Abstract
The brain or central nervous system forms a network composed of so many neurons and their function is based on complex interactions among electric neural activities, intracellular calcium signals, intercellular communications by neurotransmitter, and so on. For multi-point measurement of neural activities, fluorescent-imaging technique using voltage-sensitive dyes or calcium-sensitive dyes can be a powerful technique. This technique has been applied to measure spatiotemporal neural activities involved in the olfactory processing of the land slug Limax. In Limax, 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, and the phase of the LFP 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 wave propagation speed. In this article, we review the previous studies, as well as our recent studies, on the spatiotemporal neural activities of the land slug Limax using fluorescent-imaging technique.
Keywords
- land slug
- olfactory processing
- spatiotemporal neural activity
- fluorescent-imaging technique
- voltage-sensitive dye
- calcium-sensitive dye
1. Introduction
The conventional electrophysiological methods have been well used for the measurement of neural activities of vertebrate and invertebrate. The direct measurement from single neurons and neural areas is possible by the conventional methods using microelectrodes. However, the brain or central nervous system forms a network composed of so many neurons and 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 acquired into a computer as a series of images.
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
In this article, we review the previous studies, as well as our recent studies, on the spatiotemporal neural activities of the land slug
2. Fluorescent-imaging technique
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 activities.
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 non-permeable 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 their weak activity of intracellular esterase.
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 spatiotemporal neural activities of the land slug
3. Neural systems for olfactory processing of the land slug
The land slug
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 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
4. Fluorescent imaging of spatiotemporal neural activities involved in the olfactory processing of the land slug
As mentioned earlier, periodic waves of neural activity propagate from the distal to proximal regions in the PC lobe of the land slug
They next examined the responses of spatiotemporal neural activity in the PC lobe to odor stimuli (Figure 7). The stimuli were applied to the epithelium of the superior tentacle. The phase difference of neural activity between the distal and proximal regions was not changed in response to the moist air (Figure 7A). However, in response to a component of natural potato odor (2-ethyl-3-methoxypyrazine; EMOP), a known appetitive stimulus to the naïve slugs, the phase difference was dramatically changed (Figure 7B–F). The stimulus caused a collapse of the phase difference, that is, an initial difference of Δ
Thereafter, other groups have also studied the spatiotemporal neural activities of the PC lobe using fluorescent voltage imaging. Kimura et al. examined the responses of the PC lobe of
Watanabe et al. measured neural activities of other areas in the cerebral ganglion, metacerebrum/mesocerebrum (MC) (Figure 9), together with the PC lobe by fluorescent voltage imaging, and they analyzed the relationship between them using the correlation analysis [13]. In this study, di-4-ANEPPS was also used as the voltage-sensitive dye. Figure 11 shows the fluorescent optical recording of spontaneous activity of the cerebral ganglion of
The spatiotemporal neural activities in the PC lobe were also measured by fluorescent calcium imaging using calcium-sensitive dyes. Inoue et al. used Oregon Green-1, rhod-2, or Ca Orange bonded with AM group as the calcium-sensitive dye, and compared the fluorescent calcium imaging using each of them with the florescent voltage imaging using di-4-ANEPPS [11]. In fluorescent calcium imaging, propagation waves of fluorescence change were also observed in the PC lobe of
Fluorescent calcium imaging has been limited with respect to the selectivity of staining of the dyes. Bath application of the dyes stains both B neurons and NB neurons. Even when we intend to observe the activity of NB neurons, which is thought to be involved in odor coding [33], it is obscured by stronger calcium signals from B neurons that exhibit periodic bursting activity. To observe the activity of NB neurons, Watanabe et al. attempted selective fluorescent calcium imaging for the PC lobe of
As described above, fluorescent-imaging technique is applicable for the measurement of the spatiotemporal neural activities involved in the olfactory processing of the land slug. Through the studies using it, especially, the function and computational roles of the propagation waves of neural activity in the PC lobe on odor discrimination and learning have been elucidated. Meanwhile, the experimental apparatus has also been improved. In the earlier reports of the fluorescent voltage imaging, consecutive images were acquired at a flame rate of about 100 ms for about 100 × 100 pixels in size [6, 7], while they can be acquired at a flame rate of about 10 ms for about 1000 × 1000 pixels in size in our recent apparatus as described in Section 5.
5. Our recent fluorescent-imaging apparatus and its application
We have also measured the spatiotemporal neural activities in the PC lobe of the land slug
Figure 17 shows the propagating waves of fluorescence change in the PC lobe of
We also examined the involvement of nitric oxide (NO) in the odor-induced changes in wave propagation by our apparatus. NO, which is a gaseous neurotransmitter, has been reported to function specifically in the PC lobe. Gelperin et al. showed that the excitability of B neurons is modulated by NO [34] and suppression of NO synthesis blocks the LFP oscillation and wave propagation in the PC lobe [35]. In our study [36], the stimulation of another aversive odor, hexanol, to the epithelium of the superior tentacle increased NO concentration measured by an NO electrode applied to the PC lobe of
6. Conclusions
Fluorescent voltage imaging is applicable for the measurement of the spatiotemporal neural activities involved in the olfactory processing of the land slug
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