Quantitative Characterization of Output from the Directionally Selective Visual Interneuron H1 in the Grey Flesh Fly Sarcophaga bullata.

H1, a very well-studied insect visual interneuron, has a panoramic receptive field and is directionally selective in responding to optic flow. The synaptic basis for the directional selectivity of the H1 neuron has been studied using both theoretical and cellular approaches. Extracellular single-unit recordings are readily obtained by beginning students using commercially available adults of the grey flesh fly Sarcophaga bullata. We describe an apparatus which allows students to present a series of moving visual stimuli to the eye of the restrained, minimally dissected adult Sarcophaga, while recording both the single unit responses of the H1 neuron and the position and velocity of the moving stimulus. Students obtain quantitative and reproducible responses of H1, probing the response properties of the neuron by modulating stimulus parameters such as: direction and speed of movement, visual contrast, spatial wavelength, or the extent of the visual field occupied. Students learn to perform quantitative analysis of their data and to generate graphical representations of their results characterizing the tuning and receptive field of this neuron. This exercise demonstrates the utility of single unit recording of an identified interneuron in an awake restrained insect and promotes interpretation of these results in terms of the visual stimuli normally encountered by freely flying flies in their natural environment.

Reproducible Quantitative Stimulation Allows New Analysis of Crayfish Muscle Receptor Organ Responses.

The crustacean muscle receptor organ (MRO) has provided a particularly accessible preparation for the study of sensory coding, which has been widely used in introductory laboratory courses incorporating extracellular recording from sensory nerves in living preparations. We describe three innovations to the standard laboratory exercise using the MRO: (1) a new form of suction electrode to facilitate extracellular recording; (2) a new, Arduino-driven actuator to allow reproducible and quantifiable mechanical stimulation of the MRO; and (3) a new approach to the crayfish abdomen preparation that allows linear extension of the MRO muscles. These novel approaches allow the collection of data sets comprised of sensory cell spike trains under software control as important mechanical stimulus parameters are varied systematically through software. This additional level of user control facilitates a more robust quantitative approach to the analysis of MRO sensory neuron spike trains, which is facilitated by training in data analysis using python.

Visualization of nitric oxide production in the mouse main olfactory bulb by a cell-trappable copper(II) fluorescent probe.

We report the visualization of NO production using fluorescence in tissue slices of the mouse main olfactory bulb. This discovery was possible through the use of a novel, cell-trappable probe for intracellular nitric oxide detection based on a symmetric scaffold with two NO-reactive sites. Ester moieties installed onto the fluorescent probe are cleaved by intracellular esterases to yield the corresponding negatively charged, cell-impermeable acids. The trappable probe Cu(2)(FL2E) and the membrane-impermeable acid derivative Cu(2)(FL2A) respond rapidly and selectively to NO in buffers that simulate biological conditions, and application of Cu(2)(FL2E) leads to detection of endogenously produced NO in cell cultures and olfactory bulb brain slices.

Odor sampling strategies in mice with genetically altered olfactory responses.

Peripheral sensory cells and the central neuronal circuits that monitor environmental changes to drive behaviors should be adapted to match the behaviorally relevant kinetics of incoming stimuli, be it the detection of sound frequencies, the speed of moving objects or local temperature changes. Detection of odorants begins with the activation of olfactory receptor neurons in the nasal cavity following inhalation of air and airborne odorants carried therein. Thus, olfactory receptor neurons are stimulated in a rhythmic and repeated fashion that is determined by the breathing or sniffing frequency that can be controlled and altered by the animal. This raises the question of how the response kinetics of olfactory receptor neurons are matched to the imposed stimulation frequency and if, vice versa, the kinetics of olfactory receptor neuron responses determine the sniffing frequency. We addressed this question by using a mouse model that lacks the K+-dependent Na+/Ca2+ exchanger 4 (NCKX4), which results in markedly slowed response termination of olfactory receptor neuron responses and hence changes the temporal response kinetics of these neurons. We monitored sniffing behaviors of freely moving wildtype and NCKX4 knockout mice while they performed olfactory Go/NoGo discrimination tasks. Knockout mice performed with similar or, surprisingly, better accuracy compared to wildtype mice, but chose, depending on the task, different odorant sampling durations depending on the behavioral demands of the odorant identification task. Similarly, depending on the demands of the behavioral task, knockout mice displayed a lower basal breathing frequency prior to odorant sampling, a possible mechanism to increase the dynamic range for changes in sniffing frequency during odorant sampling. Overall, changes in sniffing behavior between wildtype and NCKX4 knockout mice were subtle, suggesting that, at least for the particular odorant-driven task we used, slowed response termination of the odorant-induced receptor neuron response either has a limited detrimental effect on odorant-driven behavior or mice are able to compensate via an as yet unknown mechanism.

Speed-accuracy tradeoff in olfaction.

The basic psychophysical principle of speed-accuracy tradeoff (SAT) has been used to understand key aspects of neuronal information processing in vision and audition, but the principle of SAT is still debated in olfaction. In this study we present the direct observation of SAT in olfaction. We developed a behavioral paradigm for mice in which both the duration of odorant sampling and the difficulty of the odor discrimination task were controlled by the experimenter. We observed that the accuracy of odor discrimination increases with the duration of imposed odorant sampling, and that the rate of this increase is slower for harder tasks. We also present a unifying picture of two previous, seemingly disparate experiments on timing of odorant sampling in odor discrimination tasks. The presence of SAT in olfaction provides strong evidence for temporal integration in olfaction and puts a constraint on models of olfactory processing.

Dynamics of odor sampling strategies in mice.

Mammalian olfactory receptor neurons in the nasal cavity are stimulated by odorants carried by the inhaled air and their activation is therefore tied to and driven by the breathing or sniffing frequency. Sniffing frequency can be deliberately modulated to alter how odorants stimulate olfactory receptor neurons, giving the animal control over the frequency of odorant exposure to potentially aid odorant detection and discrimination. We monitored sniffing behaviors and odorant discrimination ability of freely-moving mice while they sampled either decreasing concentrations of target odorants or sampled a fixed target odorant concentration in the presence of a background of increasing odorant concentrations, using a Go-NoGo behavioral paradigm. This allowed us to ask how mice alter their odorant sampling duration and sampling (sniffing) frequency depending on the demands of the task and its difficulty. Mice showed an anticipatory increase in sniffing rate prior to odorant exposure and chose to sample for longer durations when exposed to odorants as compared to the solvent control odorant. Similarly, mice also took more odorant sampling sniffs when exposed to target odorants compared to the solvent control odorant. In general, odorant sampling strategies became more similar the more difficult the task was, e.g. the lower the target odorant concentration or the lower the target odorant contrast relative to the background odorant, suggesting that sniffing patterns are not preset, but are dynamically modulated by the particular task and its difficulty.

Neural and molecular mechanisms of microcognition in Limax.

Various non-mammalian model systems are being explored in the search for mechanisms of learning and memory storage of sufficient generality to contribute to the understanding of mammalian learning mechanisms. The terrestrial mollusk Limax maximus is one such model system in which mammalian-quality learning has been documented using odors as conditioned stimuli. The Limax odor information-processing circuits incorporate several system design features also found in mammalian odor-processing circuits, such as the use of cellular and network oscillations for making odor computations and the use of nitric oxide to control network oscillations. Learning and memory formation has been localized to a particular central circuit, the procerebral lobe, in which selective gene activation occurs through odor learning. Since the isolated Limax brain can perform odor learning in vitro, the circuits and synapses causally linked to learning and memory formation are assessable for further detailed analysis.

Methods in Rodent Chemosensory Cognition.

Olfactory information processing and learning are highly developed computational abilities of rodents. These attributes can be exploited to ask questions at several levels of complexity, from aspects of odorant binding by olfactory receptors to higher order learning about the predictive consequences of odorant stimulus presentation. Quantitative understanding of rodent odorant sampling patterns, both baseline nasal breathing and odorant-stimulated sniffing, is critical to elucidating mechanisms of olfactory information processing, from primary olfactory receptors to cortical centers that synthesize olfactory percepts from preprocessed multimodal inputs. This chapter outlines an innovative new method for measuring breathing and sniffing rates in unrestrained mice while the mice are performing odor-guided tasks in a computer controlled olfactometer.The method described here involves implantation of a wireless pressure sensor in the mouse that reports on thoracic pressure transients caused by breathing and sniffing. Recordings of pressure sensor outputs are made simultaneously with optically-sensed nose pokes by the mouse into an odor delivery port or a water delivery port. Odorant delivery timing and water reward delivery are also recorded simultaneously. This method allows for breathing and sniffing dependent thoracic pressure transients to be recorded with high temporal precision before, during, and after the mouse approaches an odor delivery port, samples the delivered odor, and obtains a water reward contingent on the identity of the odor that was presented and sampled.

Sparse odor coding in awake behaving mice.

Responses of mitral cells represent the results of the first stage of odor processing in the olfactory bulb. Most of our knowledge about mitral cell activity has been obtained from recordings in anesthetized animals. We compared odor-elicited changes in firing rate of mitral cells in awake behaving mice and in anesthetized mice. We show that odor-elicited changes in mitral cell firing rate were larger and more frequently observed in the anesthetized than in the awake condition. Only 27% of mitral cells that showed a response to odors in the anesthetized state were also odor responsive in the awake state. The amplitude of their response in the awake state was smaller, and some of the responses changed sign compared with their responses in the anesthetized state. The odor representation in the olfactory bulb is therefore sparser in awake behaving mice than in anesthetized preparations. A qualitative explanation of the mechanism responsible for this phenomenon is proposed.

Volatile metabolic monitoring of glycemic status in diabetes using electronic olfaction.

The increased incidence of Type I and Type II diabetes among adults and adolescents is a growing public health concern worldwide. The primary objective of diabetes mellitus management involves keeping glycemia levels within the euglycemic range to prevent a variety of serious health complications. Unfortunately, daily self-monitoring is both a requirement and a problem for many patients with diabetes, particularly children and adolescents. Studies have shown that as many as 43% of adolescents and 30% of children (<14 years old) regularly forget to use glycemic tests and are significantly poorer at recognizing and reporting symptoms and signs of hypoglycemia/hyperglycemia. For this reason, methods for noninvasive, continuous monitoring that can signal glycemic status to a parent, teacher, or other caregiver would improve the care and management of symptoms of diabetes among these individuals. The goal of this review is to describe and evaluate electronic olfaction technology ("electronic nose") for monitoring the presence and levels of volatile chemicals from human body and breath that can be used to evaluate status of diabetes. The review is organized in four sections. The first section reviews the chemistry of the volatile signals that are produced by the body that are indicative of metabolic status. The second section provides an overview of novel sensor technology, e.g., "electronic olfaction," that mimics the biological olfactory system and can be used to monitor and identify complex plumes of volatiles that are signatures of metabolic states. The third section reviews studies that have employed electronic "nose" technology for diagnosis and monitoring of diabetes via urine and breath, and the final section discusses needed future directions for the development of olfactory-based metabolic monitoring, particularly among noncompliant populations.

The nitric oxide/cyclic GMP pathway in the olfactory processing system of the terrestrial slug Limax marginatus.

To examine the distribution of nitric oxide (NO)-generative cells and NO-responsive cells in the tentacles and procerebral lobes (olfactory processing center) of terrestrial slugs, we applied NADPH diaphorase (NADPH-d) histochemistry and NO-induced cyclic GMP (cGMP)-like immunohistochemistry. We found that NADPH-d reactive cells/fibers and cGMP-like immunoreactive cells/fibers were different, but they were localized adjacent to each other, in both the tentacles and the procerebral lobes. Then, we measured the concentration of NO that was generated around the procerebral lobes using an NO sensitive electrode, when the olfactory nerve was electrically stimulated as a replacement for an odorant stimulus. Stimulation of the olfactory nerve evoked an increase in NO concentration at nanomolar levels, suggesting that binding of nanomolar concentrations of NO to the prosthetic heme group activates soluble guanylyl cyclase. Taken together with previously reported physiological data, our results, therefore, showed that the NO/cGMP pathways are involved in slug olfactory processing.

Comparing thoracic and intra-nasal pressure transients to monitor active odor sampling during odor-guided decision making in the mouse.

BACKGROUND: Recording of physiological parameters in behaving mice has seen an immense increase over recent years driven by, for example, increased miniaturization of recording devices. One parameter particularly important for odorant-driven behaviors is the breathing frequency, since the latter dictates the rate of odorant delivery to the nasal cavity and the olfactory receptor neurons located therein. NEW METHOD: Typically, breathing patterns are monitored by either measuring the breathing-induced temperature or pressure changes in the nasal cavity. Both require the implantation of a nasal cannula and tethering of the mouse to either a cable or tubing. To avoid these limitations we used an implanted pressure sensor which reads the thoracic pressure and transmits the data telemetrically, thus making it suitable for experiments which require a freely moving animal. RESULTS: Mice performed a Go/NoGo odorant-driven behavioral task with the implanted pressure sensor, which proved to work reliably to allow recording of breathing signals over several weeks from a given animal. COMPARISON TO EXISTING METHOD(S): We simultaneously recorded the thoracic and nasal pressure changes and found that measuring the thoracic pressure change yielded similar results compared to measurements of nasal pressure changes. CONCLUSION: Telemetrically recorded breathing signals are a feasible method to monitor odorant-guided behavioral changes in breathing rates. Its advantages are most significant when recording from a freely moving animal over several weeks. The advantages and disadvantages of different methods to record breathing patterns are discussed.

Electrochemical sensing of nitric oxide with functionalized graphene electrodes.

The intrinsic electrocatalytic properties of functionalized graphene sheets (FGSs) in nitric oxide (NO) sensing are determined by cyclic voltammetry with FGS monolayer electrodes. The degrees of reduction and defectiveness of the FGSs are varied by employing different heat treatments during their fabrication. FGSs with intermediate degrees of reduction and high Raman ID to IG peak ratios exhibit an NO oxidation peak potential of 794 mV (vs 1 M Ag/AgCl), closely matching values obtained with a platinized Pt control (791 mV) as well as recent results from the literature on porous or biofunctionalized electrodes. We show that the peak potential obtained with FGS electrodes can be further reduced to 764 mV by incorporation of electrode porosity using a drop-casting approach, indicating a stronger apparent electrocatalytic effect on porous FGS electrodes as compared to platinized Pt. Taking into consideration effects of electrode morphology, we thereby demonstrate that FGSs are intrinsically as catalytic toward NO oxidation as platinum. The lowered peak potential of porous FGS electrodes is accompanied by a significant increase in peak current, which we attribute either to pore depletion effects or an amplification effect due to subsequent electrooxidation reactions. Our results suggest that the development of sensor electrodes with higher sensitivity and lower detection limits should be feasible with FGSs.

Regulatory mechanisms and the role of calcium and potassium channels controlling supercontractile crop muscles in adult Phormia regina.

Bioassays and electrophysiological recordings were conducted in the adult blowfly Phormia regina to provide new insights into the regulatory mechanisms governing the crop filling and emptying processes of the supercontractile crop muscles. The cibarial pump drives ingestion. Simultaneous multisite extracellular recordings show that crop lobe (P5) distension during ingestion of a 4.7 µl sugar meal does not require muscle activity by any of the other pumps of the system. Conversely, pumping of fluids toward the anterior of the crop system during crop emptying is brought about by active muscle contraction, in the form of a highly coordinated peristaltic wave starting from P5 and progressively propagating to P6, P4 and P3 pumps, with P5 contracting with a frequency about 3.4 times higher than the other pumps. The crop contraction rate is also modulated by hemolymph-borne factors such as sugars, through ligand recognition at a presumptive receptor site rather than by an osmotic effect, as assessed by both behavioural and electrophysiological experiments. In this respect, sugars of equal osmolarity produce different effects, glucose being inhibitory and mannose ineffective for crop muscles, while trehalose enhances crop activity. Finally, voltage and current clamp experiments show that the muscle action potentials (mAPs) at the P4 pump are sustained by a serotonin-sensitive calcium conductance. Serotonin enhances calcium entry into the muscle cells and this could lead, as an indirect modulatory effect, to activation of a Ca(2+)-activated K(+) conductance (IK(Ca)), which sustains the following mAP repolarization phase in such a way that further mAPs can be generated early and the frequency consequently increased.

Biomimetic chemical sensors using nanoelectronic readout of olfactory receptor proteins.

We have designed and implemented a practical nanoelectronic interface to G-protein coupled receptors (GPCRs), a large family of membrane proteins whose roles in the detection of molecules outside eukaryotic cells make them important pharmaceutical targets. Specifically, we have coupled olfactory receptor proteins (ORs) with carbon nanotube transistors. The resulting devices transduce signals associated with odorant binding to ORs in the gas phase under ambient conditions and show responses that are in excellent agreement with results from established assays for OR-ligand binding. The work represents significant progress on a path toward a bioelectronic nose that can be directly compared to biological olfactory systems as well as a general method for the study of GPCR function in multiple domains using electronic readout.

Neural basis of olfactory perception.

The neural basis of olfactory information processing and olfactory percept formation is a topic of intense investigation as new genetic, optical, and psychophysical tools are brought to bear to identify the sites and interaction modes of cortical areas involved in the central processing of olfactory information. New methods for recording cellular interactions and network events in the awake, behaving brain during olfactory processing and odor-based decision making have shown remarkable new properties of neuromodulation and synaptic interactions distinct from those observed in anesthetized brains. Psychophysical, imaging, and computational studies point to the orbitofrontal cortex as the likely locus of odor percept formation in mammals, but further work is needed to identify a causal link between perceptual and neural events in this area.

Presynaptic muscarinic receptors enhance glutamate release at the mitral/tufted to granule cell dendrodendritic synapse in the rat main olfactory bulb.

The mammalian olfactory bulb receives multiple modulatory inputs, including a cholinergic input from the basal forebrain. Understanding the functional roles played by the cholinergic input requires an understanding of the cellular mechanisms it modulates. In an in vitro olfactory bulb slice preparation we demonstrate cholinergic muscarinic modulation of glutamate release onto granule cells that results in gamma-aminobutyric acid (GABA) release onto mitral/tufted cells. We demonstrate that the broad-spectrum cholinergic agonist carbachol triggers glutamate release from mitral/tufted cells that activates both AMPA and NMDA receptors on granule cells. Activation of the granule cell glutamate receptors leads to calcium influx through voltage-gated calcium channels, resulting in spike-independent, asynchronous GABA release at reciprocal dendrodendritic synapses that granule cells form with mitral/tufted cells. This cholinergic modulation of glutamate release persists through much of postnatal bulbar development, suggesting a functional role for cholinergic inputs from the basal forebrain in bulbar processing of olfactory inputs and possibly in postnatal development of the olfactory bulb.

Volatile compounds characteristic of sinus-related bacteria and infected sinus mucus: analysis by solid-phase microextraction and gas chromatography-mass spectrometry.

Volatile compounds from human breath are a potential source of information for disease diagnosis. Breath may include volatile organic compounds (VOCs) originating in the nasal sinuses. If the sinuses are infected, disease-specific volatiles may enter exhaled air. Sinus infections are commonly caused by several known bacteria. We examined the volatiles characteristic of infectious bacteria in culture using solid-phase microextraction to collect and gas chromatography-mass spectrometry as well as gas chromatography with flame photometric detection to separate and analyze the resulting VOCs. Infected sinus mucus samples were also collected and their VOCs examined. Similar characteristic volatiles were seen from both cultures of individual "pure" bacteria and several mucus samples. However, the relative amounts of characteristic VOCs from individual bacteria differ greatly between cultures and sinus mucus. New compounds, not seen in culture were also seen in some mucus samples. Our results suggest an important role for growth substrate and environment. Our data further suggests that in some sinus mucus samples identification of bacteria-specific volatiles is possible and can suggest the identity of an infecting organism to physicians. Knowledge of these bacteria-related volatiles is necessary to create electronic nose-based, volatile-specific sensors for non-invasive examination for suspected sinus infection.