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Methods for investigating neurons

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Insect nervous systems consist of relatively small numbers of neurons compared to vertebrates. This feature is an advantage for investigating nervous systems from the cellular level to the level of the entire nervous system . Electrophysiological recordings, genetic engineering manipulations, and voltage- and calcium-imaging techniques for investigating neuronal structures and functions are all well-established. Besides, synthetic approaches such as neuronal network simulation and the evaluation of nervous system performance using robotics, are now available. These techniques and approaches help us to comprehensively understand structures and functions of all neurons in individuals. In this section we describe several methods for investigating structures and functions of insect neurons.



■Intracellular recording and staining (a single neuron) techniques

■calcium- and voltage- imaging techniques

■genetic engineering techniques

■engineering method



■Intracellular recording and staining techniques for single neurons

Intracellular recording with a sharp electrode is used to detect the action potentials and small graded potential changes in the local membrane caused by synaptic events in a single neuron. In general, a signal is detected by a microelectrode (sharp electrode), which is connected to an amplifier, an oscilloscope, and a computer. The oscilloscope presents a visual display of the membrane potential over time. The signal is also sent to a computer that records data and graphically represents the results of an experiment online. A set up of intracellular recording includes equipment to hold the microelectrode and correctly position it to record from a neuron of interest. A microscope is used to ensure proper placement of the electrode for in vitro and in vivo recording of insect neurons. A micromanipulator is a device that allows fine movements, preferrably in the X, Y, and Z axes, permitting precise positioning of the microelectrode in the tissue. In this subsection, we briefly describe the application of intracellular recordings to an insect, especially silkmoth.

In an insect brain, cell bodies of neurons are located on the brain surface, forming clusters. Isolated brains can be used for the intracellular recordings. The isolated brain is immersed in saline dropped onto a slide glass where it is immobilized. The slide glass is mounted on the microscope. We usually use a differential interference contrast (DIC) microscope with long working distance objective lens. Using the DIC microscope, each of the cell cluster can be easily visualized (Fig. 1B, C). The image of visualized cell cluster is fed into a video monitor through a coupled charged device (CCD) camera (C2741-79, Hamamatsu Photonics, Japan) so that we can penetrate a glass microelectrode into a cell body using a micromanipulator (MHW-3, Narishige ) while monitoring the cell image on the monitor (Fig. 1B, C). The glass electrode used has 10~100 MW resistance.

A neuron can be stained by iontophoresis of a fluorescent dye through a glass microelectrode. For example, Lucifer Yellow CH or Alexa Fluor 568 (neurobiotin is not fluorescent and describing the detection reaction only complicates things) may be used. A number of neurons in a brain can be simultaneously stained using the same  dyes. Besides, using  immunohistochemical staining for a specific neurotransmitter in the same sample, a candidate neurotransmitter of the neuron can be identified. Using a dye-loaded glass microelectrode, the penetration of the electrode into a neuron can be judged by measuring the membrane potential of the neuron and detecting the injected dye excited by epifluorescencence illumination (Fig. 1B.C). These methods are suitable for identifying neuronal networks.

Using these methods, we can accumulate data from a large number of neurons in the silkmoth brain and develop a database. Information in the database allows us to analyze and understand the neural basis of the pheromone source orientation behavior of the silkmoth.




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Fig. 1 The silkmoth brain and its neuron.

(A)Frontal view of the isolated silkmoth brain. (B, C) Method for visualizing a neuron. Each cell body can be visualized by illumination with near-infrared light using an IR-DIC microscope. The arrow indicates a glass microelectrode filled with Lucifer Yellow. The arrowhead indicates a cell body (B ). Penetrating a cell body with the glass microelectrode , injecting fluorescent dye through the electrode, and illumination with blue light allow for visualizing a neuron(C). (D) A single neuron stained with Lucifer Yellow. (E) The sample shown in (D) was afterwards stained immunohistochemically. In this sample, an anti-GABA antibody was used that was additionally detected by a red-fluorescent antibody. Candidate GABAergic neurons are displayed in magenta. Scale: 1 mm in (A), 100mm in (B-E).




■calcium- and voltage- imaging techniques

We addressed above a method of measuring single neuron activities. Intracellular recording has good temporal and spatial resolution. However, intracellular recording can record only at one point of the neuron and thus recording neural activity at multiple sites in a single neuron is not possible. In addition, in order to record multiple neurons several electrodes and amplifiers are needed and at most a few neurons can be recorded simultaneously by this method. The methods for visualizing neural activities below are alternatives to record multiple neurons and/or multiple sites in a single neuron simultaneously. The temporal resolution is typically on the order of milliseconds to seconds, depending on the specific technique. These methods can be used to visualize activity in dissociated cells in culture, tissue slices , and even intact brains.

Visualizing neural activity depends on specialized fluorescent probes that report changes in calcium concentration, or membrane potential. These probes can be organic dyes that are introduced into a neural system prior to performing an experiment or they can be genetically encoded fluorescent proteins stably expressed in transgenic animals. Dyes tend to exhibit better temporal properties and signal-to-noise characteristics than proteins. However, genetically encoded proteins can be targeted to specific cell types in the brain, allowing for the observation of activity in genetically defined neurons.

Calcium-imaging

Intracellular calcium is central to many physiological processes, including neurotransmitter release, ion channel gating, and second messenger pathways. In neurons, calcium dynamics link electrical activity and biochemical events. Thus, changes in calcium concentration can indirectly indicate changes in electrical activity. Fluorescent calcium indicators are dyes, such as Fluo-4 and Fura-2, and fluorescent proteins, such as aequorin and variants of GFP. Data from calcium imaging experiments typically show changes in fluorescence intensity or the ratio of fluorescence intensity at different wavelengths over time, normalized on the initial level of fluorescence. Calcium indicator dyes can be categorized as ratiometric or nonratiometric dyes. Ratiometric dyes are excited by or emit at slightly different wavelength when they are free of Ca2+ compared to when they are bound to Ca2+. Thus they can report changes in Ca2+ through changes in the ratio of their fluorescence intensity at distinct wavelengths. Ratiometric dyes allow investigators to correct for background changes in fluorescence unrelated to calium dynamics, such as artifacts related to photobleaching, variations in illumination intensity, or differences in dye concentration. However, data acquision and measurements are more complicated than with nonratiometric dyes. Nonratiometric dyes report changes in Ca2+ directly though changes in fluorescence intensity. The common nonratiometric indicators Fluo-4 and Calcium Green-1 exhibit predictable increases in fluorescence intensity with increases in calcium concentration. While the direct relationship between fluorecesnce intensity and calcium concentration is sensitive for detecting changes due to calcium binding, the measurement is prone to detecting changes based on dye concentration and experiment-specific conditions. However, nonratiometric dyes tend to be easier to use.Genetically encoded calcium sensors take advantage of the conformational changes that occur in certain endogenous calcium-binding proteins when they bind to calcium. G-CaMP is an example for a nonratiometric probe reporting through direct changes in fluorescence intensity caused by calcium-sensitive changes in the structure of the fluorophore. We use Calcium Green-1 for measuring neuronal activities in antennal lobe, first olfactory center, of the silkmoth.

Voltage- imaging

Techniques that visualize changes in membrane potential are the closest analogs to electrophysiological recordings, as they report voltage changes in neuronal membranes with high temporal resolution. Voltage-sensitive dye imaging (VSDI) is currently the primary method by which scientists visualize changes in transmembrane voltage spatio-temporally. Voltage-sensitive dyes shift their absorption or emission fluorescence based on the membrane potential, allowing a scientist to gauge the global electrical state of a neuron. Unlike with extracellular electrophysiological techniques, it is possible to detect subthreshold synaptic potentials in addition to spiking activity. These dyes also allow simultaneous activity measurements in large populations. Most dyes exhibit small signal changes; the fractional intensity change is in the range of 10-4 to 10-3. Thus, noise has to be minimized to detect a reliable signal. Furthermore, activity-dependent changes in the intrinsic optical absorption and reflection properties of the brain itself can interfere with voltage-sensitive dye measurements. We succeeded in measuringspatio-temporal activity in the silkmoth antennal lobe in response to electrical stimulation of the antennal nerve (Fig.XX; Hill et al., 2003). This activity is augmented by application of serotonin (5-HT), thus it reveals that sensitivity of the pheromone response is modulated in the silkmoth central nervous system (Fig. 19; Hill et al., 2003).




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■Genetic engineering techniques

Due to recent advances in large-scale DNA sequencing techniques, genome sequences up to the entire genetic information of organisms have been determined in many species. The silkmoth genome project has almost been completed and the silkmoth genome sequence has been comprehensively elucidated (Mita et al., 2004; Qiangyou et al., 2004). There are several methods to visualize neurons in a region of interest in the nervous system, which are based on the genomic information mentioned above; In situ hybridization method for detecting mRNAs in tissue of interest, immunohistochemical detection methods for specific proteins based on cloned gene products, and genetic engineering methods utilizing transgenic techniques, in particular the use of information on specific promoters and the expression of reporter genes.

In situ hybridization (ISH) is used to visualize the localization of nucleic acids containing specific sequences. The method is usually used for detecting mRNA, permitting a scientist determine when and where a specific gene is expressed in the nervous system. This technique is suitable for identifying which neurons express a gene, although it does not reveal where the functional protein product is localized within the cell. Briefly, a complementary sequence, that would bind to an mRNA sequence of interest by hybridization, i.e. formation of a double-stranded product held together by hydrogen bonds (such as in the DNA), is tagged with some means for visualization, for example a fluorescent compound.

Immunohistochemistry (IHC) is used to visualize the presence of proteins and other biomolecules. This technique depends on antibodies to recognize and bind to specific epitopes (antigens) on proteins or other molecules. For the production of antibodies, the purified antigen is necessary and a good way to obtain purified proteins or peptides in quantity is to clone the gene sequence in question and to have the antigen or a modified protein bearing a wanted epitope produced by an expression system. The antigen can then by purified in large quantities sufficient for immunization of the animals used to produce the antibodies.

In situ hybridization and immunohistochemical methods are only applicable post-mortem because processes such as enzymatic reactions and diffusion barriers that are present in living organisms interfere with these procedures. On the contrary, genetic engineering methods can be used for visualization in living tissues in vivo. Furthermore, these methods can be used to manipulate neural activity of interest.

A reporter gene is a nonendogenous gene encoding an enzyme or fluorescent protein whose expression is controlled by a promoter for a separate (endogenous) gene of interest. Therefore, it is possible to examine the spatial and temporal expression of the gene by measuring the expression of the reporter.

In addition to visualizing neural activity of interest using genetic engineering meth a scientist can also stimulate or inhibit neural activity using genetic engineering methods in combination with optical methods. There are many advantages in using the genetic engineering methods in combination with optical methods of manipulation over electrophysiological or magnetic methods: the genetic engineering methods can be targeted to specific cell types within the neural tissue, large populations of neurons can be simultaneously controlled, and off-target effects from neighboring brain regions are easier to avoid. Both electrical microstimulation and optical stimulation of light-sensitive ion channels, such as channelrhodopsin-2 (ChR2) allow investigators to control neural activity at short time scales. However, optical stimulation has distinct advantages over electrical microstimulation. Stimulation of neurons using light eliminates artifacts that can be caused by electrical stimulation. Also, because light-activated channels are genetically encoded, investigators have the possibility to selectively target specific cell types, enhancing the ability to distinguish the contribution of activity from particular populations of neurons. For example, ChR2 and halorhodopsin (NpHR) allow fast neuronal activation and inactivation, respectively. ChR2 is a cation channel that allows sodium ions to enter the cell upon illumination by ~470 nm blue light, while NpRH is a chloride pump that activates upon illumination by ~580 nm yellow light. Both constructs rapidly activate upon illumination with the appropriate wavelength of light, and rapidly turn off without proper illumination.

The fruit fly Drosophila melanogaster is the insect that is mostly used to analyze the nervous system using the genetic engineering methods because of the long tradition of genetic manipulation techniques in this fly species. Tamura et al. (2000) developed methods for producing transgenic silkmoths, which allow scientists to apply a large grid of genetic engineering methods to the silkmoth nervous system. In the following paragraphs, we briefly describe the examples of how to apply the genetic engineering methods to the silkmoth nervous system.

The process of creating a transgenic silkmoth takes advantage of transposons which are mobile genetic elements that move by way of transition between chromosomes. It usually encodes a specific enzyme called a transposase which acts on a specific DNA sequence at each end of the transposon, causing it to be inserted into a new target DNA site. The production of a transgenic silkmoth is established using the transposon piggyBac (Tamura et al., 2000). The first step in producing a transgenic silkmoth is to use recombinant DNA technology to make a transgenic construct. This construct must contain (1) the DNA sequence encoding the transgene and (2) the necessary promoter sequences for the expression of the gene (Fig. 7). Simply inserting a gene into the genome will not cause it to be expressed: endogenous cellular machinery must recognize a promoter sequence in order to transcribe the DNA into mRNA. Promoters control the expression of genes so that they are expressed in specific cells and tissues at specific time. Next, a plasmid containing a DNA construct is inserted into piggyBac. Then the transgenic construct is injected into a very young silkmoth embryo along with a separate plasmid containing the gene encoding the transposase. If this technique is performed correctly, the injected gene enters the germ line as the result of a transposition event. As reporter genes, we use green fluorescent protein (GFP) or red fluorecent protein (DsRed) to visualize neurons of interest because GFP and DsRed can be detected in living silkmoths. Fig. ? shows an example in which artificial promoter, 3xP3 and DaRed as the reporter gene are used to visualize sensory neurons in a silkmoth embryo and the larval ocelli. These reporters are distributed homogeneously in the cytoplasm so that the neurons expressing the reporters can be visualized. Neuronal activity of interest can be visualized for exampe by using G-Camp as a reporter gene. Because the same transgenic lineage is genetically homogenous the same identified neurons can be labeled once making the transgenic lineage





Fig.7 Schematic diagram of reporter gene expression systems in transgenic silkmots.

Schematic diagram of reporter gene expression systems in transgenic silkmots. (A) In the promoter-reporter system, promoter sequence is placed immediately upsteam or reporter gene and directly activates reporter gene expression. (B) In the GAL4/UAS system, reporter gene expression is activated via the GAL4/UAS yeast transcriptional system.




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Specific regions of interest in neurons , such as synaptic terminals can be visualized in fruit fly using fluorescent protein provided with an intracellular signal molecule (sequence?). This technique will be applied in the silkmoth in the near future.

The simplest method of making a transgenic animal is to use a single DNA construct, as just desicribed, in which a promoter directly regulates the expression of a transgene. An alternative approach is a binary expression system that uses multiple constructs to further refine the spatial and temporal expression of a transgene. This is typically done by creating two different lines of transgenic animals, each line expressing one of the required constructs, and then mating the animals to produce offspring that express both constructs. These flexible expression systems have numerous advantages. The main advantage is that they can provide control over the timing and location of transgene expression. Another primary advantage is the ability to mix and match combinations of transgenes, saving time and effort in making new transgenic constructs.

The Gal4/UAS system is a binary expression system primarily used in Drosophila, though it has been applied to silkmoth (Imamura et al., 2003). In one construct, a cell-specific promoter drives the expression of the gene encoding Gal4, a transcription factor normally expressed in yeast. In a second construct, a transgene of interest is regulated by a promoter sequence called an upstream activation sequence (UAS). The Gal4 protein binds to the UAS sequence and leads to high expression in cells defined by the promoter regulating Gal4 (Fig.7 B). Imamura et al., (2003) reported that the Gal4/UAS system normally functions in the body tissue (Imamura et al., 2003). In order to verify that the Gal4/UAS system can normally function in the brain tissue of the silkmoth, we made two kinds of transgenic silkmoth, each of which has different neuropeptide promoter sequences on Gal4 upstream region and observed that endocrine cells producing the neuropeptides were labeled by GFP in the F1 that was obtained by crossing the two kinds of transgenic silkmoths with a UAS-GFP line (Yamagata et al., 2008) . This results shows that the Gal4/UAS system is useful to visualize silkmoth brain neurons of interest.

In order to investigate the spatio-temporal pattern of pheromone input from antenna to brain, we made two transgenic silkmoth strains each of which expresses Gal4 under the control of promoters for bombykol receptors, detecting the major component of silkmoth sex pheromone, and the other one for bombykal receptors, detecting the minor pheromone component. Besides, we made an UAS-GFP line separately. Then we obtained the F1 from crosses of each of the GAL4 lines and the UAS-GFP line to confirm that information of bombykol and bombykal are transmitted to different regions in the antennal lobe, the first olfactory center in the brain (Sakurai et al., unpublished). In addition, we succeed in calcium imaging of antennal lobe input activity in response to pheromone stimulation using the F1s of the above Gal4 lines and a UAS-GCaMP line.

Now that genetic engineering is available in the silkmoth, we can hope to understand the structures and functions of neurons related to the pheromone information processing in the silkmoth. We aim to make UAS systems expressing genes to induce cell death or to inhibit neural transmission by toxic gene products. Using these UAS systems, we can infer the functional role of neuronal circuits of interest in pheromone orientation behavior at individual level and even change the function of individual neurons. For example, changing the neuronal characteristics at genetic level by introducing ion channels and/or ligand-gated channels into neurons would allow us to change the neuronal circuit underling the pheromone orientation behavior and even to altersilkmoth behavior artificially in well-controlled ways.




Reference

Carter M, Shieh J. (2010) Guide to Research Techniques in Neuroscience. Academic Press, san Diegeo

Hill, E.S., Okada, K., Kanzaki, R. (2003) Visualization of modulatory effects of serotonin in the silkmoth antennal lobe. J. Exp. Biol., 206: 345-352.

Imamura M, Nakai J, Inoue S, Quan GX, Kanda T, Tamura T.(2003) Targeted gene expression using the GAL4/UAS system in the silkworm Bombyx mori, Genetics, 165 : 1329-1340.

Mita K, Kasahara M, Sasaki S, Nagayasu Y, Yamada T, Kanamori H, Namiki N, Kitagawa M, Yamashita H, Yasukochi Y, Kadono-Okuda K, Yamamoto K, Ajimura M, Ravikumar G, Shimomura M, Nagamura Y, Shin-I T, Abe H, Shimada T, Morishita S, Sasaki T. (2004) The genome sequence of silkworm, Bombyx mori. DNA Res., 11:27-35.

Qiangyou X., et al. (2004) A draft sequence for the genome of the domesticated silkworm (Bombyx mori). Science, 306: 1937-1940.

Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G, Shirk P, Fraser M, Prudhomme JC, Couble P. (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector, Nat. Biotechnol., 18: 81?84.

Yamagata T, Sakurai T, Uchino K, Sezutsu H, Tamura T and Kanzaki R (2008) GFP Labeling of Neurosecretory Cells with the GAL4/UAS System in the Silkmoth Brain Enables Selective Intracellular Staining of Neurons. Zool Sci., 25: 509-516

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