**3. Neural systems for olfactory processing of the land slug**

their function is based on complex interactions among electric neural activities, intracellular calcium signals, intercellular communications by neurotransmitter, etc. The electrophysiological methods are not suitable for the simultaneous measurement from many neurons or neural areas. For multi-point measurement of neural activities, fluorescent-imaging technique using voltage-sensitive dyes or calcium-sensitive dyes can be a powerful technique. In this technique, the dyes, which change their fluorescent intensities due to the voltage or calcium ion concentration change [1], are loaded into the cells, and their fluorescence changes are

The multi-point measurement using fluorescent-imaging technique has been reported in vertebrates [2–5] and invertebrates [6–16]. In invertebrates, for example, this technique has been applied to measure spatiotemporal neural activities involved in the olfactory processing of the land slug *Limax* [6, 7, 9–11, 13–16]. In *Lima*x, the procerebral (PC) lobe, which is the olfactory center located in the lateral part of each cerebral ganglion, spontaneously produces a periodic oscillation of local field potential (LFP) of about 1 Hz. The LFP oscillation is well synchronized over the entire PC lobe, but the phase of oscillation is advanced at the distal region, resulting in periodic propagating waves of neural activity from the distal to proximal regions. The previous studies showed that odor stimuli change the LFP frequency and the

In this article, we review the previous studies, as well as our recent studies, on the spatiotem-

In fluorescent-imaging technique, the dyes, which change their fluorescence intensities due to the voltage or calcium ion concentration change [1], are loaded into cells, and their fluorescence changes are acquired into a computer as a series of images. However, the voltage-sensitive dyes generally exhibit a relatively small change in the fluorescence intensities, resulting in a low signal-to-noise (S/N) ratio. On the other hand, the calcium-sensitive dyes exhibit a larger change in the fluorescence intensities than that of the voltage-sensitive dyes. Therefore, their fluorescence changes can be detected easily, which enables us to indirectly measure neural activities because the intracellular calcium concentration often increases with neural

As the calcium-sensitive dyes, those bonded with the acetoxymethyl (AM) group are commonly used because they are permeable into cells. After the AM-bonded dyes permeate into cells, the AM group is dissociated by intracellular esterase, and then the dyes can be nonpermeable and loaded into cells. Vertebrate neurons are well known to be easily loaded with the AM-bonded calcium-sensitive dyes. In invertebrates, however, the AM-bonded dyes cannot be easily loaded into neurons because the AM group is difficult to be dissociated due to

For these reasons, it has not been determined yet which type of dyes is better for fluorescent imaging of neural activities in invertebrates. In Section 4, we introduce some studies on the

poral neural activities of the land slug *Limax* using fluorescent-imaging technique.

acquired into a computer as a series of images.

wave propagation speed.

120 Organismal and Molecular Malacology

activities.

**2. Fluorescent-imaging technique**

their weak activity of intracellular esterase.

The land slug *Limax* has two pairs of tentacles, the superior and inferior tentacles. **Figure 1** shows a photograph of the central nervous system of *Limax* and the schematic illustration of the cerebral ganglion and superior tentacle. Each tentacle has an olfactory epithelium, which receives odors, at its tip. Olfactory receptor neurons are located at the tip regions of the digitate extensions of the tentacular ganglion (TG) (referring as "digitate ganglion" in **Figure 1B**) beneath the epithelium and terminate to synapse projection neurons in the TG that is the primary center of olfactory information processing (**Figure 1B**). The TG contains interneurons, as well as projection neurons that project to the cerebral ganglion [17]. The TG shows a spontaneous oscillation of LFP at about 2 Hz, which is usually measured by extracellular recording [18]. The oscillation is modulated by odor and air stimuli [19]. Several neurotransmitters such as serotonin, acetylcholine, glutamate, and gamma-aminobutyric acid (GABA), exist in the TG and modulate the spontaneous oscillation [20–22]. In addition, the olfactory epithelium also shows an oscillatory electro-olfactogram (EOG) in response to odor and air stimuli. The EOG oscillation interacts with the LFP oscillation in the TG [19].

TG neurons project to the cerebral ganglion via the tentacle or olfactory nerves. The major target of projection is the procerebral lobe, which is the lateral part of the cerebral ganglion (**Figure 1**). The PC lobe is a division of the cerebral ganglion unique to terrestrial slugs and snails that is specialized for the processing of olfactory information [23–25]. The PC lobe consists of three layers, the cell mass (CM), terminal mass (TM), and internal mass (IM) (**Figure 2**). The CM contains a large number of cell bodies of neurons, which are generally

**Figure 1.** (A) Photograph of the central nervous system of the land slug *Limax* Color online and (B) schematic illustration of the cerebral ganglion and superior tentacle. This figure (B) is reproduced from Ref. [7] with permission.

**Figure 2.** (A) Schematic illustration of the PC and (B) structure of the neural network of the PC. B neuron: bursting neuron, NB neuron: nonbursting neuron; CM: cell mass, TM: terminal mass, IM: internal mass. These figures are reproduced from Ref. [14] with permission.

small (5–8 μm) [23]. The TM and IM are neuropile layers. The TM receives input from the tentacle nerves [9]. The PC lobe spontaneously produces a periodic slow oscillation of LFP at about 1 Hz, which is usually measured by extracellular recording [26]. The LFP oscillation is well synchronized over the entire PC, but the phase of the oscillation is advanced at the distal region. This results in periodic propagating waves of neural activity from the distal to proximal regions, which has been clearly shown by fluorescent-imaging technique [6, 7, 9–11, 13–16].

Patch-clamp recording from single neurons showed that the neurons in the PC lobe are categorized as either bursting (B) or nonbursting (NB) neurons [6, 7] (**Figure 2**). B neurons are characterized by periodic bursting activity, and NB neurons are silent or fire at a low frequency. NB neurons are more numerous (about 90%) and have projection of neurites to the TM and IM [27]. NB neurons receive olfactory input from the tentacle nerves in the TM [28]. B neurons have extensive projection of neurites within the CM and inhibit NB neurons, while NB neurons excite B neurons. The spontaneous bursting activities of B neurons and inhibitory synaptic potentials in NB neurons are synchronized with the LFP oscillation.

The propagating waves of neural activity have also been found in mammalian cortex. However, the function and computational role are still unclear even in the land slug *Limax*. To elucidate it, fluorescent-imaging technique is expected to be a powerful technique as described in the subsequent section.
