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Papers by Ricardo Delgado

Neuron, 2000

Valdivia terminal during sustained neuronal activity, we asked the following questions: what is t... more Valdivia terminal during sustained neuronal activity, we asked the following questions: what is the size of vesicle pools? Chile † Escuela de Postgrado What is the rate of vesicle mobilization from these pools? And to what extent vesicle recycling is contributing in Fac. de Ciencias Universidad de Chile maintaining synaptic transmission during prolonged tetanic stimulation? Combining measurements of nerve- ‡ Gunma University School of Medicine 3-39-22 Showa-machi evoked synaptic currents and imaging of FM1-43 in synaptic boutons, these questions can be answered by Maebashi 371-8511 Japan taking advantage of a temperature-sensitive paralytic mutant, shibire ts (shi ts ). In shi ts endocytosis ceases and synaptic vesicles are completely depleted after synaptic activation at nonpermissive temperatures, while synap-Summary tic transmission at room temperature is normal (Koenig and Ikeda, 1989, 1996; Koenig et al., 1989; Estes et al., Two vesicle pools, readily releasable (RRP) and re -1996). This mutant provides a means to estimate the serve (RP) pools, are present at Drosophila neuromussize of vesicle pools and their rates of mobilization at cular junctions. Using a temperature-sensitive mutant, the larval neuromuscular junction. The contribution of shibire ts , we studied pool sizes and vesicle mobilizarecycling for synaptic transmission can be examined in tion rates. In shibire ts , due to lack of endocytosis at wild-type larvae at steady state during repetitive nerve nonpermissive temperatures, synaptic currents constimulation.

Neuron, 1998

Phosphoinositides function as important second messengers in a wide range of cellular processes. ... more 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.”

Developmental Biology, 2001

To fertilize, mammalian sperm must complete a maturational process called capacitation. It is tho... more To fertilize, mammalian sperm must complete a maturational process called capacitation. It is thought that the membrane potential of sperm hyperpolarizes during capacitation, possibly due to the opening of K ؉ channels, but electrophysiological evidence is lacking. In this report, using patch-clamp recordings obtained from isolated mouse spermatogenic cells we document the presence of a novel K ؉ -selective inwardly rectifying current. Macroscopic current activated at membrane potentials below the equilibrium potential for K ؉ and its magnitude was dependent on the external K ؉ concentration. The channels selected K ؉ over other monovalent cations. Current was virtually absent when external K ؉ was replaced with Na ؉ or N-methyl-D-glucamine. Addition of Cs ؉ or Ba 2؉ (IC 50 of ϳ15 M) to the external solution effectively blocked K ؉ current. Dialyzing the cells with a Mg 2؉ -free solution did not affect channel activity. Cytosolic acidification reversibly inhibited the current. We verified that the resting membrane potential of mouse sperm changed from ؊52 ؎ 6 to ؊66 ؎ 9 mV during capacitation in vitro. Notably, application of 0.3-1 mM Ba 2؉ during capacitation prevented this hyperpolarization and decreased the subsequent exocytotic response to zona pellucida. A mechanism is proposed whereby opening of inwardly rectifying K ؉ channels may produce hyperpolarization under physiological conditions and contribute to the cellular changes that give rise to the capacitated state in mature sperm.

Proceedings of The Royal Society B: Biological Sciences, 1992

The effects of K(+)-channel blockers on synaptic transmission in dunce (dnc), a Drosophila learni... more The effects of K(+)-channel blockers on synaptic transmission in dunce (dnc), a Drosophila learning and memory mutant, were investigated. Larvae dnc mutants lack facilitation and post-tetanic potentiation (PTP) at their motor end-plates; dnc 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.

Brain Research Reviews, 2004

Our knowledge on the Drosophila neuromuscular synapse is rapidly expanding. Thus, this synapse of... more Our knowledge on the Drosophila neuromuscular synapse is rapidly expanding. Thus, this synapse offers an excellent model for studies of the molecular mechanism of synaptic transmission and synaptic plasticity. Two synaptic vesicle (SV) pools have been identified and characterized using a fluorescent styryl dye, FM1-43, to stain SVs. They are termed the exo/endo cycling pool (ECP), which corresponds to the readily releasable pool (RRP) defined electrophysiologically, and the reserve pool (RP). These two pools were identified first in a temperature-sensitive paralytic mutant, shibire, and subsequently confirmed in wild-type larvae. The ECP participates in synaptic transmission during low frequency firing of presynaptic nerves and locates in the periphery of presynaptic boutons in the vicinity of release sites, while SVs in the RP spread toward the center of boutons and are recruited only during tetanic stimulation. These two pools are separately replenished by endocytosis. Cyclic AMP facilitates recruitment of SVs from the RP to the ECP. Activation of presynaptic metabotropic glutamate receptors recruits SVs from the RP and enhances SV release by elevation of the cAMP level. Memory mutants that have defects in the cAMP/PKA cascade, dunce and rutabaga, exhibit reduced levels of recruitment of synaptic SVs from the RP to the ECP and have limited short-term synaptic plasticity. SV mobilization between the two pools could be a key step for changes in synaptic efficacy. Since a variety of mutants that have distinct defects in synaptic transmission are available for detailed studies of synaptic function, this direction of approach in Drosophila seems promising.

Proceedings of The National Academy of Sciences, 1991

Single-channel recording from longitudinal ventrolateral Drosophila larval muscle reveals the pre... more Single-channel recording from longitudinal ventrolateral Drosophila larval muscle reveals the presence of a potassium-selective channel that is directly and reversibly activated by cAMP in a dose-dependent fashion. Activation is specific and it cannot be mimicked by a series of agents that include AMP, cGMP, ATP, inositol trisphosphate, and Ca2 . Channel current records obtained from larval muscle in different dunce mutants possessing abnormally high levels of cAMP show that, in the mutants, the channel displays an

Neuron, 2000

Valdivia terminal during sustained neuronal activity, we asked the following questions: what is t... more Valdivia terminal during sustained neuronal activity, we asked the following questions: what is the size of vesicle pools? Chile † Escuela de Postgrado What is the rate of vesicle mobilization from these pools? And to what extent vesicle recycling is contributing in Fac. de Ciencias Universidad de Chile maintaining synaptic transmission during prolonged tetanic stimulation? Combining measurements of nerve- ‡ Gunma University School of Medicine 3-39-22 Showa-machi evoked synaptic currents and imaging of FM1-43 in synaptic boutons, these questions can be answered by Maebashi 371-8511 Japan taking advantage of a temperature-sensitive paralytic mutant, shibire ts (shi ts ). In shi ts endocytosis ceases and synaptic vesicles are completely depleted after synaptic activation at nonpermissive temperatures, while synap-Summary tic transmission at room temperature is normal (Koenig and Ikeda, 1989, 1996; Koenig et al., 1989; Estes et al., Two vesicle pools, readily releasable (RRP) and re -1996). This mutant provides a means to estimate the serve (RP) pools, are present at Drosophila neuromussize of vesicle pools and their rates of mobilization at cular junctions. Using a temperature-sensitive mutant, the larval neuromuscular junction. The contribution of shibire ts , we studied pool sizes and vesicle mobilizarecycling for synaptic transmission can be examined in tion rates. In shibire ts , due to lack of endocytosis at wild-type larvae at steady state during repetitive nerve nonpermissive temperatures, synaptic currents constimulation.

Neuron, 1998

Phosphoinositides function as important second messengers in a wide range of cellular processes. ... more 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.”

Developmental Biology, 2001

To fertilize, mammalian sperm must complete a maturational process called capacitation. It is tho... more To fertilize, mammalian sperm must complete a maturational process called capacitation. It is thought that the membrane potential of sperm hyperpolarizes during capacitation, possibly due to the opening of K ؉ channels, but electrophysiological evidence is lacking. In this report, using patch-clamp recordings obtained from isolated mouse spermatogenic cells we document the presence of a novel K ؉ -selective inwardly rectifying current. Macroscopic current activated at membrane potentials below the equilibrium potential for K ؉ and its magnitude was dependent on the external K ؉ concentration. The channels selected K ؉ over other monovalent cations. Current was virtually absent when external K ؉ was replaced with Na ؉ or N-methyl-D-glucamine. Addition of Cs ؉ or Ba 2؉ (IC 50 of ϳ15 M) to the external solution effectively blocked K ؉ current. Dialyzing the cells with a Mg 2؉ -free solution did not affect channel activity. Cytosolic acidification reversibly inhibited the current. We verified that the resting membrane potential of mouse sperm changed from ؊52 ؎ 6 to ؊66 ؎ 9 mV during capacitation in vitro. Notably, application of 0.3-1 mM Ba 2؉ during capacitation prevented this hyperpolarization and decreased the subsequent exocytotic response to zona pellucida. A mechanism is proposed whereby opening of inwardly rectifying K ؉ channels may produce hyperpolarization under physiological conditions and contribute to the cellular changes that give rise to the capacitated state in mature sperm.

Proceedings of The Royal Society B: Biological Sciences, 1992

The effects of K(+)-channel blockers on synaptic transmission in dunce (dnc), a Drosophila learni... more The effects of K(+)-channel blockers on synaptic transmission in dunce (dnc), a Drosophila learning and memory mutant, were investigated. Larvae dnc mutants lack facilitation and post-tetanic potentiation (PTP) at their motor end-plates; dnc 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.

Brain Research Reviews, 2004

Our knowledge on the Drosophila neuromuscular synapse is rapidly expanding. Thus, this synapse of... more Our knowledge on the Drosophila neuromuscular synapse is rapidly expanding. Thus, this synapse offers an excellent model for studies of the molecular mechanism of synaptic transmission and synaptic plasticity. Two synaptic vesicle (SV) pools have been identified and characterized using a fluorescent styryl dye, FM1-43, to stain SVs. They are termed the exo/endo cycling pool (ECP), which corresponds to the readily releasable pool (RRP) defined electrophysiologically, and the reserve pool (RP). These two pools were identified first in a temperature-sensitive paralytic mutant, shibire, and subsequently confirmed in wild-type larvae. The ECP participates in synaptic transmission during low frequency firing of presynaptic nerves and locates in the periphery of presynaptic boutons in the vicinity of release sites, while SVs in the RP spread toward the center of boutons and are recruited only during tetanic stimulation. These two pools are separately replenished by endocytosis. Cyclic AMP facilitates recruitment of SVs from the RP to the ECP. Activation of presynaptic metabotropic glutamate receptors recruits SVs from the RP and enhances SV release by elevation of the cAMP level. Memory mutants that have defects in the cAMP/PKA cascade, dunce and rutabaga, exhibit reduced levels of recruitment of synaptic SVs from the RP to the ECP and have limited short-term synaptic plasticity. SV mobilization between the two pools could be a key step for changes in synaptic efficacy. Since a variety of mutants that have distinct defects in synaptic transmission are available for detailed studies of synaptic function, this direction of approach in Drosophila seems promising.

Proceedings of The National Academy of Sciences, 1991

Single-channel recording from longitudinal ventrolateral Drosophila larval muscle reveals the pre... more Single-channel recording from longitudinal ventrolateral Drosophila larval muscle reveals the presence of a potassium-selective channel that is directly and reversibly activated by cAMP in a dose-dependent fashion. Activation is specific and it cannot be mimicked by a series of agents that include AMP, cGMP, ATP, inositol trisphosphate, and Ca2 . Channel current records obtained from larval muscle in different dunce mutants possessing abnormally high levels of cAMP show that, in the mutants, the channel displays an