABSTRACT
Cysteine string proteins (CSPs) are conserved secretory vesicle proteins involved in regulating neurotransmitter and peptide release. While the function of the J-domain has been studied in detail, little is known about other conserved regions. We have constructed mutant genes coding for proteins with modified cysteine string, linker region or C terminus and transformed them into Csp null-mutant Drosophila: In the living animal, mutated CSP lacking all cysteines fails to associate with membranes, does not concentrate in synaptic terminals, and cannot rescue adult temperature-sensitive paralysis and short life span, both prominent null mutant phenotypes. A mutant protein with 5 instead of 11 string cysteines appears to be normally targeted but cannot rescue paralysis at 37 degrees C. We propose that the cysteine string, in addition to its role in targeting, may be essential for a function of CSP that is dependent on the number of cysteines in the string. A deletion in the linker region or the C terminus does not affect CSP targeting, and function in adults is only marginally impaired.
