Effect of serotonin and calcium on the supercontractile muscles of the adult blowfly crop.

Bioassays and electrophysiological recordings were conducted to determine the role of serotonin and calcium on the supercontractile pump muscles of the diverticulated crop of adult blowflies. Using in situ crop preparations, serotonin was found to significantly increase the rates of contractions of a specific pump in the crop wall, pump P4. The addition of the serotonin antagonist, mianserin, or calcium free saline, both significantly reduced the contraction rates of this pump. Recordings, using suction electrodes from pump P4, confirm the in situ bioassay data and show that serotonin promotes muscle activity in empty crops in which no pump activity is normally observed. Moreover, our data indicate the crucial role of extracellular calcium ions in crop pump contractile activity. These results provide new information on how the crop of adult dipterans is modulated and suggest that serotonin, possibly supplied by neurons in the thoracico-abdominal neural plexus, may be involved in modulating the pumping of crop contents into the midgut for digestion or triggering antiperistalsis from the foregut in the process known as regurgitation or 'bubbling'.

Neural computations with mammalian infochemicals.

The mammalian olfactory system is the most sensitive and discriminating molecular recognition system known, able to detect a few dozen critical molecules in the face of strong and variable background odorants. The set of information-containing volatile molecules used to transmit information within and between mammalian species shows both great molecular and informational diversity. Chemosensory neuroscientists that apply traditional reductionist methods to the analysis of information processing and computational principles in the olfactory system find great value in understanding the ecological and ethological context in which mammalian olfactory communication occurs. This review highlights a subset of the molecular armamentarium and information transmissions relevant to understanding the uses of olfactory communication by mammals in an ecological context.

Tonic and stimulus-evoked nitric oxide production in the mouse olfactory bulb.

Nitric oxide (NO) has been long assumed to play a key role in mammalian olfaction. This was based largely on circumstantial evidence, i.e. prominent staining for nitric oxide synthase (NOS) and cyclic guanosine 3',5'-cyclic monophosphate (cGMP) or soluble guanylyl cyclase, an effector enzyme activated by NO, in local interneurons of the olfactory bulb. Here we employ innovative custom-fabricated NO micro-sensors to obtain the first direct, time-resolved measurements of NO signaling in the olfactory bulb. In 400 microm thick mouse olfactory bulb slices, we detected a steady average basal level of 87 nM NO in the extracellular space of mitral or granule cell layers. This NO 'tone' was sensitive to NOS substrate manipulation (200 microM L-arginine, 2 mM N(G)-nitro-L-arginine methyl ester) and Mg(2+) modulation of N-methyl-D-aspartate (NMDA) receptor conductance. Electrical stimulation of olfactory nerve fibers evoked transient (peak at 10 s) increments in NO levels 90-100 nM above baseline. In the anesthetized mouse, NO micro-sensors inserted into the granule cell layer detected NO transients averaging 55 nM in amplitude and peaking at 3.4 s after onset of a 5 s odorant stimulation. These findings suggest dual roles for NO signaling in the olfactory bulb: tonic inhibitory control of principal neurons, and regulation of circuit dynamics during odor information processing.

Nanotube-based sensor arrays for clinical breath analysis.

Instrumentation for clinical breath analysis is being developed using a variety of sensor technologies and information processing strategies. One type of instrumentation for clinical breath analysis uses an array of sensors for detection of volatile analytes in breath and pattern recognition and categorization algorithms able to learn and store information about the constant and variable components of human breath samples. This later approach is known as electronic olfaction. We present a new sensor technology for electronic olfaction that offers the potential to develop miniature sensor chips deploying hundreds of diverse and sensitive sensors based on DNA-decorated semiconducting single-walled carbon nanotubes. We have made sensor devices sensitive to some components of human breath (organic acids, trimethylamine). Screening of further DNA oligomers for their ability to render DNA-coated nanotubes sensitive to additional volatile components of human breath is needed to fully exploit this new sensor technology for clinical breath analysis.

Model for olfactory discrimination and learning in Limax procerebrum incorporating oscillatory dynamics and wave propagation.

We extend our model of the procerebral (PC) lobe of Limax, which is comprised of a layer of coupled oscillators and a layer of memory neurons, each layer 4 rows by 20 columns, corresponding to the cell body layer (burster cells) and neuropil layer (nonburster cells) of the PC lobe. A gradient of connections in the layer of model burster cells induces periodic wave propagation, as measured in the PC lobe. We study odor representations in the biological PC lobe using the technique of Kimura and coworkers. Lucifer yellow injection into intact Limax after appetitive or aversive odor learning results in a band or patch of labeled cells in the PC lobe with the band long axis normal to the axis of wave propagation. Learning two odors yields two parallel bands of labeled PC cells. We introduce olfactory input to our model PC lobe such that each odor maximally activates a unique row of four cells which produces a short-term memory trace of odor stimulation. A winner-take-all synaptic competition enabled by collapse of the phase gradient during odor presentation produces a single short-term memory band for each odor. The short-term memory is converted to long-term memory if odor stimulation is followed by activation of an input pathway for the unconditioned stimulus (US) which presumably results in release of one or more neuromodulatory amines or peptides in the PC lobe.

Initiation and propagation of calcium-dependent action potentials in a coupled network of olfactory interneurons.

Coherent oscillatory electrical activity and apical-basal wave propagation have been described previously in the procerebral (PC) lobe, an olfactory center of the terrestrial slug Limax maximus. In this study, we investigate the physiological basis of oscillatory activity and wave propagation in the PC lobe. Calcium green dextran was locally deposited in the PC lobe; this led to cellular uptake and transport of dye by bursting and nonbursting neurons of the PC lobe. The change of intracellular calcium concentration was measured at several different positions in neurites of individual bursting neurons in the PC lobe with a two-photon laser-scanning microscope. Fluorescence measurements were also made from neurons intracellularly injected with calcium green-1. Two different morphological classes of bursting neurons were found, varicose (VB) and smooth (SB). Our results from concurrent optical and intracellular recordings suggest that Ca2+ is the major carrier for the inward current during action potentials of bursting neurons. Intracellular recordings from bursting neurons with nystatin perforated-patch electrodes made while simultaneously recording the local field potential (LFP) with extracellular electrodes indicate that the burster spikes are precisely phase-locked to the periodic LFP events. By referencing successive calcium measurements to the common LFP signal, we could therefore accurately determine the relative timing of calcium transients at different points along a neurite. Measuring the relation of temporal to spatial differences allowed us to estimate the velocity of action potential propagation, which was 4.3 +/- 0.2 (SE) mm/s in VBs, and 1.3 +/- 0.2 mm/s in SB.

In vivo recordings of spontaneous and odor-modulated dynamics in the Limax olfactory lobe.

The major central site of olfactory information processing in the terrestrial slug Limax maximus is the procerebral lobe of the cerebral ganglion, which exhibits oscillatory dynamics of its local field potential and propagates activity waves from its apex to its base, as determined by multisite optical and electrical measurements in vitro. The learning-dependent uptake of Lucifer yellow into procerebral neurons suggests that the procerebral lobe may form learned representations of odors. To determine the role of the procerebral lobe in odor processing and odor learning, we developed procedures to implant fine wire electrodes in the lobe, which allowed recordings of local field potential in freely behaving slugs. The procerebral lobe displays oscillatory dynamics of its local field potential in vivo; however the amplitude and frequency of the local field potential are much more variable in vivo than in vitro. Odor presentation leads to increased frequency and amplitude of the local field potential signal. Several lines of evidence indicate that the variations in the local field potential signal recorded in vivo are not due to movement artifacts or activity in adjacent muscles. Multiple amine, gaseous, and peptide neuromodulators known to be present in the procerebral lobe provide pathways by which activity or coupling of bursting neurons in the procerebral lobe could be altered, resulting in the observed amplitude and frequency modulation of the local field potential.

Lateralized memory storage and crossed inhibition during odor processing by Limax.

After odor conditioning intact Limax maximus and injecting LY into their haemocoel, labeled groups of neurons are found in either the right or left procerebral lobe but never in both procerebral lobes. This suggests that a competitive interaction occurs between right and left odor processing pathways of which the procerebral lobe is a part. We use the nerve discharge in the external peritentacular nerve evoked by applying a puff of conditioned odor to the nose to document crossed inhibition between left and right odor processing pathways. Responses in the external peritentacular nerve evoked by stimulating one superior nose with a conditioned odor are strongly lateralized as responses occur only on the stimulated side. Stimulating both superior noses simultaneously with the same conditioned odor yields responses in both external peritentacular nerves that resemble the sum of responses to unilateral stimulation. Simultaneously stimulating both superior noses, each with a different conditioned odor leads to strong inhibition of both external peritentacular nerve responses. The crossed inhibition is also evident if both superior and inferior noses on the same side are stimulated simultaneously. A lateral inhibitory mechanism, situated postsynaptic to odor recognition, appears to inhibit external peritentacular nerve responses if the two noses receive conflicting sensory inputs.

Nitric oxide and carbon monoxide modulate oscillations of olfactory interneurons in a terrestrial mollusk.

Spontaneous or odor-induced oscillations in local field potential are a general feature of olfactory processing centers in a large number of vertebrate and invertebrate species. The ubiquity of such oscillations in the olfactory bulb of vertebrates and analogous structures in arthropods and mollusks suggests that oscillations are fundamental to the computations performed during processing of odor stimuli. Diffusible intercellular messengers such as nitric oxide (NO) and carbon monoxide (CO) also are associated with central olfactory structures in a wide array of species. We use the procerebral (PC) lobe of the terrestrial mollusk Limax maximus to demonstrate a role for NO and CO in the oscillatory dynamics of the PC lobe: synthesizing enzymes for NO and CO are associated with the PC lobes of Limax, application of NO to the Limax PC lobe increases the local field potential oscillation frequency, whereas block of NO synthesis slows or stops the oscillation, the bursting cells of the PC lobe that drive the field potential oscillation are driven to higher burst frequency by application of NO, the nonbursting cells of the PC lobe receive trains of inhibitory postsynaptic potentials, presumably from bursting cells, due to application of NO, and application of CO to the PC lobe by photolysis of caged CO results in an increase in oscillation frequency proportional to CO dosage.

Oscillatory dynamics and information processing in olfactory systems

Oscillatory dynamics is a universal design feature of olfactory information-processing systems. Recent results in honeybees and terrestrial slugs suggest that oscillations underlie temporal patterns of olfactory interneuron responses critical for odor discrimination. Additional general design features in olfactory information-processing systems include (1) the use of central processing areas receiving direct olfactory input for odor memory storage and (2) modulation of circuit dynamics and olfactory memory function by nitric oxide. Recent results in the procerebral lobe of the terrestrial slug Limax maximus, an olfactory analyzer with oscillatory dynamics and propagating activity waves, suggest that Lucifer Yellow can be used to reveal a band-shaped group of procerebral neurons involved in the storage of an odor memory. A model has been constructed to relate wave propagation and odor memory bands in the procerebral lobe of L. maximus and to relate these findings to glomerular odor representations in arthropods and vertebrates.

Olfactory oscillations augment odor discrimination not odor identification by Limax CNS.

There is great interest in the function of synchronous oscillations in olfactory centers, as documented in a wide variety of species. In Limax procerebral (PC) lobe, local field potential oscillations are ongoing and altered by odor stimulation. Recordings from external peritentacular nerves (ePTNs) reveal a neural correlate of tentacle positioning, a response signifying recognition of a conditioned odor. Using the odor-elicited ePTN response we found no clear difference in the dynamics of the PC oscillations which precedes and predicts the occurrence of ePTN responses. Reversibly blocking the oscillations did not impair recognition of conditioned odors but reduced the differential nerve response to related odors. PC oscillations may play a role in odor discrimination but are not necessary for odor recognition.

Minimal model of oscillations and waves in the Limax olfactory lobe with tests of the model's predictive power.

Propagating waves are observed in the olfactory or procerebral (PC) lobe of the terrestrial mollusk, Limax maximus. Wave propagation is altered by cutting through the various layers of the PC lobe both parallel and transverse to the direction of wave propagation. We present a model for the PC lobe based on two layers of coupled cells. The top layer represents the cell layer of the PC lobe, and the bottom layer corresponds to the neuropil of the PC lobe. To get wave propagation, we induce a coupling gradient so that the most apical cells receive a greater input from neighbors than the basal cells. The top layer in the model is composed of oscillators coupled locally, whereas the bottom layer is comprised of oscillators with global coupling. Odor stimulation is represented by an increase in the strength of coupling between the two layers. This model allows us to explain a number of experimental observations: 1) the intact PC lobe exhibits regular propagating waves, which travel from the apical to the basal end; 2) there is a gradient in the local frequency of slices cut transverse to the axis of wave propagation, with apical slices oscillating faster than basal slices; 3) with partial cuts through the cell layer or the neuropil layer, the apical and basal ends remain tightly coupled; 4) removal of the neuropil layer does not prevent wave propagation in the cell layer; 5) odor stimulation causes the waves to collapse and the cells in the PC lobe oscillate synchronously; and 6) by allowing a single parameter to vary in the model, we capture the reversal of waves in low chloride medium.

Vital staining from dye-coated microprobes identifies new olfactory interneurons for optical and electrical recording.

A versatile technique for dye application in living tissue is described, which results in labeling of viable cells from which electrophysiological or optical recordings can be obtained. The dye-coated surface of a glass microelectrode tip is used to apply anatomical tracers or calcium sensitive probes with spatial precision. A total of three types of dyes have been applied in this way to find and record from olfactory interneurons in the terrestrial mollusc Limax maximus. Crystals of 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) formed on the tips of glass microelectrodes were placed in the procerebral lobe, the major olfactory processing center of Limax. Somata in buccal and pedal ganglia with processes extending several 100 microm to the procerebral lobe were stained within 4-6 h. Intracellular recordings from DiI stained buccal (B(PC)) and pedal (P(PC)) cells were obtained. Cross correlograms of the oscillatory field potential in the procerebral lobe and spontaneous action potentials in P(PC) or B(PC) show that P(PC) activity is weakly coupled to the oscillation in the procerebral lobe, while B(PC) activity is clearly coupled to the oscillation. Stimulation of the procerebral lobe with nitric oxide activated P(PC) cells but suppressed activity in B(PC) cells. Calcium green-10Kdextran coated electrodes were used to place calcium green in the cell body layer of the procerebral lobe. Bursting and nonbursting procerebral neurons incorporated and transported the calcium green-dextran. Optical recordings of changes in fluorescence signals from several bursting cells recorded simultaneously were used to test alternative mechanisms of bursting cell coupling. Application of biotin 3Kdextran to the midline of the cerebral ganglion revealed a group of cells in each procerebral lobe with processes crossing the midline of the cerebral ganglion. These cells may couple right and left procerebral lobe activity during odor processing.

Central and reflex neuronal responses elicited by odor in a terrestrial mollusk.

1. We studied the responses to odor of a central olfactory processing organ and subsequent central outputs in the terrestrial mollusk Limax maximus. We used extracellular recording techniques and optical recording from preparations stained with a voltage-sensitive dye to characterize network responses in the central organ and whole nerve recording to characterize central odor-elicited outputs. 2. The central olfactory organ, the procerebral (PC) lobe, is a highly interconnected network of local olfactory interneurons that receives input from primary olfactory receptors. In the absence of odor the PC network is known to exhibit periodic waves of excitation and inhibition at a frequency of approximately 0.7 Hz. Here we study how different odor inputs affect the intrinsic oscillatory dynamics. 3. Odor stimulation causes the propagation of electrical activity along the lobe to transiently switch from the state with propagating waves, with typical phase shifts of one half cycle along the lobe, to a state with few or no phase differences along the lobe. The collapse of the phase gradient typically occurs without spatially localized changes in the amplitude of the oscillation, at least on the scale of our optical resolution, approximately 0.1 times the length of the lobe. In some trials, however, we resolved spatial nonuniformities in the magnitude of excitation across the lobe. 4. The collapse of the phase gradient along the lobe in response to odor stimulation is robust on a trial-by-trial basis. Further, the change in phase gradient can occur with little or no change in the frequency of oscillation, as occasionally observed in response to weak odor stimulation. 5. Typically odor stimulation causes changes in the frequency of the oscillation. Two odors, one attractive (potato) and one repellent (amyl acetate), produced different patterns of change; potato induced a transient increase in frequency, whereas amyl acetate produced an initial decrease in frequency followed by a transient increase in frequency. We do not yet know whether these frequency change patterns are unique to these specific odors or to their behavioral meaning. 6. Previous work demonstrated direct connections from the PC lobe to the buccal and pedal ganglia, centers controlling feeding and locomotion, respectively. To establish a correlation between odor-induced changes in the PC lobe and activation of such centers and subsequently effector organs, we recorded from selected central connectives and peripheral nerve roots. The dependence of odor-elicited activity recorded in connectives and nerve roots on PC integrity was assessed by measurements of odor-elicited activity before and after PC ablation. 7. Odor stimulation caused activation of multiple units in the cerebrobuccal connective. One output of the buccal ganglion, the salivary nerve, also showed odor-elicited activation of an identified unit, the slow burster. The necessity of the PC lobe for activation of the slow burster was established by measurements of odor-elicited activity before and after PC ablation. 8. Odor stimulation also caused activation of multiple units in the buccal mass retractor nerve. Activation of a fraction of these units (3 of 10) was dependent on an intact PC lobe, like the slow burster neuron in the salivary nerve. 9. Our results clearly show how stimuli may lead to changes in the spatial-temporal pattern of activity in a central circuit without changing the overall average level of activity in that circuit.

Oscillations and gaseous oxides in invertebrate olfaction.

Olfactory systems combine an extraordinary molecular sensitivity with robust synaptic plasticity. Central neuronal circuits that perform pattern recognition in olfaction typically discriminate between hundreds of molecular species and form associations between odor onsets and behavioral contingencies that can last a lifetime. Two design features in the olfactory system of the terrestrial mollusk Limax maximus may be common elements of olfactory systems that display the twin features of broad molecular sensitivity and rapid odor learning: spatially coherent oscillations in the second-order circuitry that receives sensory input; and involvement of the interneuronal messengers nitric oxide (NO) and carbon monoxide (CO) in sensory responses and circuit dynamics of the oscillating olfactory network. The principal odor processing center in Limax, the procerebrum (PC) of the cerebral ganglion, contains on the order of 10(5) local interneurons and receives both direct and processed input from olfactory receptors. Field potential recordings in the PC show an oscillation at approximately 0.7 Hz that is altered by odor input. Optical recordings of voltage changes in local regions of the PC show waves of depolarization that originate at the distal pole and propagate to the base of the PC. Weak odor stimulation transiently switches PC activity from a propagating mode to a spatially uniform mode. The field potential oscillation in the PC lobe depends on intercellular communication via NO, based on opposing effects of reagents that decrease or increase NO levels in the PC. Inhibition of NO synthase slows the field potential oscillation, while application of exogenous NO increases the oscillation frequency. A role for CO in PC dynamics is suggested by experiments in which CO liberation increases the PC oscillation frequency. These design features of the Limax PC lobe odor processing circuitry may relate to synaptic plasticity that subserves both connection of new receptors throughout the life of the slug and its highly developed odor learning ability.

Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy.

Light scattering by brain tissue and phototoxicity are major obstacles to the use of high-resolution optical imaging and photo-activation ('uncaging') of bioactive compounds from inactive ('caged') precursors in intact and semi-intact nervous systems. Optical methods based on 2-photon excitation promise to reduce these obstacles (Denk, 1994; Denk et al., 1990, 1994). Here we show a range of imaging modes based on 2-photon laser scanning microscopy (TPLSM) as applicable to problems in neuroscience. Fluorescence images were taken of neurons labeled with ion-sensitive and voltage-sensitive dyes in invertebrate ganglia, mammalian brain slices, and from the intact mammalian brain. Scanning photochemical images with whole-cell current detection (Denk, 1994) show how the distribution of neurotransmitter receptors on the surface of specific cells can be mapped. All images show strong optical sectioning and usable images can be obtained at depths greater than 100 microns below the surface of the preparation.

Dynamics of propagating waves in the olfactory network of a terrestrial mollusk: an electrical and optical study.

1. The procerebral (PC) lobe of the terrestrial mollusk Limax maximus contains a highly interconnected network of local olfactory interneurons that receives ipsilateral axonal projections from superior and inferior noses. This network exhibits an approximately 0.7-Hz intrinsic oscillation in its local field potential (LFP). 2. Intracellular recordings show that the lobe contains at least two classes of neurons with activity phase locked to the oscillation. Neurons in one class produce periodic bursts of spikes, followed by a period of hyperpolarization and subsequently a depolarizing afterpotential. There is a small but significant chance for a second burst to occur during the depolarizing afterpotential; this leads to a double event in the LFP. Bursting neurons constitute approximately 10% of the neurons in the lobe. 3. Neurons in the other class fire infrequently and do not produce periodic bursts of action potentials. However, they receive strong, periodic inhibitory input during every event in the LFP. These nonbursting cells constitute the major fraction of neurons in the lobe. There is a clear correlation between the periodic burst of action potentials in the bursting neurons and the hyperpolarization seen in nonbursting neurons. 4. Optical techniques are used to image the spatially averaged transmembrane potentials in preparations stained with voltage-sensitive dyes. The results of simultaneous optical and electrical measurements show that the major part of the optical signal can be interpreted as a superposition of the intracellular signals arising from the bursting and nonbursting neurons. 5. Successive images of the entire PC lobe show waves of electrical activity that span the width of the lobe and travel its full length along a longitudinal axis. The direction of propagation in the unperturbed lobe is always from the distal to the proximal end. The wavelength varies between preparations but is on the order of the length of the preparation. 6. One-dimensional images along the longitudinal axis of the lobe are used to construct a space-time map of the optical activity, from which we calculate the absolute contribution of bursting and nonbursting neurons to the optical signal. The contribution of the intracellular signals from the two cell types appears to vary systematically across the lobe; bursting cells dominate at middle and proximal locations, and nonbursting cells dominate at distal locations. 7. The direction and form of the waves can be perturbed either by microsurgical manipulation of the preparation or by chemical modulation of its synaptic and neuronal properties.(ABSTRACT TRUNCATED AT 400 WORDS)