Research reportIdentification and characterization of an output neuron from the oscillatory molluscan olfactory network
Introduction
The terrestrial slug Limax marginatus locates food by olfaction and has a high capacity for associative learning of odors and tastes [3], [18]. The olfactory system is accordingly well developed, and the main part of the slug’s central nervous system is dedicated to olfaction. Limax senses odors with two pairs of tentacles, the superior and inferior tentacles; the olfactory information then is transmitted to the olfactory center, the procerebrum (PC), and also to other regions in the cerebral ganglion, the metacerebrum and the mesocerebrum.
Coherent oscillation of membrane potentials has been observed in the PC of Limax[4], [11]. The PC is a highly developed neural network consisting of about 105 neurons that are classified into two types, bursting and nonbursting neurons, and the structure of the PC appears uniform [13], [22]. Coherent oscillations are also found in the olfactory bulb of mammals [1] and the mushroom bodies of insects [14], and they are thought to play a critical role in olfactory information processing. Several studies have suggested roles for synchronous oscillation in these olfactory centers. Direct evidence for the involvement of oscillations in cognitive function has been presented in the mushroom body of the honeybee by Stopfer et al. [19], who showed by blocking the oscillation with a GABAA receptor antagonist that synchronous membrane potential oscillation is related to odor discrimination. A similar result has been reported for Limax[20]. Several studies on Limax have shown that odor-induced frequency modulation in the PC is correlated with the value of the odorant [7], [12]. However, how the coherent membrane potential oscillations are decoded and affect behavior is not well understood. The characterization of output neuronal activities is an important step toward understanding the mechanisms of information processing in oscillatory sensory centers. Since neither bursting nor nonbursting PC neurons have been shown to extend their neurites outside the PC [22], the output neurons of the PC should be located outside the PC and should make connections with PC neurons within the PC. Until now, only a few output neurons from the PC have been identified in the buccal and pedal ganglia [5]. These neurons extend their neurites into the PC and exhibit membrane potential oscillations that are synchronous with the local field potential (LFP) of the PC. However, when more complex behavioral responses to odors are required, such as memory-guided behavioral decisions, it would be reasonable to speculate that the PC is required to interact with higher central circuits that may exist in the cerebral ganglion. We therefore looked for an output neuron from the PC in the cerebral ganglion and identified a neuron morphologically and physiologically that has connections with the PC. This neuron could be the key to further analysis of information processing in the oscillatory central neural systems
Section snippets
Preparation
The slugs were collected in the open field and maintained in our laboratory at 20°C. Each slug was anesthetized by injection of a high Mg2+ solution which contained (in mM) 57.6 MgCl2, 5 glucose, and 5 HEPES (pH 7.6); it was then dissected in a petri dish filled with a dissection solution which contained (in mM) 35 NaCl, 2 KCl, 4.9 CaCl2, 28 MgCl2, 5 glucose, and 5 HEPES (pH 7.6). The cerebral ganglion with the tentacle nerves attached was isolated and the sheath covering the cerebral ganglion
Exploration of candidates for an output neuron
Neurons projecting to the PC were searched for by applying the lipophilic dye DiI to the neuropil region that was exposed on the cut end of the PC. This strategy resulted in staining of 3–8 neurons on the anterior side of the metacerebrum close to the PC (data not shown). After random injection of sulforhodamine B with the whole-cell configuration into neurons in the metacerebrum region (which was frequently stained), a neuron that extended its neurites into the PC was found. This neuron was
Discussion
In the present work, we identified a neuron that appears to convey information from the oscillatory olfactory center to other regions of the cerebral ganglion. We have shown that the identified neuron, which we named the MPN, receives a periodic excitatory input that is synchronous with the LFP oscillation of the PC. In addition, there are varicosities in the neurites in the metacerebrum. Although Wang et al. [21] have shown that PC neurons lacking varicosities on their neurite are synaptically
Acknowledgements
This study was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (11771408, 12048209, 12210048, and 12307053) and a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences, Japan.
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