Ricardo Delgado

Synaptic Defects and Compensatory Regulation of Inositol Metabolism in Inositol Polyphosphate 1-Phosphatase Mutants

Phosphoinositides function as important second messengers in a wide range of cellular processes. Inositol polyphosphate 1-phosphatase (IPP) is an enzyme essential for the hydrolysis of the 1-phosphate from either Ins(1,4)P2 or Ins(1,3,4)P3. This enzyme is Li+ sensitive, and is one of the proposed targets of Li+ therapy in manic-depressive illness. Drosophila ipp mutants accumulate IP2 in their system and are incapable of metabolizing exogenous Ins(1,4)P2. Notably, ipp mutants demonstrate compensatory upregulation of an alternative branch in the inositol-phosphate metabolism tree, thus providing a means of ensuring continued availability of inositol. We demonstrate that ipp mutants have a defect in synaptic transmission resulting from a dramatic increase in the probability of vesicle release at larval neuromuscular junctions. We also show that Li+ phenocopies this effect in wild-type synapses. Together, these results support a role for phosphoinositides in synaptic vesicle function in vivo and mechanistically question the lithium hypothesis.

DLGS97/SAP97 Is Developmentally Upregulated and Is Required for Complex Adult Behaviors and Synapse Morphology and Function

The synaptic membrane-associated guanylate kinase (MAGUK) scaffolding protein family is thought to play key roles in synapse assembly and synaptic plasticity. Evidence supporting these roles in vivo is scarce, as a consequence of gene redundancy in mammals. The genome of Drosophila contains only one MAGUK gene, discs large (dlg), from which two major proteins originate: DLGA [PSD95 (postsynaptic density 95)-like] and DLGS97 [SAP97 (synapse-associated protein)-like]. These differ only by the inclusion in DLGS97 of an L27 domain, important for the formation of supramolecular assemblies. Known dlg mutations affect both forms and are lethal at larval stages attributable to tumoral overgrowth of epithelia. We generated independent null mutations for each, dlgA and dlgS97. These allowed unveiling of a shift in expression during the development of the nervous system: predominant expression of DLGA in the embryo, balanced expression of both during larval stages, and almost exclusive DLGS97 expression in the adult brain. Loss of embryonic DLGS97 does not alter the development of the nervous system. At larval stages, DLGA and DLGS97 fulfill both unique and partially redundant functions in the neuromuscular junction. Contrary to dlg and dlgA mutants, dlgS97 mutants are viable to adulthood, but they exhibit marked alterations in complex behaviors such as phototaxis, circadian activity, and courtship, whereas simpler behaviors like locomotion and odor and light perception are spared. We propose that the increased repertoire of associations of a synaptic scaffold protein given by an additional domain of protein–protein interaction underlies its ability to integrate molecular networks required for complex functions in adult synapses.

Unitary Recordings of TRP and TRPL Channels From Isolated Drosophila Retinal Photoreceptor Rhabdomeres: Activation by Light and Lipids

Transient receptor potential (TRP) channels play key roles in sensory transduction. The TRP family founding members, the Drosophila light-dependent channels, were previously studied under voltage clamp, but had not been characterized in intact rhabdomeres at single-channel level. We report patch-clamp recordings from intact isolated photoreceptors of wt and mutant flies lacking TRP (trp(343)), TRPL (trpl(302)), or both channels (trp(313); trpl(302)). Unitary currents were activated by light in rhabdomere-attached patches. In excised rhabdomeral patches, the channels were directly activated by molecules implicated in phototransduction, such as diacylglycerol and polyunsaturated fatty acids. Currents recorded from trpl photoreceptors are blocked by external Ca(2+), Mg(2+) (1 mM), and La(3+) (20 muM), whereas those from trp photoreceptors are not. Rhabdomeric patches lacked voltage-dependent activity. Patches from trp;trpl mutants were devoid of channels. These characteristics match the macroscopic conductances, suggesting that the unitary currents from Drosophila trpl and trp photoreceptors correspond to TRP and TRPL.

K+ -channel blockers restore synaptic plasticity in the neuromuscular junction of dunce, a Drosophila learning and memory mutant

The effects of K+-channel blockers on synaptic transmission in dunce (dne ), a Drosophila learning and memory mutant, were investigated. Larvae dne mutants lack facilitation and post-tetanic potentiation (PTP) at their motor end-plates; dne mutants are also deficient in a form of phosphodiesterase, and exhibit abnormally high levels of cyclic adenosine 3, 5 -monophosphate (cAMP). A two-microelectrode voltage-clamp was used to record end-plate currents and spontaneous end-plate currents from longitudinal ventrolateral third-instar larval muscle. The K+-channel blockers 3, 4-diaminopyridine (3, 4-DAP) and tetraethylammonium (TEA), at micromolar concentrations, caused a reversible decrease in end-plate current amplitudes both in wild-type and mutant end-plates. In the presence of blockers, a period of high-frequency stimulation (tetanus) of the nerve gave way to a transient increase in the end-plate currents of dnc mutants resembling facilitation and PTP in normal end-plates; 3, 4-DAP and TEA also restored facilitation and PTP in normal end-plates after incubation with a non-hydrolysable analogue of cAMP (8Br-cAMP). It is suggested that a specific K+ conductance might be relevant to the lack of synaptic plasticity at the dnc neuromuscular synapses.

Scaffolding proteins in highly purified rat olfactory cilia membranes.

Odour-mediated signal transduction is a complex process that occurs in the cilia of olfactory sensory neurons. To gain insight in to the molecular organization of the odour transduction machinery, we developed a procedure to purify olfactory cilia membranes by differential centrifugation of rat olfactory epithelium extracts. We tested whether known scaffolding proteins that might participate in the assembly of the complex chemotransduction apparatus are present in the purified membrane fraction. Utilizing immunoblotting and immunohistochemistry, we show that the multidomain scaffolding proteins ProSAP/Shanks and calcium/calmodulin-dependent serine protein kinase CASK are present in the olfactory cilia. Ion channels involved in chemotransduction could be reconstituted into planar lipid bilayers for electrophysiological recordings. Our procedure should allow the identification of further chemotransduction-related proteins.

Presynaptic DLG regulates synaptic function through the localization of voltage-activated Ca2+ Channels

The DLG-MAGUK subfamily of proteins plays a role on the recycling and clustering of glutamate receptors (GLUR) at the postsynaptic density. discs-large1 (dlg) is the only DLG-MAGUK gene in Drosophila and originates two main products, DLGA and DLGS97 which differ by the presence of an L27 domain. Combining electrophysiology, immunostaining and genetic manipulation at the pre and postsynaptic compartments we study the DLG contribution to the basal synaptic-function at the Drosophila larval neuromuscular junction. Our results reveal a specific function of DLGS97 in the regulation of the size of GLUR fields and their subunit composition. Strikingly the absence of any of DLG proteins at the presynaptic terminal disrupts the clustering and localization of the calcium channel DmCa1A subunit (Cacophony), decreases the action potential-evoked release probability and alters short-term plasticity. Our results show for the first time a crucial role of DLG proteins in the presynaptic function in vivo.