We thank A. Oba for her technical assistance. This research was partially supported by Grant-in-Aid for Scientific Research (C) 17K01982 to K.Y.

All experimental procedures were approved by the RIKEN committee on the care and use of animals in experiments. Mice were kept in the animal facility of the RIKEN Center for Brain Science under well-controlled temperature (23&#8211;25 &#176;C) and humidity (45%&#8211;65%) conditions. Both male and female WT mice (C57BL/6, 3&#8211;6 months) were used.
1. Preparation of Solutions Used in the Experiments
NOTE: All solutions should be made in ultrapure wa...

<ol>
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Differences among the four protocols
In LTD-inducing protocols 1 and 2, Cjs 300 times at 1 Hz is sufficient to induce cerebellar LTD. Stimulation frequency of the CF seemed to be in a physiological range, because the complex spike firing rate in alert adult mice (P60) was reported to be 1.25 Hz36. However, the CF stimulation alone did not cause long-term plasticity in the PF-CF synapse, as used in protocols 1 and 2 (Figure 4, <strong class="x...

The synaptic organization of the elaborated neuronal networks of the cerebellar cortex, composed of PCs, molecular layer interneurons (basket and stellate cells), Golgi cells, PFs from granule cells, mossy fibers and climbing fibers (CFs), have been elucidated in terms of excitation/inhibition and divergence/convergence, and the well-organized circuitry diagram has suggested that the cerebellum is a &#8220;neuronal machine&#8221;1, though there was previously no idea about purpose of this &#8220;machine&#8221;. Later Marr proposed that the PFs input to PCs constitute a triple layer associative learning network2. He also suggested that each CF conveys a cerebral instruction for elemental movement2. He assumed that simultaneous activation of PFs and CF would enhance PF-PC synapse activity, and cause long-term potentiation (LTP) of the PF-PC synapse. On the other hand, Albus assumed that synchronous activation of PFs and CF resulted in LTD at the PF-PC synapses3. Both the above studies interpret the cerebellum as a unique memory device, the incorporation of which into the cerebellar cortical network leads to the formation of the Marr&#8211;Albus model learning machine model.
Following these theoretical predictions, two lines of evidence suggest the presence of synaptic plasticity in the cerebellum. The first line of evidence was suggested by the anatomical organization of the flocculus; here MF pathways of vestibular organ origin and CF pathways of retinal origin converge on the PCs4. This unique convergence pattern suggests that a synaptic plasticity occurring in the flocculus causes the remarkable adaptability of the vestibulo-ocular reflex. Second, the recording of the PCs response in the flocculus and the lesioning of the flocculus also supported the above hypothesis5,6,7. Furthermore, the PC discharge pattern during adaptation of a monkey&#8217;s hand movement8 supported the synaptic plasticity hypothesis, especially Albus&#8217;s LTD-hypothesis3.
To determine the nature of the synaptic plasticity directly, repeated conjunctive stimulation (Cjs) of a bundle of PFs and the CF that specifically innervates the PC in vivo was shown to induce LTD for the transmission efficacy of the PF&#8211;PC synapses9,10,11. In the subsequent in vitro explorations using a cerebellar slice12 and cultured PCs, conjunction of co-cultured granule cell stimulation and olive cell stimulation13 or conjunction of iontophoretically applied glutamate and somatic depolarization14,15 caused LTD. The signal transduction mechanism underlying the LTD-induction was also intensively investigated using in vitro preparations16,17.
Adaptations of the VOR and the OKR were often used for quantitative evaluation of gene-manipulation effects on cerebellar motor learning, because the vestibule-cerebellar cortex was proven to be the essential origin in the adaptive learning of the VOR18,19,20 and the OKR19,21 The correlation between failure of LTD-induction and impairment of behavioral motor learning has been taken as evidence that LTD plays an essential role in motor learning mechanisms22. These views are collectively referred to as the LTD hypothesis of motor learning, or Marr-Albus-Ito hypothesis23,24,25,26.
Adaptive learning of eye movement was measured using similar protocols, while various experimental conditions were used to induce LTD in slice preparation27,28,29,30,31. Recently, Schonewille et al.26 reported that some gene-manipulated mice demonstrated normal motor learning, but the cerebellar slices did not show LTD, and thereby concluded that LTD was not essential for motor learning. However, the induction of LTD was only attempted using one type of protocol at room temperature. Hence, we used several types of LTD-inducing protocols under recording conditions at around 30 &#176;C, and we confirmed that the LTD was reliably induced in the gene-manipulated mice by using these protocols at near physiological temperatures32.
However, there remain some questions regarding the basic properties of conjunctive stimulation. The first is the relationship between the complex spike&#8217;s shape and the amplitude of LTD. Second, in conjunction with PF-stimulation and somatic depolarization, whether the number of stimuli used were necessary or not was elusive. In the present study, these questions were investigated using wild type (WT) mice.

<table>
	<tbody>
		<tr>
			<td>Amplifier</td>
			<td>Molecular Devices-Axon</td>
			<td>Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillary</td>
			<td>Sutter</td>
			<td>BF150-110-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitizer</td>
			<td>Molecular Devices-Axon</td>
			<td>Digidata1322A</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode puller</td>
			<td>Sutter</td>
			<td>Model P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>26675-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolator</td>
			<td>A.M.P.I.</td>
			<td>ISOflex</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear slicer</td>
			<td>Dosaka EM</td>
			<td>PRO7N</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>NIKON</td>
			<td>Eclipse E600FN</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson</td>
			<td>MP1 Single Channel Pump</td>
			<td></td>
		</tr>
		<tr>
			<td>Picrotoxin</td>
			<td>Sigma-Aldrich</td>
			<td>P1675</td>
			<td></td>
		</tr>
		<tr>
			<td>Pure water maker</td>
			<td>Merck-Millipore</td>
			<td>MilliQ 7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for experiment</td>
			<td>Molecular probe-Axon</td>
			<td>pClamp 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for statistics</td>
			<td>KyensLab</td>
			<td>KyPlot 5.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
			<td>WPI</td>
			<td>DS8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Warner</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Tocris</td>
			<td>1078</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of long-term depression induction in adult cerebellar slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

Four protocols were used in this study to induce cerebellar LTD. In the first two protocols (protocol 1 and 2), the conjunction of the PF-stimulation and the CF-stimulation was applied under current-clamp conditions. In the other two protocols (protocol 3 and 4), somatic depolarization was substituted for the CF-stimulation under voltage-clamp conditions. Voltage-traces or current-traces during conjunctive stimulation were compared (Figure 2).
Conjunction of 1 PF-...

Watch this Scientific Journal Video about Assessment of Long-term Depression Induction in Adult Cerebellar Slices at JoVE.com

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

When a particular protein function is modified by gene manipulation, coordination of irritative failure and motor running impairment is considered a necessary condition for causal relationship between LTD and motor learning. Here we demonstrate the use of multiple protocols to assess LTD which can be induced via compensatory mechanisms in gene-manipulated animals. Before harvesting the brain, chill and oxygenate two 50 milliliter beakers of ACSF on ice.When the solution temperature reaches below four degrees celsius, add 50 microliters of one millimolar tetrodotoxin to one of the ice cold beakers. To harvest the brain, hold the head and use ophthalmological scissors to cut the superficial skin along the midline. Manually retract the skin to widely expose the skull surface and use the scissors to cut along the skull horizontally from the major spinocerebellar hole to just above the eyes and ears before cutting the skull along a line above both eyes.Use a scalpel to cut the brain at the middle of the cerebrum and isolate the caudal part of the brain, including the cerebellum, from the skull. Immerse the sample in the ice cold beaker of ACSF and adjust the bubble tubing so that it does not stir the brain block in the beaker. After at least seven minutes, use a spatula to pick up the brain block and use a piece of filter paper to absorb any excess ACSF.Mount the tissue ventral side down onto a two by two centimeter piece of agar with an appropriate medical adhesive. Use a blade to cut out the right hemisphere of the brain tissue as parallel to the dendritic plane of the Purkinje cells as possible. Cut and remove the other side of the hemisphere and cut the brain between the superior and inferior colliculi.Cut off the spinal cord and glue the right side of the trimmed cerebellum with the agar block onto the pre-chilled specimen tray. Then, tilting the specimen tray, pour ACSF onto the sample to fix the tissue and to wash away any excess glue. To slice the brain sample, orient the specimen such that the dorsal side of the cerebellum is at the front and pour enough of the ice cold cutting ACSF supplemented with tetrodotoxin to completely immerse the cerebellum.Place a gas tube into the cutting solution and initiate bubbling with an oxygen-carbon dioxide gas mixture. Using fine tweezers and magnifiers, remove the arachnoid mater and excise the cerebellar peduncle. After removing the brainstem and agar block, rotate the tray 180 degrees so that the dorsal surface of the cerebellum faces the razor blade of the vibratome and adjust the first cutting location.Set the vibratome amplitude to 5.5 and frequency to 85 Hertz, the speed to three to four, and the slice thickness to 300 micrometers. As the cerebellar slices are obtained, use a nylon net to transfer the sections to an acrylic incubator in a 26 degree celsius water bath and completely immerse the samples in fresh oxygenated ACSF for at least one hour. For whole cell patch clamp reporting, dissolve one micromolar picrotoxin in ACSF by ultasonication for three minutes before perfusing a 30 degree celsius recording chamber with the toxin solution at a two milliliters per minute flow rate.After a few minutes, transfer a cerebellar slice to the recording chamber and fix the tissue with a platinum weight and nylon threads. Then fill a stimulating electrode with fresh ACSF. To stimulate the parallel fibers, place the stimulating electrode on the surface of the molecular layer about 50 micrometers from the Purkinje cell layer.For stimulation of the climbing fibers, place the stimulating electrode at the bottom of the Purkinje cell layer and use a microloader to fill a recording electrode with eight microliters of 0.45 micrometer filtered potassium or cesium based internal solution. Apply a weak positive pressure to the recording electrode before immersing the electrode into the ACSF. The electrode resistance should be two to four megaohms and the liquid junctional potential should be corrected.Approach the healthy bright cell body of a Purkinje cell with the recording electrode and push the surface of the Purkinje cell slightly. Then stop applying the positive pressure and apply negative pressure until a gigaohm seal is formed. Then use negative pressure to establish a whole cell configuration, maintaining the membrane potential at minus 70 millivolts and applying a minus two millivolt 100 millisecond pulses at 0.1 hertz, continuous monitoring of the input resistance, series resistance, and input capacitance.For long term depression induction, stimulate the molecular layer with a 0.1 millisecond pulse and apply a double pulse stimulus to identify the parallel fiber excitatory post-synaptic currents. A paired pulse facilitation and gradual increase in amplitude relative to the increase in stimulation intensity should be observed. To record the test response, apply a single 0.1 hertz pulse and adjust the intensity of the stimulus so that the evoked amplitude is around 200 picoamps.Stimulate the climbing fibers at the bottom of the Purkinje cell layer and to identify the parallel fiber excitatory post-synaptic currents elicited by the climbing fiber activation, apply a double pulse stimulus. A paired pulse depression should be observed in an all or none manner in correlation with the increase in stimulation intensity. In this representative experiment, a conjunction of one parallel fiber stimulation and one climbing fiber stimulation under current clamp conditions were used for the slice preparation.The shape of the complex spike elicited by the conjunctive stimulation was similar to that elicited by the climbing fiber stimulation alone with the first steep spikelet followed by two to three spikelets. A similarly shaped complex spike was observed when one parallel fiber stimulation was followed 50 milliseconds later by a conjunctive second parallel and climbing fiber stimulation. In this test under voltage clamp conditions using a cesium-based internal solution, a parallel fiber stimulation was followed 50 milliseconds later by a concomitant application of a second parallel fiber stimulation and somatic depolarization.An inward current was elicited upon somatic depolarization from minus 70 to zero millivolts and the tail current was also evoked after repolarization. Finally, five parallel fiber stimuli at 100 hertz were given simultaneously with the somatic depolarization under voltage clamp conditions. Again, a repetitive generation of inward currents was elicited during depolarization and the tail current was induced after the repolarization.To assess the relationship between cerebellar LTD and motor learning in gene-manipulated animals, multiple protocols should be used to induce LTD under cozy physiological conditions. If cerebellar LTD is visualized in gene-manipulated animals after motor learning, the causal relationship between them can be more directly examined.

assessment-long-term-depression-induction-adult-cerebellar

Research

JoVE Journal

Neuroscience

6.8K ビュー.  RIKEN. 特定のタンパク質機能が遺伝子操作によって変わる場合、刺激性障害と運動走行障害の調整は、LTDと運動学習との因果関係に必要な条件と考えられる。ここでは、遺伝子操作された動物の補償機構を介して誘導することができるLTDを評価するための複数のプロトコルの使用を実証する。脳を収穫する前に、氷の上にACSFの2つの50ミリリットルビーカーを冷やして酸素化す?...

ビデオ: Assessment of Long-term Depression Induction in Adult Cerebellar Slices

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are...

a modified roller tube method for the long-term culture of rodent brain slices

This video demonstrates a modified roller tube method for the long-term culturing of brain slices from a rodent model. First, the brain slices are attached to coverslips. Then, flat-sided roller tubes with a hole on the flat side are taken, and the coverslips are positioned on the hole using adhesive discs. Finally, a nutrient medium and a carbon dioxide and air mixture are added to the tube, and the tubes are incubated with a rolling...

A Modified Roller Tube Method for the Long-Term Culture of Rodent Brain Slices

isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat...

improved preparation and preservation of hippocampal mouse slices for a very stable and reproducible recording of long-term potentiation

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of...

With the swiveling deburring tool, smooth the edges of the hole and make four grooves on the inside edge of the hole to facilitate draining of the hole during rotation. Next, rinse the tubes with 70% ethanol, and then air-dry them in a biological safety cabinet. Sterilize the tubes in some 12-millimeter punched adhesive silicone rubber disks for 40 minutes under the UV...

modified roller tube method for precisely localized and repetitive intermittent imaging during long-term culture of brain slices in an enclosed system

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are...

long-term sensory conflict in freely behaving mice

Long-term Sensory Conflict in Freely Behaving Mice

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home...

ex vivo culture of chick cerebellar slices and spatially targeted electroporation of granule cell precursors

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

long-term continuous electroencephalography monitoring in a rodent pup

The video demonstrates long-term continuous electroencephalography (EEG) monitoring in rodent pups using a wireless transmitter system. The transmitter contains two recording electrodes and one reference electrode extending to the brain&#39;s surface. Upon placing the pup in a recording chamber, EEG can be continuously monitored throughout the pup&#39;s development into...

Long-Term Continuous Electroencephalography Monitoring in a Rodent Pup

preparation of human myocardial tissue for long-term cultivation

Preparation of Human Myocardial Tissue for Long-Term Cultivation

We present a protocol for ex vivo cultivation of human ventricular myocardial tissue. It allows for detailed analysis of contraction force and kinetics, as well as the application of pre- and afterload to mimic the in vivo physiological environment more closely.

a molecular readout of long-term olfactory adaptation in c. elegans

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

long term chronic pseudomonas aeruginosa airway infection in mice

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

serial enrichment of spermatogonial stem and progenitor cells (sscs) in culture for derivation of long-term adult mouse ssc lines

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here.  The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

minimizing hypoxia in hippocampal slices from adult and aging mice

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and...

long-term in vivo tracking of inflammatory cell dynamics within drosophila pupae

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene...

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

induction of myocardial infarction in adult zebrafish using cryoinjury

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian...

a model of long-term ventricular fibrillation in isolated rat hearts

A Model of Long-Term Ventricular Fibrillation in Isolated Rat Hearts

This protocol presents a model of long-term ventricular fibrillation in rat hearts induced by continuous stimulation with low-voltage alternating current. This model has a high success rate, is stable, reliable, and reproducible, has a low impact on cardiac function, and causes only mild myocardial...

measurement of oxygen consumption rate in acute striatal slices from adult mice

Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice

Oxygen consumption rate (OCR) is a common proxy for mitochondrial function and can be used to study different disease models. We developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice that is more physiologically relevant than other...

isolation method for long-term and short-term hematopoietic stem cells

Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells

We present a step-by-step protocol for the isolation of long-term hematopoietic stem cells (LT-HSCs) and short-term HSCs (ST-HSCs) using the Hoxb5 reporter...

long-term continuous measurement of renal blood flow in conscious rats

Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats

The present protocol describes a long-term continuous measurement of renal blood flow in conscious rats and simultaneously recording blood pressure with implanted catheters (fluid-filled or by telemetry).

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-&#946; (A&#946;) peptide.

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...