A PHS Grant (MH51393) supported this work. Some of the Aplysia we use are provided by the National Resource for Aplysia of the University of Miami under Grant RR10294 from the National Center for Research Resources, NIH.

1. Preparation
<ol>
 <li>Anesthetize the animal by injecting 75-100 ml isotonic magnesium chloride solution. The Aplysia we use for imaging are generally 150-200 grams and are obtained from Marinus Scientific. </li>
 <li>Pin the anesthetized animal to a wax covered dish. Syringe needles work well for this purpose; sterile techniques are not necessary. Using gross forceps and standard scissors make an incision in the animal's foot and expose the buccal mass. Locate the buccal ganglion. Using spring scissors and fine forceps, carefully free the ganglion by cutting all buccal nerves. </li>
 <li>Remove the ganglion and place it in a Sylgard coated dish containing artificial seawater. Place a few insect pins through the buccal nerves to stabilize the ganglion. Desheath the buccal ganglion using ultrafine forceps and spring scissors, so as to expose the neurons for intracellular recording. Then re-pin the ganglion using about 15 fine insect (minutien) pins. The ganglion must be held firmly in place as any movement will be problematic during imaging. </li>
</ol>
2. Prepare Electrodes
<ol>
 <li>Pull electrodes using glass capillary tubing with filament (e.g. WPI TW100F-4) and a puller. Settings should be individually determined to create electrodes of the required resistance. When filled with 3 M KAc (potassium acetate) our electrode resistance is generally about 10 MOhms if the electrodes are to be beveled, or about 5 MOhms if the beveling step is omitted. </li>
 <li>Fill one set of electrodes with a solution containing 3 M KAc/30 mM KCl using a syringe and microfil needle. These electrodes will be used for electrophysiology. </li>
 <li>Fill a second set of electrodes with the calcium indicator dye by dipping the back end of the electrode in dye, previously reconstituted with approx. 40 &mu;l distilled water for 500&mu;g dye. When the tip of the electrode has filled, back fill about 1/4 inch of the end of the electrode with 200 mM KCl to ensure good electrical contact. </li>
 <li>It is beneficial to bevel the electrodes, as this facilitates dye injection and reduces the damage done by multiple membrane penetrations. We use a custom beveler that generates a stream of a salt water / alumina powder suspension. The electrode is mounted in a holder and its tip enters the stream at a 45&deg; angle. Electrode resistance is continuously monitored and the beveling terminated when the resistance reaches about 5 MOhm for KAc filled electrodes. The dye filled electrodes are beveled for an equal amount of time and typically have resistances of 15-20 MOhm. </li>
</ol>
3. Loading the Calcium Indicator Dye
<ol>
 <li>Place the preparation on an electrophysiology rig and visually locate the presynaptic neuron of interest. Many of our experiments are done with an identified sensory neuron utilized during feeding, B21 5, 6. The relatively fixed position, size (~100 &mu;m), and elongated shape of B21's soma make it easy to locate.</li>
 <li>Impale the neuron of interest with an electrode containing the calcium indicator dye. B21's identity can be verified by impaling the post-synaptic neuron B8. Depolarization of B21 with ~8 nA current will trigger spikes and result in facilitating PSPs in B8 7. </li>
 <li>Inject dye in the presynaptic neuron of interest by passing hyperpolarizing pulses (typically -15 nA, 1 Hz, 75% duty cycle) for approximately 30 minutes. If dye loading is successful, the neuron's soma will acquire a faint pink color. Remove the dye-containing electrode by slowly withdrawing it from the neuron. </li>
 <li>Leave preparations for approximately 30 minutes to allow the dye to diffuse into the fine processes that make synaptic contact with follower neurons. </li>
</ol>
4. Calcium Imaging and Electrophysiological Recording 
<ol>
 <li>Place the preparation on the imaging microscope. The dish with the preparation is held on a cooling platform with pieces of Permagum sealing cord which, unlike clay, remains sticky when wet. The fixed stage microscope focuses by moving the objective. The stage remains stationary, which allows attaching magnet mounted manipulators. As the high magnification makes vibrations (especially lateral movement) a problem, the entire imaging setup should be placed on a vibration isolation platform. </li>
 <li>Impale presynaptic and postsynaptic neurons with electrodes containing normal electrolyte solution. </li>
 <li>Select a filter block suitable to image Calcium Orange (excitation 549 nm, emission 576 nm) 8 and adjust the camera. Most scientific CCD cameras allow the investigator to choose a subset of CCD cells that are read out. Additionally, cells can be combined (binned). Selecting a smaller subset increases speed, while binning increases sensitivity. These two settings, together with the exposure time, determine the frame rate i.e. how many images can be acquired per second. Suitable settings will image an area of 580 x 350 &mu;m2 with 500 x 300 pixels (using a 10x lens and a 0.5x camera adapter). Add neutral density filters to the light path to adjust the illumination intensity to the minimal amount that provides correct exposure with an exposure time &lt;25 ms. These settings should result in a frame rate of about 30 Hz - which in Aplysia is fast enough to image single spike evoked calcium alterations. Finally, close the field aperture so that only the imaged area is illuminated. </li>
 <li>In the imaging software mark the neuron's regions you want to measure. In the Elements AR software this is done in the "Time Measurement" section by placing regions of interest (ROIs). B21 is a bipolar neuron and previous work has demonstrated that its lateral process contacts the postsynaptic neuron of interest 9. We generally image primary, secondary, and tertiary branches of the lateral process by placing a ROI over each part of the neuron. An additional ROI next to the dye filled neuron is measured to provide a background value that is later subtracted from all other data points.</li>
 <li>Instruct the electrophysiology acquisition (in these experiments Spike II) to wait for a trigger signal provided by the "frame readout" signal of the camera. Start the image acquisition (Elements AR); Spike II will now automatically start recording when the camera takes its first frame. You can also use a TTL output of the Power 1401 to briefly turn on a LED mounted in the microscope's camera port. This will provide a signal to subsequently synchronize imaging and electrophysiological data. </li>
 <li>Stimulate the presynaptic neuron by injecting a series of brief depolarizing current pulses at the desired frequency. It is important to monitor each pulse (e.g. with a stimulus triggered oscilloscope) to determine whether an action potential is successfully generated. While Calcium Orange is very photo-stable compared to other dyes, it is a good idea to record a control trial without stimulation to check how much dye bleaching occurs. </li>
 <li>In experiments in which intracellular calcium concentrations are manipulated, a drug such as EGTA is introduced into the presynaptic neuron and the stimulation and data acquisition steps described above are repeated. </li>
</ol>
5. Analysis of Representative Data
<ol>
 <li>Export the imaging data (the list of measured intensity values for each ROI) by writing it into a text file. A custom script in Spike II can then import the text file. This script also normalizes the values with the individual probe's area, and subtracts the value of the background probe. The data points are then plotted alongside the electrophysiology data as a "virtual RealWave" channel. The "channel process / time shift function" in Spike II is used to precisely align the imaging data points, using the LED signal as reference. </li>
 <li>To obtain values as percent change, analyze the imaging data by calculating the relative change in fluorescence as dF/Fo= (F-Fo)/ Fo. Fo denotes the fluorescence level just before the stimulus and F the fluorescence during the stimulus. </li>
</ol>
6. Representative Results
<img src="/files/ftp_upload/3907/3907fig1.jpg" alt="Figure 1" /> 
Figure 1. Overall scheme of the experiment. Experiments are conducted in a ganglion preparation of Aplysia. Changes in intracellular calcium are imaged with a fluorescent dye, which is iontophoretically introduced into the presynaptic neuron. Pre- and postsynaptic neurons are then impaled with sharp electrodes so that synaptic transmission can be induced and monitored.
<img src="/files/ftp_upload/3907/3907fig2.jpg" alt="Figure 2" /> 
Figure 2. Calcium imaging and intracellular recording from identified neurons in the buccal ganglion of Aplysia. (A) Photo of the imaged lateral branch of neuron B21. The box indicates the measured region of interest. (B1) Intracellular stimulation of B21 evokes action potentials (bottom trace) and facilitating postsynaptic potentials (PSPs) in the postsynaptic follower neuron B8 (middle trace) causing it to also fire action potentials. Increases in presynaptic calcium fluorescence are shown in the top trace. (B2) Effect of presynaptic EGTA on homosynaptic facilitation. EGTA was intracellularly injected into B21 ~ 15 minutes before intracellular stimulation. It is thought that EGTA is a slow acting calcium chelator, which at low concentrations is not fast enough to suppress synaptic transmission. Note the reduction in the widespread calcium signal and the corresponding decrease in PSP amplitude. (C) The PSP amplitude correlates with the presynaptic calcium signal. <a href="https://www.jove.com/files/ftp_upload/3907/3907fig2large.jpg" target="_blank"> Click here to view larger figure</a>.

<ol>
	<li>Zucker, R. S., Regehr, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+synaptic+plasticity.">Short-term synaptic plasticity.</a> Annu. Rev. Physiol. 64, 355-405 (2002).</li><li>Eberhard, M., Erne, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+binding+to+fluorescent+calcium+indicators:+Calcium+green,+calcium+orange+and+calcium+crimson.">Calcium binding to fluorescent calcium indicators: Calcium green, calcium orange and calcium crimson.</a> Biochem. Biophysical Res. Comm. 180, 209-215 (1991).</li><li>Escobar, A. L., Velez, P., Kim, A. M., Cifuentes, F., Fill, M., Vergata, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+properties+of+DM-nitrophen+and+calcium+indicators:+rapid+transient+response+to+flash+photolysis.">Kinetic properties of DM-nitrophen and calcium indicators: rapid transient response to flash photolysis.</a> Eur. J. Physiol. 434, 615-631 (1997).</li><li>Ivanov, A. I., Calabrese, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+spike-mediated+synaptic+transmission+by+presynaptic+background+Ca2++in+leech+heart+interneurons.">Modulation of spike-mediated synaptic transmission by presynaptic background Ca2+ in leech heart interneurons.</a> J. Neurosci. 23, 1206-1218 (2003).</li><li>Rosen, S. C., Miller, M. W., Evans, C. G., Cropper, E. C., Kupfermann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+synaptic+connections+between+peptidergic+radula+mechanoafferent+neurons+and+neurons+in+the+feeding+system+of+Aplysia.">Diverse synaptic connections between peptidergic radula mechanoafferent neurons and neurons in the feeding system of Aplysia.</a> J. Neurophysiol. 83, 1605-1620 (2000).</li><li>Ludwar, B. C. h., Evans, C. G., Jing, J., Cropper, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+mechanisms+mediate+potentiating+effects+of+depolarization+on+synaptic+transmission.">Two distinct mechanisms mediate potentiating effects of depolarization on synaptic transmission.</a> J. Neurophysiol. 102, 1976-1983 (2009).</li><li>Evans, C. G., Ludwar, B. C. h., Askansas, J., Cropper, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+holding+potential+on+the+dynamics+of+homosynaptic+facilitation.">Effect of holding potential on the dynamics of homosynaptic facilitation.</a> J. Neurosci. 31, 11039-11043 (2011).</li><li>Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Handbook. , (2005).</li><li>Borovikov, D., Evans, C. G., Jing, J., Rosen, S. C., Cropper, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+proprioceptive+role+for+an+exteroceptive+mechanoafferent+neuron+in+Aplysia.">A proprioceptive role for an exteroceptive mechanoafferent neuron in Aplysia.</a> J. Neurosci. 20, 1990-2002 (2000).</li><li>Goldberg, J. H., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+38:+A+practical+guide:+Two-photon+calcium+imaging+of+spines+and+dendrites.">Chapter 38: A practical guide: Two-photon calcium imaging of spines and dendrites.</a> Imaging in Neuroscience and Development. , (2005).</li></ol>

We have nothing to disclose.

We demonstrate techniques that can be used to simultaneously monitor the intracellular calcium concentration and evaluate the efficacy of synaptic transmission. These techniques are useful for determining how various forms of short-term plasticity are mediated. 
The imaging is carried out with a fluorescence microscope and CCD camera. These equipment requirements are relatively modest when compared to most functional imaging set-ups. The technique is simple and easy to learn. While imaging wit...

<table border="1">
<tbody>
 <tr>
 <td>Reagent Name</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>Calcium Orange</td>
 <td>Invitrogen</td>
 <td>C-3013</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td>Sigma</td>
 <td>E-4378</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Calcium calibration buffer kit</td>
 <td>Invitrogen </td>
 <td>C-3008MP</td>
 <td>useful for testing the sensitivity and dynamic range of the 			signal</td>
 </tr>
 <tr>
 <td>Magnesium chloride hexahydrate</td>
 <td>Sigma </td>
 <td>M0250</td>
 <td>used in 0.33 M solution to anesthetize animal</td>
 </tr>
 </tbody>
</table>
Table 1. Reagents used.
<table border="1">
<tbody>
 <tr>
 <td>Equipment Name</td>
 <td>Company</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>FN-1 upright fluorescence microscope</td>
 <td>Nikon Instruments</td>
 <td>with Narishige ITS-FN1 stage</td>
 </tr>
 <tr>
 <td>NMN-21 manipulators</td>
 <td>Narishige</td>
 <td>mounted on stage with magnets</td>
 </tr>
 <tr>
 <td>CoolSNAP HQ2 CCD camera</td>
 <td>Photometrics</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NIS elements AR 
	(version 3.22)</td>
 <td>Nikon Instruments</td>
 <td>imaging software used to acquire fluorescence data</td>
 </tr>
 <tr>
 <td>10X/0.3w Plan Fluor objective</td>
 <td>Nikon Instruments</td>
 <td>this water immersion lens has a very long working distance of 			3.5 mm</td>
 </tr>
 <tr>
 <td>X-Cite 120 PC metal halide lamp</td>
 <td>EXFO</td>
 <td>used for fluorescence imaging</td>
 </tr>
 <tr>
 <td>LS-DWL 
	halogen lamp</td>
 <td>Sumica</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ET-CY3 filter set</td>
 <td>Chroma Technology</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Power 1401 A/D converter</td>
 <td>Cambridge Electronic Design </td>
 <td>sampling was done at 3 kHz</td>
 </tr>
 <tr>
 <td>Spike II 
	(version 7.07)</td>
 <td>Cambridge Electronic Design</td>
 <td>software used to acquire electrophysiology data</td>
 </tr>
 <tr>
 <td>SEC-10 LX amplifier</td>
 <td>NPI electronics</td>
 <td>used with a 10X headstage</td>
 </tr>
 <tr>
 <td>Model 410 amplifier</td>
 <td>Brownlee precision</td>
 <td>used to amplify and filter the signal</td>
 </tr>
 <tr>
 <td>WS-4</td>
 <td>minus k Technology</td>
 <td>vibration isolation for imaging</td>
 </tr>
 <tr>
 <td>cooling platform</td>
 <td>custom made</td>
 <td>brass plate through which ice water is pumped at a variable 			rate</td>
 </tr>
 </tbody>
</table>
Table 2. Equipment used.

monitoring changes in the intracellular calcium concentration and synaptic efficacy in the mollusc aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Watch this Scientific Journal Video about Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia at JoVE.com

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

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10.9K Views.  Mt. Sinai School of Medicine. We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

Video: Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.