Characterizing complex chemosensors: information-theoretic analysis of olfactory systems.

The mechanisms that underlie a wine lover's ability to identify a favorite vintage and a dog's ability to track the scent of a lost child are still deep mysteries. Our understanding of these olfactory phenomena is confounded by the difficulty encountered when attempting to identify the parameters that define odor stimuli, by the broad tuning and variability of neurons in the olfactory pathway,and by the distributed nature of olfactory encoding. These issues pertain to both biological systems and to newly developed 'artificial noses' that seek to mimic these natural processes. Information theory, which quantifies explicitly the extent to which the state of one system (for example, the universe of all odors) relates to the state of another (for example, the responses of an odor-sensing device),can serve as a basis for analysing both natural and engineered odor sensors. This analytical approach can be used to explore the problems of defining stimulus dimensions, assessing strategies of neuronal processing, and examining the properties of biological systems that emerge from interactions among their complex components. It can also serve to optimize the design of artificial olfactory devices for a variety of applications, which include process control, medical diagnostics and the detection of explosives.

Inhibitory glutamate receptor channels.

Inhibitory glutamate receptors (IGluRs) are a family of ion channel proteins closely related to ionotropic glycine and gamma-aminobutyric acid (GABA) receptors; They are gated directly by glutamate; the open channel is permeable to chloride and sometimes potassium. Physiologically and pharmacologically, IGluRs most closely resemble GABA receptors; they are picrotoxin-sensitive and sometimes crossdesensitized by GABA. However, the amino acid sequences of cloned IGluRs are most similar to those of glycine receptors. Ibotenic acid, a conformationally restricted glutamate analog closely related to muscimol, activates all IGluRs. Quisqualate is not an IGluR agonist except among pulmonate molluscs and for a unique multiagonist receptor in the crayfish Austropotamobius torrentium. Other excitatory amino acid agonists are generally ineffective. Avermectins have several effects on IGluRs, depending on concentration: potentiation, direct gating, and blockade, both reversible and irreversible. Since IGluRs have only been clearly described in protostomes and pseudocoelomates, these effects may mediate the powerful antihelminthic and insecticidal action of avermectins, while explaining their low toxicity to mammals. IGluRs mediate synaptic inhibition in neurons and are expressed extrajunctionally in striated muscles. The presence of IGluRs in a neuron or muscle is independent of the presence or absence of excitatory glutamate receptors or GABA receptors in the cell. Generally, extrajunctional IGluRs in muscle have a higher sensitivity to glutamate than do neuronal synaptic receptors. Some extrajunctional receptors are sensitive in the range of circulating plasma glutamate levels, suggesting a role for IGluRs in regulating muscle excitability The divergence of the IGlu/GABA/Gly/ACh receptor superfamily in protostomes could become a powerful model system for adaptive molecular evolution. Physiologically and pharmacologically, protostome receptors are considerably more diverse than their vertebrate counterparts. Antagonist profiles are only loosely correlated with agonist profiles (e.g., curare-sensitive GABA receptors, bicuculline-sensitive AChRs), and pharmacologically identical receptors may be either excitatory or inhibitory, and permeable to different ions. The assumption that agonist sensitivity reliably connotes discrete, homologous receptor families is contraindicated. Protostome ionotropic receptors are highly diverse and straightforward to assay; they provide an excellent system in which to study and integrate fundamental questions in molecular evolution and adaptation.

Imprinted Rasgrf1 expression in neonatal mice affects olfactory learning and memory.

Rasgrf1 is genomically imprinted; only the paternally inherited allele is expressed in the neonatal mouse brain until weaning, at which time expression becomes biallelic. Whereas Rasgrf1 has been implicated in learning and memory via knockout studies in adult mice, the effect of its normal imprinted expression on these phenotypes has not yet been examined. Neonatal mice with experimentally manipulated patterns of imprinted Rasgrf1 expression were assessed on an associative olfactory task. Neonates lacking the normally expressed wild-type paternal allele exhibited significant impairment in olfactory associative memory. Adult animals in which neonatal imprinting had been manipulated were also behaviorally assessed; while neonatal imprinting significantly affects body weight even into adulthood, no learning and memory phenotype attributable to imprinting was observed in adults. Additional analyses of neonates showed imprinted Rasgrf1 transcript selective to olfactory bulb even in mice that were null for Rasgrf1 in the rest of the brain and showed that Rasgrf1 affects Ras and Rac activation in the brain. Taken together, these results indicate that Rasgrf1 expression from the wild-type paternal allele contributes to learning and memory in neonatal mice.

Glutamate-gated inhibitory currents of central pattern generator neurons in the lobster stomatogastric ganglion.

Inhibitory glutamatergic neurotransmission is an elemental "building block" of the oscillatory networks within the crustacean stomatogastric ganglion (STG). This study constitutes the initial characterization of glutamatergic currents in isolated STG neurons in primary culture. Superfusion of 1 mM L-glutamate evoked a current response in 45 of 65 neurons examined. The evoked current incorporated two kinetically distinct components in variable proportion: a fast desensitizing component and a slower component. The current was mediated by an outwardly rectifying conductance increase and reversed at -48.8 +/- 5.3 mV. Reducing the external chloride concentration by 50% deflected the glutamate equilibrium potential (Eglu) by +14 mV, while increasing external potassium threefold shifted Eglu by up to +6 mV. Ibotenic acid fully activated both components of the glutamate response. Saturating concentrations of glutamate completely occluded neuronal responses to ibotenic acid, indicating that ibotenic acid was activating the same receptor(s) as glutamate. Millimolar concentrations of quisqualic acid, kainate, AMPA, and NMDA each failed to evoke any response. Picrotoxin (10(-4)M) completely blocked the glutamate response. Niflumic acid (100 microM) blocked > 80% of the desensitizing component and congruent to 50% of the sustained component. Reduction or elimination of extracellular calcium did not abolish the response. This study extends the ionic and pharmacological analysis of glutamatergic conductances in STG neurons. The currents described are consistent with glutamatergic inhibitory synaptic and agonist-evoked responses previously described in situ. We discuss their pharmacology, ionic mechanisms, and functional significance.

How spike synchronization among olfactory neurons can contribute to sensory discrimination.

Recent studies in honeybees have demonstrated that, when odor-evoked action potentials in antennal lobe neurons are pharmacologically desynchronized, the bees are impaired in their ability to discriminate chemically similar odor stimuli. Using a reduced computational model of the honeybee antennal lobe, we show how changes in spike-synchronization properties alone, independent of changes in overall spike-discharge rate or differences in activity levels among responsive neurons, can produce changes in associative learning similar to those observed experimentally.

Inhibitory glutamate receptor channels in cultured lobster stomatogastric neurons.

Inhibitory glutamate receptor channels (IGluRs) are ligand-gated ionotropic receptors related to ionotropic gamma-aminobutyric acid (GABA) and glycine receptors and expressed in neural and muscular tissues. In the crustacean stomatogastric ganglion (STG), IGluRs mediate recurrent synaptic inhibition central to the rhythmogenic capabilities of its embedded neural circuits. IGluRs expressed in cultured spiny lobster STG neurons exhibited an EC50 of 1.2 mM and a Hill coefficient of 1.4. They were neither cross-activated nor cross-desensitized by GABA, although a distinct GABA-gated chloride current was observed. Glycine did not evoke any current from STG neurons. The IGluR was weakly blocked by the chloride channel blocker furosemide and the excitatory glutamate receptor antagonist6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but was not inhibited by bicuculline methiodide, strychnine, kynurenic acid, gamma--glutamylglycine, or aspartate. Outside-out patch-clamp recordings were analyzed using the mean-variance histogram technique. Under excised-patch conditions, the receptor exhibited only a single open state with an estimated unitary conductance of 80 +/- 8. 6 (SD) pS. The distinct GABA receptor also displayed a single open state with a conductance of 72 +/- 10 pS.

Concentration tuning mediated by spare receptor capacity in olfactory sensory neurons: A theoretical study.

The olfactory system is capable of detecting odorants at very low concentrations. Physiological experiments have demonstrated odorant sensitivities down to the picomolar range in preparations from the sensory epithelium. However, the contemporary model for olfactory signal transduction provides that odorants bind to olfactory receptors with relatively low specificity and consequently low affinity, making this detection of low-concentration odorants theoretically difficult to understand. We employ a computational model to demonstrate how olfactory sensory neuron (OSN) sensitivity can be tuned by modulation of receptor-effector coupling and/or by other mechanisms regulating spare receptor capacity, thus resolving this conundrum. The EC10-90 intensity tuning ranges (ITRs) of whole olfactory glomeruli and postsynaptic mitral cells are considerably broader than the commensurate ITRs of individual OSNs. These data are difficult to reconcile with certain contemporary hypotheses that convergent OSNs in mammals exhibit a homogeneous population of olfactory receptors and identical tuning for odor stimuli. We show that heterogeneity in spare receptor capacities within a convergent OSN population can increase the ITR (EC10-90) of a convergent population of OSNs regardless of the presence or absence of a diversity of receptor expression within the population. The modulation of receptor-effector coupling has been observed in OSNs; other mechanisms for cellular regulation of spare receptor capacity are also highly plausible (e.g., quantitative regulation of the relative expression levels of receptor and effector proteins). We present a model illustrating that these processes can underlie both how OSNs come to exhibit high sensitivity to odorant stimuli without necessitating increased ligand-receptor binding affinities or specificities and how a population of convergent OSNs could exhibit a broader concentration sensitivity than its individual constituent neurons, even given a population expressing identical odorant receptors. The regulation of spare receptor capacity may play an important role in the olfactory system's ability to reliably detect low odor concentrations, discriminate odor intensities, and segregate this intensity information from representations of odor quality.

Dopaminergic modulation of inhibitory glutamate receptors in the lobster stomatogastric ganglion.

The intrinsic rhythmicity of the spiny lobster stomatogastric ganglion (STG) is strongly influenced by the strengths of the graded synapses between identified cells within the neural network. These synaptic strengths can be powerfully influenced by chemical neuromodulators such as dopamine and serotonin. Most of the intraganglionic chemical synapses in the STG are mediated by postsynaptic inhibitory glutamate receptors (IGluRs). To determine whether or not direct effects on these IGluRs contribute to the modulation of synaptic strength, unidentified STG neurons were extracted into primary culture and the effects of these aminergic neuromodulators on the glutamate-evoked membrane current were assessed. Dopamine (100 microM) reliably and significantly reduced the whole cell slope conductance of all IGluRs tested. Serotonin (20 microM) never affected the IGlu response, although it clearly altered other cellular membrane properties. Although all identified STG neurons may not conform to these observations, the data reveal a specific dopamine-activated modulatory pathway within cultured neurons that reduces IGluR slope conductance. The relationship between IGluR modulation and net synaptic modulation in situ contributes to an emerging model in which synaptic strengths can be multiply modulated at different functional sites, yielding a complex, distributed, and state-dependent regulatory structure.

Characterization of the synaptic properties of olfactory bulb projections.

The olfactory bulb directly projects to several diverse telencephalic structures, but, to date, few studies have investigated the physiological characteristics of most of these areas. As an initial step towards understanding the odor processing functions of these secondary olfactory structures, we recorded evoked field potentials in response to lateral olfactory tract stimulation in vivo in urethane-anesthetized Sprague-Dawley rats in the following brain structures: anterior olfactory nucleus, ventral and dorsal tenia tecta, olfactory tubercle, anterior and posterior piriform cortex, the anterior cortical nucleus of the amygdala, and lateral entorhinal cortex. Using paired-pulse stimulation with interpulse intervals of 25-1000 ms, we observed facilitation of the response to the second pulse in every structure examined, although the degree of facilitation varied among the target structures. Additionally, pulse train stimulation at three different frequencies (40, 10 and 2 Hz) produced facilitation of evoked field potentials that also varied among target structures. We discuss the potential utility of such short-term facilitation in olfactory processing.

Lobster stomatogastric neurons in primary culture. I. Basic characteristics.

1. A method for the isolation of stomatogastric neurons with neuropilar processes and an axon < or = 2 mm long is described. Isolated neurons adhered to an uncoated plastic surface and demonstrated neurite outgrowth for > or = 7-10 days in a simple medium (salt-adjusted Leibovitz-15). Neurite outgrowth started immediately after plating and was maximal during the first 2-3 days. The electrical activity of neurons and their responses to bath application of pilocarpine were studied between 2 and 10 days after plating. 2. Identified neurons [pyloric dilator (PD), pyloric (PY), and lateral pyloric (LP) neurons from the pyloric pattern generator as well as gastric mill (GM) and lateral posterior gastric (LPG) neurons from the gastric mill pattern generator], isolated with neuropilar processes and axons, behaved in general like corresponding neurons in the isolated stomatogastric ganglion (STG). PD neurons were tonically active or silent in culture; pilocarpine caused them to begin rhythmic activity, which at particular levels of imposed polarization was similar to the pyloric rhythm in vitro. PY and LP neurons were silent. Pilocarpine produced some rhythmicity in the PY neuron, whereas in LP neurons it decreased the firing threshold to depolarizing current and accentuated postinhibitory rebound. LPG neurons were tonically active. Pilocarpine depolarized the LPG neurons and accelerated their tonic activity; neuron hyperpolarization by current injection led to bursting pacemaker activity that was similar to the gastric rhythm in vitro. GM neurons were silent; pilocarpine did not cause them to generate rhythmic activity but did lower their thresholds to depolarizing current. Simultaneous recordings from the soma and axon under direct visual control demonstrated that the intrasomatic spikes (15-20 mV in amplitude) were attenuated action potentials generated in the axon. 3. Neurons isolated with short primary neurites, including those without any noticeable primary neurite (in contrast to neurons isolated with longer neuropilar processes and axons), never generated any kind of electrical activity immediately after extraction from the STG. After 2 days in culture, these "short-neurite" neurons became capable of generating different types of electrical activity (e.g., fast spikes with amplitudes of < or = 40-45 mV, plateau potentials, bursting potentials, etc.). The capability of isolated somata to generate electrical activity did not depend on whether or not the cell had adhered to the substrate and demonstrated neurite outgrowth.(ABSTRACT TRUNCATED AT 400 WORDS)

Nucleosome structural changes during derepression of silent mating-type loci in yeast.

Mutant a and alpha yeast cells were created with histone H3 containing cysteine in place of alanine 110. Because transcriptionally active nucleosomes "unfold" to reveal the histone H3-thiol groups at the center of the core, the active nucleosomes of the mutant strain can be isolated by mercury-affinity chromatography. We compared the unbound and mercury-bound nucleosomes of haploid H3-mutant strains expressing either the MAT alpha or the MATa mating-type locus. In a MAT alpha strain, the Hg-bound nucleosomes are enriched in MAT alpha DNA but lack the DNA of the transcriptionally silent HMRa mating-type locus. Conversely, in a MATa strain, the Hg-bound nucleosomes are enriched in MATa DNA sequences but deficient in HML alpha DNA. When the SIR3 gene, known to be required for silencing of the repressed mating-type loci, is mutated in the MAT alpha strain, transcription of the HMRa ensues, and its nucleosomes, as well as those of the MAT alpha locus, are retained by the organomercurial column. It follows that derepression of the silent mating-type locus, caused by the sir3 null mutation, is accompanied by an unfolding of its nucleosomes to reveal the histone H3 SH groups at their centers. Nucleosomes of the pheromone-encoding gene MFA2, a gene that is expressed in MATa cells but not in MAT alpha cells, are bound to the organomercurial column when isolated from MATa cells but not from MAT alpha cells. Thus, there is a good correlation between nucleosome unfolding and the renewed transcriptional activity at mating-type loci, and at MFA2, which had been silenced for prolonged periods. A close temporal correlation between nucleosome refolding and the cessation of transcription is not always observed in yeast, however, in contrast to observations in mammalian cells. For example, nucleosomes of the GAL1 gene are maintained in a "poised" or "primed" thiol-reactive state even when the gene is not being transcribed (Chen, T. A., Smith, M. M., Le, S., Sternglanz, R., and Allfrey, V. G. (1991) J. Biol. Chem. 266, 6489-6498). It follows that the unfolding of the nucleosome cores of the yeast H3 mutant is regulated by factors that are not temporally linked to the recruitment or traverse of the RNA polymerase complex, but which may determine the rate at which different domains of chromatin adapt to the need for transcription of the associated DNA sequences.

Recovery of transcriptionally active chromatin restriction fragments by binding to organomercurial-agarose magnetic beads. A rapid and sensitive method for monitoring changes in higher order chromatin structure during gene activation and repression.

The unfolding of nucleosomes along transcriptionally active DNA sequences uncovers previously shielded cysteinyl-thiol groups of histone H3 molecules located at the center of the nucleosome core. This change in conformation and SH reactivity of nucleosomes along transcribed DNA sequences makes it possible to separate active from inactive nucleosomes by mercury affinity chromatography. The binding of thiol-reactive nucleosomes to an organomercurial-agarose column has been shown previously to reflect, with accuracy, both the timing and extent of transcription of the associated DNA sequences (Chen, T. A., and Allfrey, V. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5252-5256). Here, we extend this experimental approach to the analysis of higher order chromatin structures. Large chromatin fragments released by treating isolated nuclei with restriction endonucleases are fractionated on mercurated agarose magnetic beads that capture nucleosomes with accessible histone H3 thiols, but do not react with the hidden H3 thiols of the compactly beaded nucleosomes of inactive genes. The SH-reactive domains of c-myc and other genes are rapidly separated from the non-SH-reactive restriction fragments by the magnetic bead technique. The new method also overcomes a major limitation of mercurated agarose column chromatography, which is not suitable for studies of higher order chromatin structure because large chromatin fragments occlude the mercury column; occlusion is not a problem in magnetic separations using suspended mercurated agarose beads. Here, we describe the synthesis of mercurated agarose magnetic beads with high capacity for SH groups and test their application to the recovery of chromatin restriction fragments of c-myc and the growth arrest gene gas1.