Name: slice it hot: acute adult brain slicing in physiological temperature
Uploaded: 2014-10-30T14:00:00
Description: Watch this Scientific Journal Video about Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature at JoVE.com

We would like to acknowledge the significant contribution Dr. Shiwei Huang (Australian National University) in validating the method. Furthermore, we would like to thank Ms. Kasia Pietrajtis for helpful comments regarding Golgi cells and Mr. Vitaly Lerner for the cortex experimental data. This work was supported by PITN-GA-2009-238686 (CEREBNET), FP7-ICT (REALNET), ELSC and ISF.

All experimental procedures described in this protocol were approved by the Hebrew University&#39;s Animal Care and Use Committee.
1. Preparing the Solutions and Tools for Slicing
<ol>
	<li>Prepare 1 L of standard physiological solution (SPS) containing the ions described in Table 1.
	<ol>
		<li>Prepare a stock solution containing the salts at 10 times the final concentration in advance in a storage glass bottle filled with 1,000 ml of deionized water (conductance of 0.055 &...

<ol>
	<li>Skrede, K., Westgaard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transverse+hippocampal+slice:+a+well-defined+cortical+structure+maintained+in+vitro.">The transverse hippocampal slice: a well-defined cortical structure maintained in vitro.</a> Brain Research. 35, 589-593 (1971).</li><li>Aghajanian, G., Rasmussen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+studies+in+the+facial+nucleus+illustrating+a+simple+new+method+for+obtaining+viable+motoneurons+in+adult+rat+brain+slices.">Intracellular studies in the facial nucleus illustrating a simple new method for obtaining viable motoneurons in adult rat brain slices.</a> Synapse. 3, 331-338 (1989).</li><li>Gueritaud, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+activity+of+rat+ocular+motoneurons+recorded+in+vitro.">Electrical activity of rat ocular motoneurons recorded in vitro.</a> Neuroscience. 24, 837-852 (1988).</li><li>Lipton, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+the+best+of+brain+slices:+comparing+preparative+methods.">Making the best of brain slices: comparing preparative methods.</a> Journal of Neuroscience Methods. 59, 151-156 (1995).</li><li>Richerson, G., Messer, 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+composition+of+experimental+solutions+on+neuronal+survival+during+rat+brain+slicing.">Effect of composition of experimental solutions on neuronal survival during rat brain slicing.</a> Experimental Neurology. 131, 133-143 (1995).</li><li>Brahma, B., Forman, R., Stewart, E., Nicholson, C., Rice, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascorbate+inhibits+edema+in+brain+slices.">Ascorbate inhibits edema in brain slices.</a> Journal of Neurochemistry. 74, 1263-1270 (2000).</li><li>Moyer, J. R., Brown, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch-clamp+techniques+applied+to+brain+slices.+In:+Patch-clamp+analysis:+advanced+techniques.">Patch-clamp techniques applied to brain slices. In: Patch-clamp analysis: advanced techniques.</a> Springer. , 135-193 (2002).</li><li>Ye, J. H., Zhang, J., Xiao, C., Kong, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch-clamp+studies+in+the+CNS+illustrate+a+simple+new+method+for+obtaining+viable+neurons+in+rat+brain+slices:+glycerol+replacement+of+NaCl+protects+CNS+neurons.">Patch-clamp studies in the CNS illustrate a simple new method for obtaining viable neurons in rat brain slices: glycerol replacement of NaCl protects CNS neurons.</a> J. Neuroscience Methods. 156, 251-259 (2006).</li><li>Bischofberger, J., Engel, D., Li, L., Geiger, J., Jonas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch-clamp+recording+from+mossy+fiber+terminals+in+hippocampal+slices.">Patch-clamp recording from mossy fiber terminals in hippocampal slices.</a> Nature Protocols. 1, 2075-2081 (2006).</li><li>Zhao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type&#8211;specific+channelrhodopsin-2+transgenic+mice+for+optogenetic+dissection+of+neural+circuitry+function.">Cell type&#8211;specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function.</a> Nature Methods. 8, 745-752 (2011).</li><li>Huang, S., Uusisaari, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+temperature+during+brain+slicing+enhances+the+quality+of+acute+slice+preparations.">Physiological temperature during brain slicing enhances the quality of acute slice preparations.</a> Front. Cell. Neurosci. 7, (2013).</li><li>Ohe, G. C., Darian-Smith, C., Garner, C. C., Heller, H. 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> The Journal of Neuroscience. 26 (41), 10590-10598 (2006).</li><li>Voets, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+principle+of+temperature-dependent+gating+in+cold-+and+heat-sensitive+TRP+channels.">The principle of temperature-dependent gating in cold- and heat-sensitive TRP channels.</a> Nature. 430, 748-754 (2004).</li><li>Coulter, D. A., Huguenard, J. R., Prince, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+currents+in+rat+thalamocortical+relay+neurones:+kinetic+properties+of+the+transient,+low-threshold+current.">Calcium currents in rat thalamocortical relay neurones: kinetic properties of the transient, low-threshold current.</a> The Journal of Physiology. 414, 587-604 (1998).</li><li>Gibb, A. J., Edward, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+recording+from+cells+in+slice+tissues.">Patch clamp recording from cells in slice tissues.</a> Microelectrode Techniques: the Plymouth workshop handbook. , (1994).</li><li>Lefler, Y., Yarom, Y., Uusisaari, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+Inhibitory+Input+to+the+Inferior+Olive+Decreases+Electrical+Coupling+and+Blocks+Subthreshold+Oscillations.">Cerebellar Inhibitory Input to the Inferior Olive Decreases Electrical Coupling and Blocks Subthreshold Oscillations.</a> Neuron. 81 (6), 1389-1400 (2014).</li><li>Bourne, J. N., Kirov, S. A., Sorra, K. E., Harris, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Warmer+preparation+of+hippocampal+slices+prevents+synapse+proliferation+that+might+obscure+LTP-related+structural+plasticity.">Warmer preparation of hippocampal slices prevents synapse proliferation that might obscure LTP-related structural plasticity.</a> Neuropharmacology. 52, 55-59 (2007).</li><li>Kirov, S. A., Sorra, K. E., Harris, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slices+have+more+synapses+than+perfusion-fixed+hippocampus+from+both+young+and+mature+rats.">Slices have more synapses than perfusion-fixed hippocampus from both young and mature rats.</a> J. Neurosci. 19, 2876-2886 (1999).</li><li>Kirov, S. A., Petrak, L. J., Fiala, J. C., Harris, K. 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We demonstrate a method for preparing acute brain slices from mice in physiological instead of ice-cold temperature.
It has been shown11 that the quality of slices obtained in warm conditions is superior when compared with those prepared with cold conditions, provided that the slicer blade has minimal vertical vibration. Slicing in physiological temperature may prevent physiological artifacts caused by the low temperature, such as those related to changes in metabolic processes...

The aim of the presented method is to get high-quality acute brain slices for in vitro electrophysiological experiments, especially when using adult or even old animals.
The acute brain slicing method, as described by Skrede and Westgaard1 in two elegant sentences, has become one of the foundations of modern neuroscience research and is employed in innumerable variations worldwide. The quality of the slices is reflected in number of living neurons per slice, the period of time during which the cells keep their electrophysiological and morphological properties as well as in the integrity of the tissue. Moreover, the maximal duration for stable recordings depends on the quality of the slices. Thus, along the decades, the original slicing method has been further developed by individual research groups to enhance slice recovery after cutting2-10, often by complex modifications of the composition of cutting or recovery solutions (such as adding ascorbate, thiourea or even H2O2) as well as intra-cardiac pre-perfusion of the animal with cooled physiological solutions.
As has been recently shown11, physiological temperature during slicing seems to be more beneficial than cooling to neuronal health; the improvement is most striking when working with adult (2-8 month) rodents. Avoiding dramatic temperature changes prevents artifacts due to temperature-dependent processes in the cells, such as plasticity13 and ion-channels kinetics13,14. Such changes could influence membrane voltage and intracellular calcium signaling, spike threshold, and spike shape.
The &#8220;hot&#8221; acute slice preparation method presented here is a general procedure for obtaining high-quality acute brain slices from any brain region, including the cerebellum, the cortex and hippocampus, brainstem nuclei16 as well as the olfactory bulb, both in rats and mice.
Notably, the physiological temperature slicing procedure requires that the cutting blade vibrates nearly perfectly horizontally and is without any structural defects. Such precision might not be attainable with older slicer models; in such cases, we recommend performing the slice preparation in freezing-cold conditions as the low temperature seems to make the tissue more resistant to mechanical damage, even if at the cost of metabolic aberrations.

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			<td height="26" style="height:26px;width:140px;">Name</td>
			<td style="width:73px;">Company</td>
			<td style="width:69px;">Catalog #</td>
			<td style="width:292px;">Comments</td>
		</tr>
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			<td height="26" style="height:26px;width:140px;">Pentobarbital</td>
			<td style="width:73px;">CTS</td>
			<td style="width:69px;">170066</td>
			<td style="width:292px;">Concentration: 60 mg / ml&#160; in physiological saline.</td>
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			<td height="40" style="height:41px;width:140px;">Big scissors&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">14001-16</td>
			<td style="width:292px;">Any large scissors or a guillotine with sufficiently sharp edges can be used for decapitation&#160;</td>
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			<td height="40" style="height:41px;width:140px;">Iris scissors&#160;</td>
			<td style="width:73px;">Prestige medical</td>
			<td style="width:69px;">48,148</td>
			<td style="width:292px;">Any fine tip scissors can be used, provided the scissor blades are not longer than 1.5 - 2 cm</td>
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			<td height="26" style="height:27px;width:140px;">Fine tip forceps&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">11254-20</td>
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			<td height="26" style="height:27px;width:140px;">Scalpel&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">91003-12</td>
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			<td height="26" style="height:27px;width:140px;">Scalpel blade #11</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">10011-00</td>
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			<td height="26" style="height:27px;width:140px;">Small spatula&#160;</td>
			<td style="width:73px;">Fisher&#160;</td>
			<td style="width:69px;">2350</td>
			<td style="width:292px;"></td>
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			<td height="26" style="height:27px;width:140px;">Filter paper</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">Any laboratory brand can be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Petri dishes</td>
			<td style="width:73px;">Duroplan</td>
			<td style="width:69px;">Z231509-1</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Glass beakers&#160;</td>
			<td style="width:73px;">SCHOT</td>
			<td style="width:69px;">10022846</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Pasteur pipette&#160;</td>
			<td style="width:73px;">Maple Leaf Brand</td>
			<td style="width:69px;">14672-029</td>
			<td style="width:292px;"></td>
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			<td height="40" style="height:41px;width:140px;">Super glue&#160;</td>
			<td style="width:73px;">LOCTITE</td>
			<td style="width:69px;">4091361/1</td>
			<td style="width:292px;"></td>
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		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Slicer</td>
			<td style="width:73px;">Campden</td>
			<td style="width:69px;">7000-smz</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Ceramic slicing blade</td>
			<td style="width:73px;">Campden</td>
			<td style="width:69px;">7550-1-C</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Magnetic heater/stirrer</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For heating up the SPS for the procedure</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Electric kettle</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For heating up water for temperature control</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:56px;width:140px;">Slice recovery chamber + heating unit</td>
			<td style="width:73px;">Warner instruments</td>
			<td style="width:69px;">&#160;BSC-HT +&#160; BSC-BUW</td>
			<td style="width:292px;">Home-built models may also be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Thermometer</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For monitoring SPS temperature during dissection and slicing</td>
		</tr>
	</tbody>
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slice it hot: acute adult brain slicing in physiological temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

Slices prepared in the described manner can be used for various electrophysiological and optogenetic experiments. In Figure 3A and 3C, we show a representative example of a horizontal cerebellar slice and a coronal cerebral cortical slice, respectively, viewed under differential interference (DIC) optics. In the cerebellar slice, several types of cerebellar neurons can be easily recognized by their location and cell body shape, allowing targeted electrophysiological recordings. In <stron...

Watch this Scientific Journal Video about Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature at JoVE.com

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

slice-it-hot-acute-adult-brain-slicing-in-physiological-temperature

Research

JoVE Journal

Neuroscience

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.3,4,5

In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.6

In this article, we examine the effects of visually relevant state dependency on TMS induced motive phosphenic presentations.

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

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 ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...