Juan Bacigalupo

Inhibitory K+ current activated by odorants in toad olfactory neurons

Odorant responses of isolated olfactory neurons from the toad Caudiverbera caudiverbera were monitored by using patch-clamp techniques. Depending on the stimulus, the same neuron responded with an increase or a decrease in action potential firing. Odorants that activate the cAMP cascade in olfactory cilia increased electrical activity, caused membrane depolarization, and triggered inward currents. In contrast, odorants that do not activate the cAMP cascade inhibited electrical activity, produced membrane hyperpolarization, and activated outward currents in a dose-dependent fashion. Such currents were carried by K+ and blocked by tetraethylammonium. Similar currents were recorded from Xenopus laevis. Our results suggest that this K+ current is responsible for odorant-induced inhibition of action potential firing in olfactory neurons.

Cyclic-GMP enhances light-induced excitation and induces membrane currents in Drosophila retinal photoreceptors

Phototransduction in the Drosophila retina appears to require the phosphoinositide signaling cascade following receptor/G-protein activation. Subsequent opening of membrane cationic channels causes excitation. The biochemical events underlying channel opening and regulation of sensitivity remain largely unknown. Evidence is mounting that phototransduction in Drosophila and other invertebrate species may additionally involve the second messenger, cyclic-GMP (cGMP). We report that exogenous cGMP influenced Drosophila retinal phototransduction in two ways. In whole cell tight-seal voltage-clamp experiments, membrane permeant cGMP analog, 8-bromo-cyclic-GMP (8-Br-cGMP), induced membrane currents and dramatically enhanced light-induced currents. The currents induced by 8-Br-cGMP possessed reversal potentials similar to those induced by light. The magnitudes of cGMP-induced currents exhibited marked dependence on intensity of background illumination. Potential direct or modulatory roles of cGMP in Drosophila phototransduction are discussed.

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.

A ciliary K+ conductance sensitive to charibdotoxin underlies inhibitory responses in toad olfactory receptor neurons.

In olfactory neurons from Caudiverbera caudiverbera, a mixture of putrid odorants trigger an inhibitory, K+‐selective current and a hyperpolarizing receptor potential. The current‐voltage relation resembles that of a Ca2+‐activated K+ conductance; their amplitude depends on extracellular Ca2+. 10 nM charibdotoxin, a blocker of K+‐selective channels, including Ca2+‐activated ones, reversibly abolished inhibitory currents and receptor potentials. Focal stimulation demonstrates that the underlying transduction mechanism is confined to the cilia. This represents the first evidence for inhibitory responses in vertebrate olfactory cells mediated by a ciliary CTX‐sensitive K+ conductance, most likely a Ca2+‐activated one.

Calcium mediates the activation of the inhibitory current induced by odorants in toad olfactory receptor neurons

In toad olfactory neurons, a putrid odorant mixture inducing inhibitory responses increases Ca2+‐activated K+ conductance, developing a hyperpolarizing receptor potential. Removal of extracellular Ca2+ or exposure to nifedipine reversibly reduced the inhibitory response, suggesting that odorants induce a Ca2+ influx. We show evidence for an odorant‐induced Ca2+ current. Using confocal microscopy, it is shown that odorants induce a nifedipine‐sensitive elevation of Ca2+ in the apical end of the cell. These results suggest an inhibitory mechanism in which an apical Ca2+ influx causes an increase in internal Ca2+, opening Ca2+‐activated K+ channels that lead to membrane hyperpolarization.

Plasma membrane Ca2+-ATPase in the cilia of olfactory receptor neurons: possible role in Ca2+ clearance

Olfactory sensory neurons respond to odorants increasing Ca2+ concentrations in their chemosensory cilia. Calcium enters the cilia through cAMP‐gated channels, activating Ca2+‐dependent chloride or potassium channels. Calcium also has a fundamental role in odour adaptation, regulating cAMP turnover rate and the affinity of the cyclic nucleotide‐gated channels for cAMP. It has been shown that a Na+/Ca2+ exchanger (NCX) extrudes Ca2+ from the cilia. Here we confirm previous evidence that olfactory cilia also express plasma membrane Ca2+‐ATPase (PMCA), and show the first evidence supporting a role in Ca2+ removal. Both transporters were detected by immunoblot of purified olfactory cilia membranes. The pump was also revealed by immunocytochemistry and immunohistochemistry. Inside‐out cilia membrane vesicles transported Ca2+ in an ATP‐dependent fashion. PMCA activity was potentiated by luminal Ca2+ (K0.5 = 670 nm) and enhanced by calmodulin (CaM; K0.5 = 31 nm). Both carboxyeosin (CE) and calmidazolium reduced Ca2+ transport, as expected for a CaM‐modulated PMCA. The relaxation time constant (τ) of the Ca2+‐dependent Cl– current (272 ± 78 ms), indicative of luminal Ca2+ decline, was increased by CE (2181 ± 437 ms), by omitting ATP (666 ± 49 ms) and by raising pH (725 ± 65 ms), suggesting a role of the pump on Ca2+ clearance. Replacement of external Na+ by Li+ had a similar effect (τ = 442 ± 8 ms), confirming the NCX involvement in Ca2+ extrusion. The evidence suggests that both Ca2+ transporters contribute to re‐establish resting Ca2+ levels in the cilia following olfactory responses.

A TRIBUTE TO FRANCISCO VARELA

he present volume of Biological Research is dedicated to the memory of Francisco Varela,renown Chilean scholar recently deceased in Paris (1). The issue contains a collection ofpapers by people who shared moments of thought and research with Francisco at differenttimes and places during his short but remarkably productive life. The variety of issues addressedby the articles contained here aims to be no more than an unpretentious modest reflection of thewide spectrum of Francisco´s intellectual interests, from Mathematics to Neuroscience, fromEpistemology to Psychology. In all of the fields that Francisco explored with his well preparedand open mind he contributed with deep novel insights, that crystallized in numerous articles,comments, books and interviews. We hope that this volume, which we have entitled “A Tributeto Francisco Varela (1946-2001)” will be informative and inspiring for colleagues and studentsall over the world.1) LETELIER JC (2001) Los derroteros cientificos de Francisco Varela (1946-2001). Biol. Res. 34:vii-xiiiJUAN BACIGALUPO AND ADRIAN PALACIOS,Santiago, March 2003.

Transduction for Pheromones in the Main Olfactory Epithelium Is Mediated by the Ca2+-Activated Channel TRPM5

Growing evidence suggests that the main olfactory epithelium contains a subset of olfactory sensory neurons (OSNs) responding to pheromones. One candidate subpopulation expresses the calcium activated cation channel TRPM5 (transient receptor potential channel M5). Using GFP driven by the TRPM5 promoter in mice, we show that this subpopulation responds to putative pheromones, urine, and major histocompatibility complex peptides, but not to regular odors or a pheromone detected by other species. In addition, this subpopulation of TRPM5-GFP+ OSNs uses novel transduction. In regular OSNs, odorants elicit activation of the cyclic nucleotide-gated (CNG) channel, leading to Ca2+ gating of Cl− channels; in TRPM5-GFP+ OSNs, the Ca2+-activated Cl− ANO2 (anoctamin 2) channel is not expressed, and pheromones elicit activation of the CNG channel leading to Ca2+ gating of TRPM5. In conclusion, we show that OSNs expressing TRPM5 respond to pheromones, but not to regular odors through the opening of CNG channels leading to Ca2+ gating of TRPM5.

Intrinsic subthreshold oscillations of the membrane potential in pyramidal neurons of the olfactory amygdala

The amygdala complex is a heterogeneous group of temporal lobe brain structures involved in the processing of biologically significant sensory stimuli and in the generation of appropriate responses to them. The amygdala has also been implicated in certain forms of emotional learning and memory. While much progress has been made in understanding neural processing in the basolateral subgroup of the amygdala, physiological studies in the cortical regions of the complex, also known as olfactory amygdala, are missing. Using a rat brain slice preparation, we conducted whole‐cell recordings on pyramidal neurons of the periamygdaloid cortex and the anterior cortical nucleus, two structures receiving direct connections from the olfactory bulb. Upon depolarization by current injection through the recording electrode, a fraction of periamygdaloid cortex and most anterior cortical nucleus layer II pyramidal neurons displayed an intermittent discharge pattern, where clusters of action potentials were interspersed by periods of membrane potential subthreshold oscillations. Oscillations frequency increased with membrane potential and correlated linearly with the cluster spiking frequency. Frequency ranged from 3 to 20 Hz, considering different cells and membrane potential values (up to approximately 30 mV above resting potentials of typically approximately −70 mV). Subthreshold oscillations were preserved after pharmacological inhibition of fast excitatory and inhibitory synaptic transmission, but were abolished by application of the sodium channel blocker tetrodotoxin. We conclude that pyramidal neurons of the olfactory cortical amygdala display intrinsically generated voltage‐dependent membrane potential rhythmic fluctuations in the theta‐low beta range, requiring the activation of a sodium conductance.

Second Messengers in Invertebrate Phototransduction

In visual transduction, the absorption of light by specialized photoreceptor cells evokes a change in voltage across the plasma membrane termed the receptor potential. The problem of how this excitation process occurs has fascinated physiologists and biochemists for over 100 years. We now know that the process involves three fundamentally different phases. In the first phase, the absorption of light by the visual pigment, rhodopsin, is transduced into a change in the conformation of the visual pigment. This phase involves no amplification because one photon alters the conformation of only one rhodopsin molecule. In the second phase, chemical amplification produces a large change in the concentration of a second messenger. In the final phase, this concentration change is detected by membrane channels which gate the flow of ions, thereby generating the receptor potential.