Biomedical subjects
G M Shepherd
Publications and source records attributed to G M Shepherd.
Calcium imaging of single stereocilia in hair cells: localization of transduction channels at both ends of tip links.
Mechanically gated "transduction" channels in inner ear hair cells are thought to be connected to tip links stretched between adjacent stereocilia. To locate active channels, calcium-green fluorescence in single stereocilia was measured with two-photon laser scanning microscopy. Bundle deflection increased fluorescence in many but not all stereocilia; the increase was blocked by depolarization. The number of stereocilia responding was proportional to the transduction current, consistent with Ca2+ influx through transduction channels. Fluorescence rose first in the tips of stereocilia and then in the bases, in agreement with channel localization at the tips. Some of the shortest stereocilia in a bundle showed a fluorescence increase, as did some of the tallest, indicating that transduction channels can be at either or both ends of tip links.
Interaction of anionic and cationic currents leads to a voltage dependence in the odor response of olfactory receptor neurons.
1. We recorded odor-induced currents from isolated olfactory receptor neurons of the land phase tiger salamander (Ambystoma tigrinum) with the whole cell patch clamp. 2. In a subset of cells the current-voltage relation for the odor-induced current showed a strong rectification with, in some cells, a negative resistance slope between about -45 and -25 mV. In these cells there was little or no odor-induced current at -55 mV, the average resting potential of olfactory neurons. 3. Depolarizing the membrane to +20 mV revealed a large outward current, and on repolarizing the membrane to -55 mV we could observe a large inward current. This current was not observed in the absence of the depolarizing step or in the absence of odor stimuli. 4. This odor-induced tail current was dependent on extracellular Ca2+ and voltage, activating with increased depolarization. The reversal potential was sensitive to the chloride equilibrium potential and it could be significantly blocked by niflumic acid, a blocker of calcium-activated chloride currents. The voltage dependence could result from either the voltage-dependent block of adenosine 3',5'-cyclic monophosphate-gated cation channels known to be activated by odorants and permeable to Ca2+, or from an inherent voltage dependence in the chloride channel gating. 5. The current appears to function as a regenerative mechanism that might increase the amplitude and duration of the odor-induced current, especially to low concentrations of stimulus.
Regulation of cyclic nucleotide-gated channels and membrane excitability in olfactory receptor cells by carbon monoxide.
1. The effect of the putative neural messenger carbon monoxide (CO) and the role of the cGMP second-messenger system for olfactory signal generation was examined in isolated olfactory receptor neurons (ORNs) of the tiger salamander. 2. With the use of whole cell voltage-clamp recordings in combination with a series of ionic and pharmological tests, it is demonstrated that exogenously applied CO is a potent activator (K1/2 = 2.9 microM) of cyclic nucleotide-gated (CNG) channels previously described to mediate odor transduction. 3. Several lines of evidence suggest that CO mediates its effect through stimulation of a soluble guanylyl cyclase (sGC) leading to formation of the second-messenger cGMP. This conclusion is based on the findings that CO responses show an absolute requirement for guanosine 5'-triphosphate (GTP) in the internal solution, that no direct effect of CO on CNG currents in the absence of GTP is detectable, and that a blocker of sGC activation, LY85383 (10 microM), completely inhibits the CO response. 4. The dose-response curve for cGMP at CNG channels is used as a calibration to provide a quantitative estimate of the CO-stimulated cGMP formation. This analysis implies that CO is a potent activator of olfactory sGC. 5. Perforated patch recordings using amphotericin B demonstrate that low micromolar doses of CO effectively depolarize the membrane potential of ORNs through tonic activation of CNG channels. This effect in turn regulates excitable and adaptive properties of ORNs and modulates neuronal responsiveness. 6. These data argue for an important role of the cGMP pathway in olfactory signaling and support the idea that CO may function as a diffusible messenger in the olfactory system.
Potential ligand-binding residues in rat olfactory receptors identified by correlated mutation analysis.
A family of G-protein-coupled receptors is believed to mediate the recognition of odor molecules. In order to identify potential ligand-binding residues, we have applied correlated mutation analysis to receptor sequences from the rat. This method identifies pairs of sequence positions where residues remain conserved or mutate in tandem, thereby suggesting structural or functional importance. The analysis supported molecular modeling studies in suggesting several residues in positions that were consistent with ligand-binding function. Two of these positions, dominated by histidine residues, may play important roles in ligand binding and could confer broad specificity to mammalian odor receptors. The presence of positive (overdominant) selection at some of the identified positions provides additional evidence for roles in ligand binding. Higher-order groups of correlated residues were also observed. Each group may interact with an individual ligand determinant, and combinations of these groups may provide a multi-dimensional mechanism for receptor diversity.
Network-based informatics support of research collaborations in the Human Genome Project and the Human Brain Project.
Sophisticated network-based informatics support will increasingly be required for collaborating biomedical laboratories located in different geographic locations, both to accommodate the massive amount of data being generated in certain fields, and to allow different types of data produced at different laboratories to be analyzed in an integrated fashion. The paper describes the experience of the Yale Center for Medical Informatics in providing informatics support for collaborative projects in gene mapping (as part of the national Human Genome Project) and neuroscience (as part of the national Human Brain Project). The paper describes the informatics needs of the two projects and the solutions being developed, describes certain lessons learned, and discusses certain broader issues that arise.
Molecular modeling of ligand-receptor interactions in the OR5 olfactory receptor.
Olfactory receptors belong to the superfamily of seven transmembrane domain, G protein-coupled receptors. In order to begin analysis of mechanisms of receptor activation, a computer model of the OR5 olfactory receptor has been constructed and compared with other members of this superfamily. We have tested docking of the odor molecule lyral, which is known to activate the OR5 receptor. The results point to specific ligand-binding residues on helices III through VII that form a binding pocket in the receptor. Some of these residues occupy sequence positions identical to ligand-binding residues conserved among other superfamily members. The results provide new insights into possible molecular mechanisms of odor recognition and suggest hypotheses to guide future experimental studies using site-directed mutagenesis.
Emerging principles of molecular signal processing by mitral/tufted cells in the olfactory bulb.
The olfactory system shares many principles of functional organization with other sensory systems, but differs in that the sensory input is in the form of molecular information carried in odor molecules. Current studies are providing new insights into how this information is processed. In analogy with the spatial receptive fields of visual neurons, the molecular receptive range of olfactory cells is defined as the range of odor molecules that will affect the firing of that cell. Olfactory receptor molecules belong to a large gene family; it is hypothesized that individual receptor molecules may have relatively broad molecular receptive ranges, and that an individual receptor cell need therefore express only one or a few different types of receptors to cover a broad range. Mitral/tufted cells have narrower molecular receptive ranges, comprising molecules with related structures (odotopes). This is believed to reflect processing through the olfactory glomeruli, each glomerulus acting as a convergence center for related inputs. Varying overlapping specificities of receptor cells, glomeruli and mitral/tufted cells appear to provide the basis for discrimination of odor molecules, in analogy with discrimination of color in the visual system.
Retinal ganglion cells express a cGMP-gated cation conductance activatable by nitric oxide donors.
We have identified a putative cGMP-gated cation conductance in rat retinal ganglion cells. Both in situ hybridization and polymerase chain reaction amplification detected transcripts in ganglion cells that were highly homologous to the cGMP-gated cation channel expressed in rod photoreceptors. Whole-cell patch-clamp recordings detected a current stimulated by cGMP due to activation of nonselective cation channels. This current had a reversal potential near 0 mV, showed some outward rectification, and could be blocked by Cd2+. The current could also be activated by a phosphodiesterase inhibitor and the nitric oxide donors sodium nitroprusside and S-nitrosocysteine. We propose that nitric oxide released from an identified subpopulation of amacrine cells may activate this channel to modulate ganglion cell activity.
Discrimination of molecular signals by the olfactory receptor neuron.
Explore the source record for details and available documents.
Cyclic nucleotide-gated ion channels and sensory transduction in olfactory receptor neurons.
Explore the source record for details and available documents.
Glycine exerts potent inhibitory actions on mammalian olfactory bulb neurons.
1. It is generally presumed that gamma-aminobutyric acid (GABA) mediates inhibition in the mammalian brain, whereas glycine is restricted to the brain stem and spinal cord. Recent immunocytochemical and molecular biological studies have demonstrated, however, a widespread distribution of glycine receptors through-out the CNS, including the olfactory bulb. To explore the functional significance of glycine receptors in the olfactory bulb we have used primary culture and whole-cell voltage-clamp recording techniques to test the hypothesis that glycine, as well as GABA, exerts inhibitory actions on olfactory bulb neurons. 2. Cultures of olfactory bulb neurons contain two primary populations of morphologically distinct neurons, mitral/tufted cells and interneurons (granule and periglomerular cells). In all mitral/tufted cells and interneurons examined, both glycine and GABA evoked concentration-dependent desensitizing currents mediated by chloride, similar to those seen in mammalian neurons elsewhere in the brain and spinal cord. 3. The median effective concentration (EC50) for glycine was 125 microM, with a Hill coefficient of 1.7, whereas the EC50 and Hill coefficient for GABA were 52 microM and 1.8, respectively. These values are similar to values previously reported for other central neurons. 4. At moderate concentrations (> 1 microM) strychnine nonselectively antagonized both glycine- and GABA-evoked currents. At low concentrations (< or = 1 microM) strychnine blocked glycine-mediated currents but had little effect on GABA-mediated currents. Similarly, picrotoxin was a nonselective antagonist for glycine- and GABA-mediated currents at high concentrations (100 microM), but was selective for GABA at low concentrations (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
Evidence for glutamate as the olfactory receptor cell neurotransmitter.
1. Synaptic transmission between olfactory receptor neurons and mitral/tufted cells was examined using a whole-cell recording technique in a hemisected preparation of the turtle olfactory bulb. To determine the olfactory receptor neuron transmitter, we isolated components of the synaptic response of mitral/tufted cells to olfactory nerve stimulation using postsynaptic receptor antagonists. 2. Low-intensity stimulation of the olfactory nerve evoked monosynaptic excitatory postsynaptic potentials in mitral/tufted cells that consisted of a rapid and prolonged depolarization with little contribution from other bulb neurons. The exogenous application of glutamate mimicked the response of mitral/tufted cells to olfactory nerve stimulation. 3. Olfactory nerve stimulation evoked in mitral/tufted cells a two component response that was reversibly blocked by glutamate receptor antagonists. The first, a rapid depolarization of short duration, was sensitive to the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX); the second, a depolarization of slower onset but longer duration, was sensitive to the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5). When DNQX and AP5 were both present the postsynaptic response was completely abolished. These results strongly support the notion that glutamate is the neurotransmitter at the olfactory nerve to mitral/tufted cell synapse.
The extent of adaptation in bullfrog saccular hair cells.
Positive deflection of the sensory hair bundle of a vertebrate hair cell opens transduction channels to depolarize the cell. In bullfrog saccular hair cells, there is a subsequent adaptation process, whereby the proportion of transduction channels that are open, and thus the receptor current, declines toward the resting value. This occurs because the sensitivity curve, relating open probability to bundle deflection, shifts along the deflection axis in response to bundle deflections, in a manner consistent with a relaxation of mechanical tension on transduction channels. In this study we determined the extent of adaptation, measured as the shift of the sensitivity curve following deflection of the hair bundle. The shift was determined both by comparison of the receptor current in the adapted state to the resting sensitivity curve, and by comparison of pre- and postadapted sensitivity curves. The adaptive shift approached steady state with a time constant of 20-30 msec, and was at steady state within 150 msec. For all positive and for small negative deflections, both methods showed a shift that was approximately 80% of the deflection. For larger negative deflections, the shift reached a fixed limit that was 100-500 nm negative to the freestanding bundle position. The limited extent of adaptation confers a time-dependent sensitivity: the cell has an instantaneous or phasic sensitivity curve that is steep, and steady-state or tonic sensitivity curve that is about five times broader. It also suggests the existence of two additional structural elements within the transduction apparatus. A revised quantitative theory accommodates these elements.
Comparison between the membrane and synaptic properties of human and rodent dentate granule cells.
We have compared the cellular and synaptic properties of rodent dentate granule cells with those of humans. The human tissue was obtained from neurosurgical procedures which necessitated removal of the hippocampus for treatment of extra-hippocampal tumors which presented clinically with seizures. The hippocampi studied here were neuroanatomically similar to autopsy controls. The present studies have demonstrated that there are few differences between rodent and human granule cells as regards either their membrane properties or their synaptic physiology and pharmacology. The differences we noted were (1) less spike frequency adaptation in the human relative to rodent cells; and (2) perforant path stimulation reliably elicited both feedforward and feedback inhibition in the rodent cells, while in the human tissue feedback inhibition appeared to predominate. It is unclear if these changes are due to the seizure experience or if they represent true species differences.
Principles of specificity and redundancy underlying the organization of the olfactory system.
Explore the source record for details and available documents.
Implications of the NO/cGMP system for olfaction.
The rapid and transient generation of pulses of either cAMP or IP3 is considered the primary reaction in olfactory signal transduction. There is some recent evidence suggesting that another second messenger system, involving cGMP, may also play an important role in olfactory signalling. High doses of odorant elicit a delayed and sustained elevation of cGMP levels due to the operation of the nitric oxide (NO) synthase/guanylyl cyclase system. The interplay of NO and cGMP is supposed to trigger molecular mechanisms, including adaptation processes, which enable the olfactory neuroepithelium to cope with strong stimuli. Furthermore, a characteristic pattern of staining for NO synthase in the olfactory bulb suggests that the NO/cGMP system may also participate in neuronal processing of the sensory input.
Signal transmission in lobster olfactory receptor cells: functional significance of electrotonic structure analysed by a compartmental model.
The electrotonic structure of lobster olfactory receptor cells was evaluated using general purpose simulation software in a compartmental model derived from electron-microscopic reconstruction. The model with non-uniform membrane resistance (Rm) was used to (i) simulate current spread and (ii) determine if the electronic structure of the cell improves signal recognition in the soma. The odor-evoked conductance change in dendrites was calculated according to the Michaelis-Menten equation with the assumption that the outer dendritic segments function as independent stimulus detectors. The inflection point of the concentration-response function measured in the soma was shifted to lower concentrations relative to that measured in the ciliary (outer dendritic) arbor. The shift, which was greater for inputs with lower efficacy (represented in the model by smaller Hill coefficients) and for the dynamic phase of the response than for the steady-state phase, effectively increased the selectivity of the somatic response. Randomized input distributed uniformly to progressively more restricted areas of the ciliary arbor showed that stimulation of larger areas (presumably the entire ciliary arbor) decreased the statistical variability of the somatic response.