for investigating neurons
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
|■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.
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.
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
Fig. 1 The silkmoth brain and its neuron.
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.
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
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.
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).
■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.
(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.
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
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
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
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.
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
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
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.
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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