Mapping of odor-related neuronal activity using a fluorescent derivative of glucose.

Activity labeling was applied to the olfactory systems of the terrestrial slug Limax valentianus using 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), a fluorescent derivative of glucose. 2-NBDG was incorporated into cultured Limax olfactory interneurons, and this was partially blocked by the presence of a high concentration of glucose in the medium, indicating that a part of the uptake of 2-NBDG is mediated by glucose transporters. Next, in order to map odor-related neuronal activity in the primary olfactory center, tentacular ganglion, we injected 2-NBDG into the body cavities of slugs and exposed them to odors or clean air (control). In the odor-stimulated animals, the cell mass region was strongly stained. The digit-like extensions and the neuropil region were also stained in some animals. The control animals showed no staining. The neurons in the cell mass are thought to be involved in generating oscillating activities in the tentacular ganglion, and their activation may imply modulation of oscillatory activity during odor processing. Our results show that 2-NBDG is useful for mapping neuronal activity in vivo.

Wavelet analysis can sensitively describe dynamics of ethanol evoked local field potentials of the slug (Limax marginatus) brain.

Odorants evoke characteristic, but complex, local field potentials (LFPs) in the molluscan brain. Wavelet tools in combination with Fourier analysis can detect and characterize hitherto unknown discrete, slow potentials underlying the conspicuous oscillations. Ethanol was one of the odorants that we have extensively studied (J. Neurosci. Methods, 119 (2002) 89). To detect new features and to elucidate their functions, we tested the wavelet tools on the ethanol-evoked LFP responses of the slug (Limax) procerebrum. Recordings were made in vitro from the neuropile and the cell layer. The present study led to the following findings: (i) Mutual exclusion. Energy concentrated mainly in two ranges, (a) 0.1-0.4 Hz and (b) 1.56-12.5 Hz, and the sum of energy remained constant throughout experiments regardless of the condition. A redistribution of relative energy within this sum seemed to occur in the course of main, possible interactions between the two components excluding each other ('mutual exclusion'). (ii) Transient signal ordering and disordering. Ethanol stimulation alternatingly evoked periods of strongly time evolving oscillation dominated by the energy of 1.56-12.5 Hz (increase of entropy=disordered or complexly ordered state) and those of near-silence were predominated by the energy of 0.1-0.4 Hz (decrease of entropy=ordered state). (iii) About 0.1 Hz slow wave oscillation. It was robust. The dominant energy oscillation and the resulting large entropy fluctuation were negatively correlated to each other, and revealed strong frequency-tuning or synchronization at this frequency. Our findings suggest that discrete slow waves play functionally important roles in the invertebrate brain, as widely known in vertebrate EEG. Wavelet tools allow an easy interpretation of several minutes of frequency variations in a single display and give precise information on stimulus-evoked complex change of the neural system describing the new state 'more ordered' or 'non-ordered or more complexly ordered'.

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.

Negative relationship between odor-induced spike activity and spontaneous oscillations in the primary olfactory system of the terrestrial slug Limax marginatus.

Although primary olfactory systems in various animals display spontaneous oscillatory activity, its functional significance in olfactory processing has not been elucidated. The tentacular ganglion, the primary olfactory system of the terrestrial slug Limax marginatus, also displays spontaneous oscillatory activity at 1-2 Hz. In the present study, we examined the relationship between odor-evoked spike activity and spontaneous field potential oscillations in the tentacular nerve, representing the pathway from the primary olfactory system to the olfactory center. Neural activity was recorded from the tentacular nerve before, during and after application of various odors (garlic, carrot, and rat chow) to the sensory epithelium and the changes in firing rate and spontaneous oscillations were analyzed. We detected the baseline amplitude of the oscillations and baseline spike activity before stimulation. Odor stimulations for 20 s or 60 s evoked a transient increase in the firing rate followed by a decrease in the amplitude of spontaneous oscillations. The decrease in the amplitude was larger in the first 8 s of stimulation and subsequently showed recovery during stimulation. The amplitude of the recovered oscillations often fluctuated. Odor-evoked spikes appeared when the amplitude of the recovered oscillations was transiently small. These results suggest that the large oscillations could inhibit spike activity whereas the first transient increase in spike activity was followed by the decrease in the oscillation amplitude. Our results indicate that there is a significant negative correlation between spontaneous oscillations and odor-evoked spike activity, suggesting that the spontaneous oscillations contribute to the olfactory processing in slugs.

Distributions of gamma-aminobutyric acid immunoreactive and acetylcholinesterase-containing cells in the primary olfactory system in the terrestrial slug Limax marginatus.

The tentacular ganglion, the primary olfactory system of terrestrial slugs, exhibits spontaneous oscillations with a spatial coherence. The digit-like extensions (digits) of the tentacular ganglion presumably house the cell bodies of the neurons underlying the oscillations. The present study was designed to identify the anatomical and physiological determinants of these oscillations with a special focus on whether the neurons located in the digits contribute to the coherent oscillations. We recorded field potentials from the spatially separated sites in the digits in the terrestrial slug Limax marginatus. We also simultaneously recorded tentacular nerve to monitor the coherent oscillations. The spatially separated regions in the digits oscillated at the same frequency as the tentacular nerve, indicating a single coherent activity. To study the neural networks underlying the coherent oscillations, we examined the distributions of acetylcholinesterase (AChE)-containing and gamma-aminobutyric acid immunoreactive (GABA-ir) neurons. AChE-containing and GABA-ir fibers were found to connect the neurons in a branch of the digits with those in other branches. We also used a vital staining technique with 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate to examine the projections of neurons in the digits. Large stained cells were detected in many branches of the digits after placing the dye on one of the cell masses located in right and left sides of the tentacular ganglion. They were detected in the cell masses and in many branches of the digits after placing the dye on a branch of the digits. Our results showed that the slug primary olfactory system has highly interconnected neural networks.

Frequency transitions in odor-evoked neural oscillations.

In many species, sensory stimuli elicit the oscillatory synchronization of groups of neurons. What determines the properties of these oscillations? In the olfactory system of the moth, we found that odors elicited oscillatory synchronization through a neural mechanism like that described in locust and Drosophila. During responses to long odor pulses, oscillations suddenly slowed as net olfactory receptor neuron (ORN) output decreased; thus, stimulus intensity appeared to determine oscillation frequency. However, changing the concentration of the odor had little effect upon oscillatory frequency. Our recordings in vivo and computational models based on these results suggested that the main effect of increasing odor concentration was to recruit additional, less well-tuned ORNs whose firing rates were tightly constrained by adaptation and saturation. Thus, in the periphery, concentration is encoded mainly by the size of the responsive ORN population, and oscillation frequency is set by the adaptation and saturation of this response.

Bilateral olfaction: two is better than one for navigation.

Do animals require bilateral input to track odors? A recent study reveals that fruit fly larvae can localize odor sources using unilateral inputs from a single functional sensory neuron, but that an enhanced signal-to-noise ratio provided by dual inputs is helpful in more challenging environments.

Olfactory learning and spike timing dependent plasticity.

Hebbian spike-timing-dependent plasticity (STDP) is widely observed in organisms ranging from insects to humans and may provide a cellular mechanism for associative learning. STDP requires a millisecond-scale temporal correlation of spiking activity in pre- and postsynaptic neurons. However, animals can learn to associate a sensory cue and a reward that are presented seconds apart. Thus, for STDP to mediate associative learning, the brain must retain information about the sensory cue as spiking activity until the reinforcement signal arrives. In our recent study, we tested this requirement in the moth Manduca sexta. We characterized the odor responses of Kenyon cells, a key neuronal population for insect olfactory learning, and conditioned moths to associate an odor with a sugar water reward. By varying the amount of temporal overlap between odor-evoked spikes and the reward presentation, we found that the most learning occurred when spiking activity had no overlap with the reward presentation; further, increasing the overlap actually decreased the learning efficacy. Thus, STDP alone cannot mediate the olfactory learning in Kenyon cells. Here, we discuss possible cellular mechanisms that could bridge the temporal gap between physiological and behavioral time scales.

Sparse odor representation and olfactory learning.

Sensory systems create neural representations of environmental stimuli and these representations can be associated with other stimuli through learning. Are spike patterns the neural representations that get directly associated with reinforcement during conditioning? In the moth Manduca sexta, we found that odor presentations that support associative conditioning elicited only one or two spikes on the odor's onset (and sometimes offset) in each of a small fraction of Kenyon cells. Using associative conditioning procedures that effectively induced learning and varying the timing of reinforcement relative to spiking in Kenyon cells, we found that odor-elicited spiking in these cells ended well before the reinforcement was delivered. Furthermore, increasing the temporal overlap between spiking in Kenyon cells and reinforcement presentation actually reduced the efficacy of learning. Thus, spikes in Kenyon cells do not constitute the odor representation that coincides with reinforcement, and Hebbian spike timing-dependent plasticity in Kenyon cells alone cannot underlie this learning.

Air movement evokes electro-olfactogram oscillations in the olfactory epithelium and modulates olfactory processing in a slug.

In many animals, neurons in the olfactory system have been shown to respond not only to odorants but also to air movements. However, the manner in which the mechanical dynamics of odor stimulation affect olfactory processing remains poorly understood. Using a series of flow rates and odor concentrations from clean air to high-concentration vapors, we systematically analyzed the effects of air movement and odor concentration on olfactory processing. We extracellularly recorded local field potentials and spike units from the olfactory epithelium (OE) and tentacular nerve (TN), which connects the first and second relay centers of olfactory information, in the terrestrial slug Limax marginatus. We found that clean air puffs at a flow rate of 0.18 ml/s (gentle wind), but not high-concentration odor puffs at lower flow rates, induced electro-olfactogram (EOG) oscillations in the OE with a constant frequency (2.5 Hz), regardless of the odor. Surgically isolated OE preparations also showed these EOG oscillations, indicating that the oscillations arose from the OE independently of the downstream circuits. The EOG oscillations entrained the slower spontaneous TN oscillations (1-2 Hz) to the fixed rhythm (2.5 Hz). Spontaneous and odor-evoked units were phase-locked to the TN oscillation peaks. This TN oscillation entrainment by the EOG oscillations caused stronger phase-locking, specifically TN oscillation peaks and EOG oscillation troughs. Taken together, these results suggest that when odors are carried by a gentle wind, the air movement induces EOG oscillations and modulates rhythmic spike patterning of olfactory outputs to the second olfactory relay center in Limax.

Modulation of two oscillatory networks in the peripheral olfactory system by gamma-aminobutyric acid, glutamate, and acetylcholine in the terrestrial slug Limax marginatus.

The digit-like extensions (the digits) of the tentacular ganglion of the terrestrial slug Limax marginatus are the cell body rich region in the primary olfactory system, and they contain primary olfactory neurons and projection neurons that send their axons to the olfactory center via the tentacular nerves. Two cell clusters (the cell masses) at the bases of the digits form the other cell body rich regions. Although the spontaneous slow oscillations and odor responses in the tentacular nerve have been studied, the origin of the oscillatory activity is unknown. In the present study, we examined the contribution of the neurons in the digits and cell masses to generation of the tentacular nerve oscillations by surgical removal from the whole tentacle preparations. Both structures contributed to the tentacular oscillations, and surgical isolation of the digits from the whole tentacle preparations still showed spontaneous oscillations. To analyze the dynamics of odor-processing circuits in the digits and tentacular ganglia, we studied the effects of gamma-aminobutyric acid, glutamate, and acetylcholine on the circuit dynamics of the oscillatory network(s) in the peripheral olfactory system. Bath or local puff application of gamma-aminobutyric acid to the cell masses decreased the tentacular nerve oscillations, whereas the bath or local puff application of glutamate and acetylcholine to the digits increased the digits' oscillations. Our results suggest the existence of two intrinsic oscillatory circuits that respond differentially to endogenous neurotransmitters in the primary olfactory system of slugs.