Name: a plate-based assay for the measurement of endogenous monoamine release in acute brain slices
Uploaded: 2021-08-11T13:00:00
Description: Watch this Scientific Journal Video about A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices at JoVE.com

This work was supported by grants Fondecyt Initiation Fund N 11191049 to J.A.P. and NIH grant DA038598 to G.E.T.

All experiments, including animal handling and tissue collection, were carried out in accordance with the University of Florida and the City College of New York Institutional Animal Care and Use Committee (IACUC), following the approved protocol 201508873 (UF) and 1071 (CCNY). For reagents and buffer please refer to the Supplementary File.
1. Prepare acute rat brain slices
NOTE: In this experiment adult male rats (250-350 g) were used. However, this s...

Monoamine release measurements have been performed for years in a number of systems such as heterologous cells, neuronal cultures, brain synaptosomes, ex vivo acute brain slices, and whole animals13,20,41,42,58,64,65,66,67...

A plethora of neurological and psychiatric diseases are associated with dysregulation or insufficient maintenance of monoamine neurotransmitter (dopamine [DA], serotonin [5-HT], norepinephrine [NE]) homeostasis1,2,3. These conditions include, but are not limited to, depression1,2, schizophrenia2, anxiety2, addiction4, menopause5,6,7, pain8, and Parkinson&#39;s disease3. For instance, several rat models of menopause have shown that the dysregulation or reduction of monoamines within the hippocampus, prefrontal cortex, and striatum may be associated with both depression and cognitive decline, which is seen in women experiencing menopause. The dysregulation of monoamines in these models have been extensively examined using HPLC-ECD, although the studies did not discriminate between measured neurotransmitter content versus neurotransmitter release5,6,7. Monoamines are classically released into the extracellular space through Ca2+-dependent vesicular release9, and are recycled back through their respective plasma membrane re-uptake system (dopamine transporter, DAT; serotonin transporter, SERT; norepinephrine transporter, NET)10,11. Conversely, data suggests that these transporters are able to release or efflux monoamines, since drugs of abuse such as amphetamine (AMPH) and 3,4-Methylenedioxymethamphetamine (MDMA) are known to release DA and 5-HT, respectively through their transporter systems12,13,14,15,16,17. Thus, a proper mechanistic understanding of monoamine release dynamics is crucial for developing specific and targeted pharmacotherapies.
A wide range of techniques have been employed to study monoamine release such as Fast Scan Cyclic Voltammetry (FSCV)18, in vivo&#160;microdialysis13, imaging19, preincubation with radiolabeled monoamines20, optogenetics, and more recently, genetically encoded fluorescent sensors and photometry21,22. FSCV and in vivo microdialysis are the primary techniques used for studying monoamine release. FSCV is used to study the stimulated exocytotic release of, primarily, DA in acute brain slices and in vivo23. Because FSCV uses electrodes to stimulate or evoke release, the primary source of neurotransmitter release is Ca2+-dependent vesicular release18,24,25,26,27,28,29,30,31. In vivo&#160;microdialysis coupled with HPLC measures changes in extracellular neurotransmitter levels using a probe placed in a brain area of interest13,32. Similar to FSCV, a major limitation to in vivo&#160;microdialysis is the difficulty in determining the source of neurotransmitter release: Ca2+ dependent vesicular release or transporter dependent. Noteworthy, both methods allow for the direct measurement of monoamine release. Through the recent advancement of optogenetics, research demonstrates detection of 5-HT and DA release in a short timespan with exquisite cell-type specificity21,22. However, these strategies require complex and costly techniques and equipment, and indirectly measure monoamine release, specifically through monoamine binding to receptors. Further, radiolabeled monoamines are also used for studying monoamine dynamics. Radiolabeled monoamines may be preloaded into various model systems such as heterologous cells overexpressing each monoamine transporter20,33,34,35,36,37,38,39,40, primary neurons20, synaptosomes33,39,41,42, and acute brain slices43,44. However, radioactivity poses potential harm to the experimenter, and the tritium-labeled analytes may not faithfully recapitulate endogenous monoamine dynamics45,46. Superfusion systems combined with off-line detection methods such as HPLC-ECD have allowed for the detection of monoamines from multiple tissue sources. Here, this protocol provides as an optimized and low-cost, simple, and precise method using acute brain slices to directly measure endogenous basal and stimulated monoamine release.
Acute brain slices allow for testing mechanistic hypotheses, primarily as they preserve the in vivo anatomical microenvironment, and maintain intact synapses47,48,49,50,51,52. In a few studies, acute brain slices or chopped brain tissue have been used in conjunction with a superfusion technique using KCl to stimulate Ca2+ mediated release53,54,55,56. Superfusion systems have been critical to advance the field&#39;s understanding of neurotransmitter release mechanisms, including monoamines. However, these systems are relatively expensive, and the number of chambers available for tissue analysis ranges from 4-12. In comparison, the method presented here is inexpensive, allows the measurement of 48 tissue samples, and may be refined to use up to 96 tissue samples. Each well within the 48-well plate contains tissue holders that use filters to separate the released product from the tissue, and released monoamines are then collected and analyzed by HPLC-ECD. Importantly, this method allows for the simultaneous measurement of 5-HT, DA, and NE release from different brain areas such as the prefrontal cortex, the hippocampus, and the dorsal striatum after treatment with pharmacological agents that modulate monoamine release. Thus, the experimenter can answer multiple questions using an inexpensive multi-well system that increases the number of samples tested and thereby reducing the number of animals used.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>48 Well plate</td>
			<td>NA</td>
			<td>NA</td>
			<td>Assay</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fischer Scientific</td>
			<td>A998-1</td>
			<td>Mobile Phase</td>
		</tr>
		<tr>
			<td>Calcium Chloride Ahydrous</td>
			<td>Sigma Aldrich</td>
			<td>C1016</td>
			<td>Modified Artifical Cerebrospinal Fluid OR Efflux Buffer</td>
		</tr>
		<tr>
			<td>Clarity Software</td>
			<td>Anetc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>Mobile Phase</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>1002608421</td>
			<td>Dissection Buffer</td>
		</tr>
		<tr>
			<td>DMF</td>
			<td>Sigma Aldrich</td>
			<td>D4551</td>
			<td>MTT Assay</td>
		</tr>
		<tr>
			<td>EDTA-Na2</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>Mobile Phase</td>
		</tr>
		<tr>
			<td>GraphPad Software</td>
			<td>Graphpad Software, Inc</td>
			<td></td>
			<td>Statistical Analysis</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G5516</td>
			<td>Lysis buffer</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td>Lysis buffer</td>
		</tr>
		<tr>
			<td>HPLC, Decade Amperometric</td>
			<td>Anetc</td>
			<td></td>
			<td>HPLC, LC-EC system</td>
		</tr>
		<tr>
			<td>HPLC</td>
			<td>Amuza</td>
			<td></td>
			<td>HPLC HTEC-510.</td>
		</tr>
		<tr>
			<td>L-Asrobic Acid</td>
			<td>Sigma Aldrich</td>
			<td>A5960</td>
			<td>Dissection Buffer</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma</td>
			<td>7487-88-9</td>
			<td>KH Buffer</td>
		</tr>
		<tr>
			<td>Microcentrifuge Filter Units UltraFree</td>
			<td>Millipore</td>
			<td>C7554</td>
			<td>Assay - 6 to fit in 48 well plate</td>
		</tr>
		<tr>
			<td>MTT</td>
			<td>Thermo Fisher</td>
			<td>M6494</td>
			<td>MTT Assay</td>
		</tr>
		<tr>
			<td>Nanosep</td>
			<td>VWR</td>
			<td>29300-606</td>
			<td>Assay; protein assay</td>
		</tr>
		<tr>
			<td>Octanesulfonic acid</td>
			<td>Sigma Aldrich</td>
			<td>V800010</td>
			<td>Mobile Phase</td>
		</tr>
		<tr>
			<td>Pargyline Clorohydrate</td>
			<td>Sigma Aldrich</td>
			<td>P8013</td>
			<td>Modified Artifical Cerebrospinal Fluid OR Efflux Buffer</td>
		</tr>
		<tr>
			<td>Phosphoric Acid</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>Mobile Phase</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>12636</td>
			<td>KH Buffer</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Sigma</td>
			<td>1001655559</td>
			<td>KH Buffer</td>
		</tr>
		<tr>
			<td>Precisonary VF-21-0Z</td>
			<td>Precissonary</td>
			<td></td>
			<td>Compresstome</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Sigma Aldrich</td>
			<td>P2714</td>
			<td>Lysis buffer.</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>Dissection Buffer</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td>Dissection Buffer</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td>KH Buffer</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Sigma Aldrich</td>
			<td>L3771</td>
			<td>Lysis buffer</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T8787</td>
			<td>MTT Assay / Lysis buffer</td>
		</tr>
	</tbody>
</table>

a plate-based assay for the measurement of endogenous monoamine release in acute brain slices

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in monoamine neurotransmitter release and uptake dynamics. Technically complex methods such as electrophysiology, Fast Scan Cyclic Voltammetry (FSCV), imaging, in vivo&#160;microdialysis, optogenetics, or use of radioactivity are required to study monoamine function. The method presented here is an optimized two-step approach for detecting monoamine release in acute brain slices using a 48-well plate containing tissue holders for examining monoamine release, and high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) for monoamine release measurement. Briefly, rat brain sections containing regions of interest, including prefrontal cortex, hippocampus, and dorsal striatum were obtained using a tissue slicer or vibratome. These regions of interest were dissected from the whole brain and incubated in an oxygenated physiological buffer. Viability was examined throughout the experimental time course, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The acutely dissected brain regions were incubated in varying drug conditions that are known to induce monoamine release through the transporter (amphetamine) or through the activation of exocytotic vesicular release (KCl). After incubation, the released products in the supernatant were collected and analyzed through an HPLC-ECD system. Here, basal monoamine release is detected by HPLC from acute brain slices. This data supports previous in vivo and in vitro results showing that AMPH and KCl induce monoamine release. This method is particularly useful for studying mechanisms associated with monoamine transporter-dependent release and provides an opportunity to screen compounds affecting monoamine release in a rapid and low-cost manner.

This technique describes the use of brain slices to measure the release of endogenous monoamines using HPLC with electrochemical detection based in a 48-well plate with an internal tissue holder. Experimental set up is depicted in Figure 1 and Figure 2. Initially, to ensure tissue viability by the end of the experimentation, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay was performed. Af...

Watch this Scientific Journal Video about A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices at JoVE.com

A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices

This method introduces a simple technique for the detection of endogenous monoamine release using acute brain slices. The setup uses a 48-well plate containing a tissue holder for monoamine release. Released monoamine is analyzed by HPLC coupled with electrochemical detection. Additionally, this technique provides a screening method for drug discovery.

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in ...

This technique is an adaptation of commonly used methods for examining monoamine release, allowing the measurement of endogenous release rather than preloaded radiolabeled monoamines under multiple pharmacological conditions at one time. The main advantages to this technique are that it is fairly cost-effective, multiple pharmacological conditions may be examined at once, and we can study endogenous monoamine release with the ability to distinguish between vesicular and transporter mediated release. To prepare brain slices from adult 250 to 350 gram male rats after harvest, immediately placed the brain and ice cold oxygenated dissection buffer, and acquire 300 micron coronal brain sections from each region of interest.For further dissection of the brain slices, carefully move the tissues to glass slides and use a rat brain atlas to identify the dorsal striatum based on its dark striated structure, and to identify the hippocampus based on its proximity to the cortex and its unique spiral structure. Separate the right and left hemispheres for their use as control and experimental slices. The dorsal striatum can be further dissected into two millimeter punches.Then use a plastic transfer pipette with a modified tip to transfer the samples into small containers immersed in oxygenated ice cold dissection buffer with oxygen bubbling. To induce monoamine release, transfer the tissue samples to each well of a custom made 48 well efflux chamber containing 0.5 to one milliliter of efflux buffer per well with constant gentle bubbling and allow the samples to recover for 30 to 50 minutes at 37 degrees Celsius. At the end of the equilibration period, move the tissue holder with brain tissue to wells containing 500 microliters of oxygenated efflux buffer with or without pharmacological agent, tapping the holder on the edge of the well to prevent the minimal transfer of buffer between wells for a 20 minute incubation at 37 degrees Celsius.At the end of the incubation, move the holder to a new set of wells containing 500 microliters of efflux buffer with or without the drug of interest and return the plate to the cell culture incubator for an additional 20 minutes. During the second incubation, transfer the solution from the wells from the first incubation into microcentrifuge tubes containing 50 microliters of one normal perchloric or phosphoric acid on ice, and label the tubes number one"At the end of the second incubation, move the tissue holder to empty wells with the plate on ice and transfer the supernatants from the second incubation to new microcentrifuge tubes containing 50 microliters of one normal perchloric or phosphoric acid on ice. Label these tubes number two"When all of the supernatants have been transferred, add one milliliter of ice cold dissection buffer to each well of tissue and use small tweezers to transfer each tissue sample to a new microcentrifuge tube.Remove the buffer from the sample tubes and store the tissues at minus 80 degrees Celsius, then transfer the collected incubation solutions into microcentrifuge filter tubes for centrifugation and place the filtrate on ice until HPLC analysis. Set the potential of the ECD and flow rate to the manufacturer's recommendation for detecting monoamines. Load and appropriate aliquot of each sample, including neuro-transmitter standards into the HPLC for auto injection and detection and allow the instrument to complete the analysis.Then use the HPLC analysis software to acquire and analyze the chromatograph data using a standard curve composed of each monoamine to obtain the area under the curve based on the manufacturer's guidelines. After six hours of functional analysis, tissue samples remain viable, while samples incubated with 1%Triton X-100 demonstrate a significant reduction in MTT transformation. Acute 20 minute treatment of hippocampal and prefrontal cortex brain sections with amphetamine induces a significant increase in the extracellular level of each monoamine.When the samples are pretreated with fluoxetine, a selective serotonin re-uptake inhibitor, the amphetamine induced increase in extracellular serotonin within the hippocampus and the prefrontal cortex does not occur, while the increase in extracellular dopamine and norepinephrine production is not affected. In addition, acute 20 minute treatment of dorsal striatum punches with amphetamine induces a 35 fold increase in extracellular dopamine level expression, while dorsal striatum punch treatment with cocaine, a monoamine transporter blocker, results in a significant inhibition of amphetamine induced extracellular dopamine. Increasing the concentration of extracellular potassium chloride to evoke membrane depolarization is sufficient to induce the exocytotic release of monoamines compared to untreated control conditions, and neither fluoxetine nor cocaine treatment blocks this deep polarization induced increase.It is imperative to have clean cut sections, a precise localization of punches, and to maintain consistent oxygenation throughout the experiment. The brain sections may be used post experimentation for further biochemical processing and analysis, such as western blotting and histology.

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Research

JoVE Journal

Neuroscience

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 ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

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 ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

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.

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 ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.