Name: combined optogenetic and freeze-fracture replica immunolabeling to examine input-specific arrangement of glutamate receptors in the mouse amygdala
Uploaded: 2016-04-15T15:00:00
Duration: 9 min 49 s
Description: Watch this Scientific Journal Video about Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala at JoVE.com

Funding was provided by the Austrian Science Fund FWF grant No. P-22969-B11 to F. Ferraguti, and by the Charitable Hertie Foundation and the Werner Reichardt Centre for Integrative Neuroscience and by the DFG (CIN-Exc. 307) to I. Ehrlich.

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C., Pare, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastic+synaptic+networks+of+the+amygdala+for+the+acquisition,+expression,+and+extinction+of+conditioned+fear.">Plastic synaptic networks of the amygdala for the acquisition, expression, and extinction of conditioned fear.</a> Physiol Rev. 90 (2), 419-463 (2010).</li><li>Sigurdsson, 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=Long-term+potentiation+in+the+amygdala:+a+cellular+mechanism+of+fear+learning+and+memory.">Long-term potentiation in the amygdala: a cellular mechanism of fear learning and memory.</a> Neuropharmacology. 52 (1), 215-227 (2007).</li><li>Bosch, D., Asede, D., Ehrlich, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex-vivo+optogenetic+dissection+of+fear+circuits+in+brain+slices.">Ex-vivo optogenetic dissection of fear circuits in brain slices.</a> J. Vis. Exp. (110), e53628 (2016).</li><li>Fenno, L., Yizhar, O., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+application+of+optogenetics.">The development and application of optogenetics.</a> Annu Rev Neurosci. 34, 389-412 (2011).</li><li>Bienvenu, T. C. M., 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=Large+intercalated+neurons+of+amygdala+relay+noxious+sensory+information.">Large intercalated neurons of amygdala relay noxious sensory information.</a> J. Neurosci. 35 (5), 2044-2057 (2015).</li><li>Sandri, C., Akert, K., Livingston, R. B., Moor, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+aggregations+at+specialized+sites+in+freeze-etched+postsynaptic+membranes.">Particle aggregations at specialized sites in freeze-etched postsynaptic membranes.</a> Brain Res. 41 (1), 1-16 (1972).</li><li>Likhtik, E., Popa, D., Apergis-Schoute, J., Fidacaro, G. 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The authors declare that they have no competing financial interests.

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy due to the development of effective ways to reveal integral membrane proteins by immunogold labeling 1, 2, namely the FRIL technique.
The FRIL technique possesses several advantages over other immunogold ultrastructural methods. First, proteins are readily accessible to antibodies increasing the sensitivity. Second, exposure of large portion of plasma membrane specializations, such as the postsynaptic membrane, on the two-dimensional surface of the replica allows the inspection of the spatial distribution and physical contiguity of molecules of interest without laborious and time-consuming reconstruction of serial ultrathin sections. Third, the availability of both halves of the plasma membrane increases the number of proteins that can be labeled for each individual structure, provided suitable antibodies are available. Upon fracturing, the hydrophobic face of the split membrane is coated with carbon-platinum that entrenches protein domains remaining on the fractured surface. This prevents access of antibodies to antigens in these domains. For instance on the P-face of a replica only epitopes facing the protoplasmic space can be detected by antibodies, whereas on the E-face only epitopes facing the exoplasmic space can be bound by antibodies (see Figure 2).
On the other hand, the FRIL technique also suffers from certain limitations 2. As fractures occur randomly, it might be difficult to target specific cells or structures. This can also lead to a sampling bias, e.g., in synapse collection, given the different probability of fracturing along the membrane of structures with different curvature (e.g., spines versus shafts). Moreover, the allocation of membrane proteins to one of the two faces is unpredictable. Therefore, the distribution of a protein to the P-face or E-face, particularly for quantitative studies, should be carefully examined using antibodies reactive to intracellular and extracellular domains. Finally, the identification in the replica of certain structures, such as presynaptic axon terminals, can be difficult when based only on morphological features. However, the use of specific antibodies for marker proteins or the transduction of tagged integral membrane proteins or channels using viral vectors offers additional tools to facilitate identification of the fractured membranes. For example, this study took advantage of the transduction of ChR2-YFP in thalamic neurons to identify their axonal efferents in the amygdala or the labeling for &#181;-opioid receptors to reveal postsynaptic membranes of ITC neurons.
In order to perform the FRIL technique successfully, particular care should be taken concerning tissue fixation. Strong tissue fixation (&#62; 2% paraformaldehyde) can result in a high rate of cross-fractures and a decrease in labeling sensitivity. On the other hand, weak fixations make the tissue handling and preparation (e.g., cutting of sections) difficult. It is also important to control that the thickness of the trimmed blocks matches the thickness of the double-sided tape. If the thickness of the specimen is lower than that of the tape, the surfaces of the tissue might not attach to the surface of the two metal carriers, consequently the frozen specimen is not fractured. If the tissue is thicker, it will be compressed with inevitable structural distortions when the sandwich of the two copper carriers is made. The temperature at which the specimen is fractured (in this protocol, -115 &#176;C) plays also an important role on the structure of the replica. Higher temperatures may produce a higher rate of artifacts such as condensation of water vapor on the surface of the tissue prior or during evaporation. Lower temperatures (&#60; -125 &#176;C) may increase the risk of splitting off of material during fracturing. This material may fall onto the surface of the specimen or stay connected to it. These flakes of material are also coated and contrasted producing dark spots on the image. Fracturing at lower temperatures can also affect the frequency of cross-fractures particularly for small fine structures such as dendritic spines. A further critical step in the preparation of replicas is the detergent-digestion. If the digestion is incomplete, the undigested tissue appears as dark patches on the replica, confounding the analysis of the structure at the TEM. Moreover, the undigested tissue can non-specifically trap or bind antibodies, increasing the background labeling. On the other hand, the use of detergents for tissue digestion can denature the molecules associated to the replica altering their secondary and tertiary structures. Therefore, for certain antigens it might be necessary to gradually dilute the concentration of SDS with additional washing steps.
For immunolabeling, the availability of different sizes of gold particle conjugated to secondary antibodies allows to detect at the same time, but only qualitatively, multiple proteins, even in specific microdomains of the plasma membrane, such as the postsynaptic specialization. However, due to steric hindrance, quantitative studies are generally limited to the detection of just one molecule. The size of the gold particle can also affect the labeling efficacy.
For the interpretation of the labeling in FRIL, it should be kept in mind that the immunogold particle can be located anywhere within a hemisphere with a radius of 20-25 nm from the antigen due to the flexible complex formed by the primary and secondary antibody 26. For further information on the theory and practice of FRIL and related techniques, we refer the reader also to other methodological articles 27, 28.
The FRIL technique has been recently used for high-resolution quantitative analyses of glutamate receptor localization in diverse synapse populations 29, 30. Moreover, the detection sensitivity of the FRIL technique for AMPA-Rs was estimated as high as one immunogold particle per one functional AMPA-R channel 29. Thus, this approach is overall very useful to quantify and analyze the pattern of postsynaptic expression of AMPA-Rs and NMDA-Rs at central synapses. Here, we demonstrated its applicability at PIN/MGn-ITC synapses, a site most likely important for relaying US information during fear conditioning. Using an antibody raised against the highly conserved extracellular amino acid residues of the AMPA receptor subunits GluA1-GluA4, we found an even distribution of gold particles within IMP clusters corresponding to postsynaptic membrane specializations. The density of AMPA-Rs in ITC spines was significantly higher compared to shaft synapses targeted by PIN/MGn thalamic afferents. At both spine and shaft synapses, a positive correlation between labeling for AMPA-Rs and postsynaptic area was detected, a feature common to other glutamatergic synapses 25. The low variance in density of AMPA-Rs in PIN/MGn-ITC synapses indicates a homogeneous distribution similar to other synapses formed by thalamic efferents 7, but different from cortical synapses 25. Conversely, the density of NMDA-Rs was more variable and did not differ between spine and shaft synapses suggesting a different regulation than AMPA-Rs. In the future, the high reproducibility of the FRIL technique will not only allow to assess the basal molecular composition of central synapses but may facilitate detection of changes in ionotropic glutamate receptor numbers and subsynaptic distribution after fear learning, complementing ex-vivo recordings of pre- and postsynaptic properties of these inputs.
In conclusion, this approach could be used by other investigators to gain insights into structure-function relationships of input-specific excitatory synapses in many other neural circuits in which disentangling the origin of the inputs and the nature and composition of postsynaptic elements is crucial but problematic.

The definition of the functional architecture of biomembranes at nanometer scale has been challenged in recent years by the development of a number of immunolabeling techniques suitable for transmission electron microscopy. However, these techniques, e.g., pre- and post-embedding immunogold, have a number of important limitations, which include poor detection of antigens and/or limited quantitative assessment of membrane-bound proteins. These limitations become particularly critical in the investigation of the fine structure of the nervous system, which is characterized by a high degree of cell diversity and synapse heterogeneity. This heterogeneity results from both structural and functional diversity dictated by the pre- and postsynaptic elements and by the differential expression, enrichment, or interaction of signalling proteins, such as receptors, transporters, and effector molecules.
A new approach for direct immunolabeling of integral or cross-linked membrane proteins in detergent-solubilized freeze-fracture replicas (FRIL) was originally introduced by Fujimoto two decades ago 1. This original method had, however, several limitations, i.e., severe fragmentation of replicas, which hampered meaningful correlations of labeled molecules with individually mapped cells in complex tissues such as the brain. Approximately 10 years ago, Shigemoto and Fukazawa progressively improved the technique 2. This was paralleled by efforts from another group of scientists at the Boulder laboratories of the Colorado State University, who also significantly improved the technique, in particular for the study of gap junctions 3.
The improvement in freeze-fracturing protocols and machines, as well as the introduction of quick freezing (under high pressure), now allows investigators to produce unbroken replicas of specimens of relatively large size and high quality images of most cellular components without the limitations and artifacts produced by strong chemical fixations.
The FRIL technique offers the great advantage of a highly quantitative in situ identification of one or more proteins (simultaneously) in histologically mapped and cytologically identified cells within complex tissues such as the brain, with the additional advantage of a planar view of pre- and postsynaptic elements in a single replica. Therefore, the FRIL technique, despite its many technical hurdles, holds the promise for a number of very significant scientific breakthroughs, particularly for the correlation of structural and functional properties of individual synapses. During the last several decades, a great deal of information has been obtained on the structure, molecular make up, and physiological function of synapses; yet synapses are morphologically and molecularly highly diverse depending on the pre and postsynaptic parent neurons 4. Only for a handful of synapse types were structure-function studies accomplished so far 5-7. This was mostly due to technical constrains that prevented a precise identification of the nature of the pre- and postsynaptic elements.
Ultrastructural analysis has provided critical insights into the variability of postsynaptic membrane specializations across distinct synaptic contacts both in terms of synaptic size and content in neurotransmitter receptors 6, which has a large impact on the strength and plasticity of synaptic transmission. Furthermore, a large body of research indicates that the number of ionotropic glutamate receptors expressed at different types of synapse is regulated in an afferent- and target-dependent fashion 7-10.
Here, a method is outlined that allows analysis of the structure and receptor composition of postsynaptic membrane specializations with defined presynaptic elements and function. This approach takes advantage of presynaptic expression of recently developed light sensitive algal proteins, such as channelrhodopsin2 (ChR2), and of the FRIL technique to analyze the pattern of postsynaptic expression of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA-Rs) and N-methyl-D-aspartate (NMDA-Rs) glutamate receptors. This is demonstrated at synapses formed by axons originating from the posterior thalamic-medial geniculate nuclei (PIN/MGn) onto neurons of the intercalated cell masses of the amygdala (ITC). ITC neurons are small spiny GABAergic cells organized in clusters surrounding the basolateral amygdaloid complex (BLA) 11, 12. ITC neurons are known to receive excitatory inputs from BLA principal neurons and to target the central nucleus (CeA), thus functioning as an inhibitory gate for the information flow between the BLA and CeA 12-15.
Recently, we demonstrated that ITC neurons located in the medio-dorsal cluster between BLA and CeA also receive direct and convergent excitatory inputs from sensory thalamic and temporal cortical regions, which are modified upon fear learning during Pavlovian auditory fear conditioning 16. Fear conditioning is one of the best understood forms of associative learning in terms of brain mechanisms. In fear conditioning, an initially neutral conditioned stimulus (CS, e.g., a tone) is paired with an aversive unconditioned stimulus (US, e.g., a mild foot shock) resulting in a CS-US association and conditioned fear response 17, 18. Excitatory inputs from both thalamic and neocortical areas, which carry information representing the CS and US, respectively, were known to converge onto pyramidal neurons of the lateral nucleus of the amygdala (LA) and to undergo plasticity 19. Our previous work revealed that sensory input information is also in parallel relayed to ITC neurons 16.
As a first step towards a mechanistic molecular analysis of individual sensory input synapses onto ITC neurons, we used an adeno-associated virus (AAV) to express ChR2 tagged with the yellow fluorescent protein (YFP). The AAV was injected into the PIN/MGn and the axon terminals were identified by their expression of ChR2-YFP. We used both faces generated by the FRIL technique to assess the density of postsynaptic AMPA-Rs and NMDA-Rs at synapses formed with ITC neurons by PIN/MGn axon terminals.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Surgery</td>
		</tr>
		<tr>
			<td>Stereotactic frame</td>
			<td>Stoelting, USA</td>
			<td>51670</td>
			<td>can be replaced by other stereotactic frame for mice</td>
		</tr>
		<tr>
			<td>Steretoxic frame mouse adaptor</td>
			<td>Stoelting, USA</td>
			<td>51625</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas anesthesia mask for mice</td>
			<td>Stoelting, USA</td>
			<td>50264</td>
			<td>no longer available, replaced by item no. 51609M</td>
		</tr>
		<tr>
			<td>Pressure injection device, Toohey Spritzer</td>
			<td>Toohey Company, USA</td>
			<td>T25-2-900</td>
			<td>other pressure injection devices (e.g. Picospritzer) can be used</td>
		</tr>
		<tr>
			<td>Kwik Fill glass capillaries</td>
			<td>World Precision Instruments, Germany</td>
			<td>1B150F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia machine, IsoFlo</td>
			<td>Eickemeyer, Germany</td>
			<td>213261</td>
			<td></td>
		</tr>
		<tr>
			<td>DC Temperature Controler and heating pad</td>
			<td>FHC, USA</td>
			<td>40-90-8D</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal Micropipette Puller Model P-1000</td>
			<td>Sutter Instruments, USA</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tool sterilizer, Sterilizator 75</td>
			<td>Melag, Germany</td>
			<td>08754200</td>
			<td></td>
		</tr>
		<tr>
			<td>rAAV-hSyn-ChR2(H134R)-eYFP (serotype 2/9)</td>
			<td>Penn Vector Core, USA</td>
			<td>AV-9-26973P</td>
			<td></td>
		</tr>
		<tr>
			<td>fast green</td>
			<td>Roth, Germany</td>
			<td>0301.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane Anesthetic, Isofuran CP (1ml/ml)</td>
			<td>CP Pharma, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antiseptic, Betadine (providone-iodine)</td>
			<td>Purdure Products, USA</td>
			<td>BSOL32</td>
			<td>can be replaced by other disinfectants</td>
		</tr>
		<tr>
			<td>Analgesic, Metacam Solution (5mg/ml meloxicam)</td>
			<td>Boehringer Ingelheim, Germany</td>
			<td></td>
			<td>can be replaced by other analgesics</td>
		</tr>
		<tr>
			<td>Bepanthen eye ointment</td>
			<td>Bayer, Germany</td>
			<td>0191</td>
			<td>can be replaced by other eye ointments</td>
		</tr>
		<tr>
			<td>Drill NM3000 (SNKG1341 and SNIH1681)</td>
			<td>Nouvag, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sutranox Suture Needle</td>
			<td>Fine Science Tools, Germany</td>
			<td>12050-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Braided Silk Suture</td>
			<td>Fine Science Tools, Germany</td>
			<td>18020-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Tissue preparation</td>
		</tr>
		<tr>
			<td>Paraformaldehyde EM grade</td>
			<td>Agar Scientific Ltd., United Kingdom</td>
			<td>AGR1018</td>
			<td></td>
		</tr>
		<tr>
			<td>Saturated picric acid solution</td>
			<td>Sigma-Aldrich, USA</td>
			<td>P6744-1GA</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4-2H20</td>
			<td>Merck Millipore, Germany</td>
			<td>1065860500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4-2H2O&#160;</td>
			<td>Merck Millipore, Germany</td>
			<td>1063451000</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Merck Millipore, Germany</td>
			<td>1064041000</td>
			<td></td>
		</tr>
		<tr>
			<td>4N NaOH</td>
			<td>Carl Roth, Germany</td>
			<td>T198.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiopental</td>
			<td>Sandoz, Austria</td>
			<td>5,133</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich, USA</td>
			<td>G5516-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>GenPure ultrapure water system</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>50131235</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>ISMATEC, Germany</td>
			<td>ISM 930C</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Paper</td>
			<td>MACHEREY-NAGEL, Germany</td>
			<td>MN 615 1/4</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibroslicer, VT1000S</td>
			<td>Leica Microsystems, Austria</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic scalpel</td>
			<td>Alcon Laboratories, USA</td>
			<td></td>
			<td>can be replaced by other ophthalmic scalpels</td>
		</tr>
		<tr>
			<td>Perfusion cannula</td>
			<td>Vieweg, Germany</td>
			<td>F560088-1</td>
			<td>can be replaced by similar items from other companies</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">High-pressure&#160; Freezing</td>
		</tr>
		<tr>
			<td>Copper carriers</td>
			<td>Engineering Office M. Wohlwend, CH</td>
			<td>528</td>
			<td></td>
		</tr>
		<tr>
			<td>Sidol Polish</td>
			<td>Henkel, Germany</td>
			<td></td>
			<td>can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>Chamois skin</td>
			<td>Household supply store</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hole punch, 1,5mm</td>
			<td>Stubai, Austria</td>
			<td></td>
			<td>can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>Denatured ethanol</td>
			<td>Donauchem, Austria</td>
			<td></td>
			<td>can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>Aceton</td>
			<td>Roth, Germany</td>
			<td>9372.5</td>
			<td>CAUTION!</td>
		</tr>
		<tr>
			<td>High Pressure Freezing Machine HPM 010</td>
			<td>BalTec, CH; now Leica Microsystems</td>
			<td>HPM010</td>
			<td>not produced any more, substituted by LeicaEM HPM100</td>
		</tr>
		<tr>
			<td>Stereo-microscope</td>
			<td>Olympus, Japan</td>
			<td>SZX10</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td>CAUTION!</td>
		</tr>
		<tr>
			<td>Cryo-vials</td>
			<td>Roth, Germany</td>
			<td>E309.1</td>
			<td>can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>CryoCane</td>
			<td>Nalge Nunc International,USA</td>
			<td>5015-0001</td>
			<td>can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>CryoSleeve</td>
			<td>Nalge Nunc International,USA</td>
			<td>5016-0001</td>
			<td>can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>Liquid nitrogen storage vessel</td>
			<td>Cryopal, France</td>
			<td>GT38</td>
			<td>can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>Non-ionic detergent (Lavocid)</td>
			<td>Werner &#38; Mertz Professional, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Freeze-fracture and Replication</td>
		</tr>
		<tr>
			<td>Sandblaster, Mikromat 200-1</td>
			<td>JOKE Joisten &#38; Kettenbaum, Germany</td>
			<td>SANDURET 2-K</td>
			<td>can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>Siliciumcarbid SIC 360, grain size 25 - 21&#181;</td>
			<td>JOKE Joisten &#38; Kettenbaum, Germany</td>
			<td>955932</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze Fracture System BAF 060</td>
			<td>BalTec, CH; now Leica Microsystems</td>
			<td>BAF060</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceramic 12 well plate</td>
			<td>Gr&#246;pel, Austria</td>
			<td>14511</td>
			<td>can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>SIGMA, USA</td>
			<td>T1503</td>
			<td>can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>Trizma hydrochloride</td>
			<td>SIGMA, USA</td>
			<td>T3253</td>
			<td>can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck, Germany</td>
			<td>1,064,041,000</td>
			<td>can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>SDS, Sodium lauryl sulfate</td>
			<td>Roth, Germany</td>
			<td>5136.1</td>
			<td>CAUTION! ;&#160; can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Merck, Germany</td>
			<td>1,076,871,000</td>
			<td>can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>TRIS</td>
			<td>Roth, Germany</td>
			<td>5429.3</td>
			<td>can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>Universal Hybridization Oven</td>
			<td>Binder, Germany</td>
			<td>7001-0050</td>
			<td>can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Immunolabelling</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>SIGMA, USA</td>
			<td>A9647</td>
			<td>can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>Anti-GFP Antibody</td>
			<td>Molecular Probes, USA</td>
			<td>A11122</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-pan-AMPAR Antibody</td>
			<td>Frontier Institute, Japan</td>
			<td>pan AMPAR-GP-Af580-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-NMDAR1 Antibody, clone 54.1</td>
			<td>Merck Millipore, Germany</td>
			<td>MAB363</td>
			<td></td>
		</tr>
		<tr>
			<td>Opioid Receptor-Mu (MOR) Antibody</td>
			<td>ImmunoStar, USA</td>
			<td>24216</td>
			<td></td>
		</tr>
		<tr>
			<td>EM goat anti-guinea pig, 5nm; secondary antibody</td>
			<td>BBInternational,</td>
			<td>EM.GAG5</td>
			<td></td>
		</tr>
		<tr>
			<td>EM goat anti-rabbit, 15nm; secondary antibody</td>
			<td>BBInternational,</td>
			<td>EM.GAR15</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit, 10nm, secondary antibody</td>
			<td>AURION, Netherlands</td>
			<td>DAR 10nm</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper grids, 100 Parallel Bar</td>
			<td>&#160;Agar scientific, UK</td>
			<td>G2012C</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Major Science, USA</td>
			<td>MO-RC</td>
			<td>can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>Pioloform Powder</td>
			<td>&#160;Agar scientific, UK</td>
			<td>R1275</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Roth, Germany</td>
			<td>3313.1</td>
			<td>CAUTION! ;&#160; can be replaced by same item from other companies</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">EM analysis</td>
		</tr>
		<tr>
			<td>Philips CM120 TEM</td>
			<td>Philips/FEI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Morada CCD camera</td>
			<td>Soft Imaging Systems, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iTEM Ver. 5.2, imaging software</td>
			<td>Soft Imaging Systems, Germany</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

combined optogenetic and freeze-fracture replica immunolabeling to examine input-specific arrangement of glutamate receptors in the mouse amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

The FRIL technique, when combined with expression of optogenetic actuators of microbial origin 21, i.e., channels integrated in the plasma membrane and effectively transported anterogradely along axons, allows to examine quantitatively the postsynaptic expression of AMPA-Rs and NMDA-Rs at a defined subgroup of synapses. This is shown here for axons originating from distinct thalamic nuclei (e.g., PIN/MGn) onto ITC neurons in the amygdala. This approach enables a molecular analysis of individual sensory input synapses onto ITC neurons, a group of cells that have been refractory to a detailed anatomical and molecular characterization so far.
Four weeks after stereotactic injection of the rAAV-ChR2-YFP into the PIN/MGn, ChR2-YFP-positive axons densely innervated the LA, the amygdalostriatal transition area (Astria) and the medial paracapsular ITC cluster in the amygdala, a pattern fully consistent with previous tracing studies 16, 22. We also detected intense gold immunolabeling for the ChR2-YFP on the P-face of axons and terminals in freeze-fracture replica from rAAV-ChR2-YFP-injected mice (Figure 5A), but not in replicas from non-injected mice. The postsynaptic membrane specialization of glutamatergic synapses in a replica can be observed as a cluster of intramembrane particles (IMPs) on the E-face of the plasma membrane 2, 23, and is often accompanied by the P-face of its presynaptic plasma membrane 7 (Figure 5B-C). These features allowed to identify the postsynaptic specialization of glutamatergic synapses formed by PIN/MGn axon terminals (Figure 5 and 6). We labeled AMPA-Rs with an antibody that recognizes all four subunits (GluA1-4), whereas NMDA-Rs were detected using an antibody against the essential NR1 subunit.
Because of the lack of tools to detect on the same replica whether these synapses were made with dendritic spines or shafts of ITC neurons, we labeled the corresponding replica face for &#181;-opioid receptors, as ITC neurons express postsynaptically high levels of these receptors 24. This required the identification of the same postsynaptic profiles in the two replicas (Figure 5C-F and Figure 6A-D) using a strategy that employs landmarks (Figure 4E).
<img alt="Figure 5" src="/files/ftp_upload/53853/53853fig5.jpg" /> 
Figure 5. Detection of ChR2-YFP and Ionotropic Glutamate Receptors on Replica by Immunogold Particles. (A) A cross-fractured axon terminal (light blue) and small portions of its P-face labeled with 15 nm gold particles detecting ChR2-YFP. Within the terminal, the membrane of numerous synaptic vesicles can be observed (sv). Note the specificity of the immunolabeling in large part restricted to the plasma membrane. Labeling for ChR2 identifies the terminal as originating from the PIN/MGn. The terminal forms an asymmetric synapse with a spine. The postsynaptic membrane specialization (PSD) on the E-face shows a characteristic cluster of intramembrane particles and is labeled with 5 nm gold particles revealing AMPA-Rs. (B) The P-face of a ChR2-expressing axon (labeled with 15 nm gold particles) is shown bordering two dendrites, one of them possessing two PSDs labeled with 5 nm gold particles revealing NMDA-Rs. (C-D) Opposite faces of the pre- and postsynaptic membranes of a PIN/MGn-ITC synapse. (C) The P-face of the terminal expresses ChR2 (labeled with 15 nm gold particles) and extends over the E-face of two dendritic shafts, one of them containing two PSDs labeled for AMPA-Rs (5 nm gold particles). (D) The corresponding P-face of the two dendrites is labeled for &#181;-opioid receptors (10 nm gold particles). (E-F) Enlarged views of the areas outlined by the dashed lines. Scale bars: 500 nm. <a href="https://www.jove.com/files/ftp_upload/53853/53853fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Immunoparticles for AMPA-Rs in the PIN/MGn-ITC synapses were found all over the IMP cluster, suggesting a homogeneous distribution within the postsynaptic specialization (Figure 5E). A significantly higher (unpaired t-test p&#60;0.018) density of AMPA-R labeling was observed in PIN/MGn synapses onto ITC spines (715&#177;38 gold particles/&#181;m2, n = 32) compared to synapses onto ITC dendrites (590&#177;44 gold particles/&#181;m2, n = 32). Overall, the density of AMPA-Rs in PIN/MGn-ITC synapses showed a relatively low variance (Coefficient of variance, CV = 0.37) consistent with a homogeneous distribution.
Immunoparticles for NMDA-Rs in the PIN/MGn-ITC synapses were often observed unevenly distributed within postsynaptic IMP clusters (Figure 5B). The density of NMDA-R labeling was similar (unpaired t-test p=0.39) between PIN/MGn synapses onto ITC spines (1070&#177;153 gold particles/&#181;m2, n=8) and ITC dendrites (812&#177;183 gold particles/&#181;m2, n=9). Unlike what was observed for AMPA-Rs, the density of NMDA-Rs in PIN/MGn-ITC synapses was highly variable (CV = 0.54).
<img alt="Figure 6" src="/files/ftp_upload/53853/53853fig6.jpg" /> 
Figure 6. AMPA-Rs and NMDA-Rs Immunolabeling at Identified PIN/MGn-ITC Synapses. 
(A-B) Opposite faces of the pre- and postsynaptic membranes of a PIN/MGn-ITC synapse made onto a dendritic spine in which the P-face of the terminal expresses ChR2 (labeled with 15 nm gold particles) and the PSD onto a dendritic spine is labeled for AMPA-Rs (5 nm gold particles). (C-D) Enlarged views of the areas outlined by the dashed line. These areas have been rotated approximately 45&#176; anticlockwise to allow a better view of the PSD. (E) Scatterplots of the number of AMPA-R particles versus synaptic area in ITC spines and dendrites. In both structures, a positive correlation has been observed. (F) Scatterplots of the number of NMDA-R particles against synaptic area in ITC spines and dendrites. A significant positive correlation was detected only in dendritic spines. Scale bars: 500 nm. <a href="https://www.jove.com/files/ftp_upload/53853/53853fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Because the P-face of the presynaptic plasma membrane often overlaid in part the postsynaptic IMP cluster, we could estimate the synaptic area of only 30% of the synapses (spines: mean area 0.032 &#181;m2, range: 0.007 to 0.063 &#181;m2, n = 8; dendrites: 0.047 &#181;m2, range: 0.024 to 0.166 &#181;m2, n = 11). These were in a similar range as previously analyzed telencephalic glutamatergic synapses 25.
In both spines and dendrites, the number of gold immunoparticles for AMPA-Rs in individual synapses was positively correlated with the synaptic area (Spearman, spines: r = 0.88, dendrites: r = 0.60, p&#60;0.0001) (Figure 6E). Conversely, the number of gold immunoparticles for NMDA-Rs was found to correlate with the synaptic area in spines (Spearman, spines: r = 0.90, p&#60;0.002) but not in dendrites (r = 0.21, p = 0.29) (Figure 6F).

Watch this Scientific Journal Video about Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala at JoVE.com

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

The goal of this procedure, which combines optogenetics with the freeze-fracture replica immunolabeling technique, is to analyze the density of ionotropic glutamate receptors at synapses in the mouse amygdala with identified pre, and post, synaptic elements. The high reproducibility and versatility of that freeze-fracture replica immunolabeling technique, when combined with optogenetics, offers a very powerful approach for the correlated analysis of the structural and functional properties of synapses. The main advantages of this procedure are:the planar view of the pre and post synaptic elements, and the quantitative analysis of proteins in these specialized micro-domains.Different technique can be adapted to a variety of different tissues to study the distribution and organization of integral membrane proteins. Generally, this combined optogenetic FRIL approach is challenging for beginners because of the large range of different apparatus and machines required. Beginning the modulation with different method is critical as some other steps are difficult to learn.Such as fracturing and replicating frozen specimens To begin this procedure glue a coronal block of mouse brain onto the holder of the vibro-slicer. Orient the tissue block so that the neo-cortex faces the vibrating blade. Next, slice the coronal sections containing the amygdala at a thickness of 140 micrometers in zero point one molar, ice-cold PB.And collect them in a six-well dish in the same buffer.Under a stereo-microscope, trim out the region of interest from the slices, in a petri dish coated with silicone elastomer and filled with zero point one molar PB.Then, transfer the trimmed block into the cryo-protection solution, and keep overnight at six degrees Celsius. In this step, prepare the copper carriers for use in the successive stages of the FRIL procedure, by polishing them with sheet of shammy skin as a tarnish remover. Under the microscope, attach a ring of double-sided tape to the copper carrier, which will serve as the holding well for the trimmed block.Then, place the trimmed block in the hole of the double-sided tape using a platinum wire loop. Remove excess cryoprotectant solution using a filter paper or a brush. Afterward, cover the holding carrier with another carrier.So that the tissue block is sandwiched between the two carriers. To freeze the specimen, insert the carrier sandwich into the specimen holder. Subsequently, insert the specimen holder into the high-pressure freezing unit.Initiate the freezing cycle by pressing the jet-auto button, remove the specimen holder immediately, and submerge the tip into an insulated box with liquid nitrogen. Then, carefully remove the carrier sandwich from the specimen holder, and place it in a pre-chilled cryovial. Store the cryovials containing the carriers in a cryotank until replication.Before inserting the electron beam gun, remove the shield with the deflector plate. Place the setting gauge, for centering the filament, into the collet chuck through the lower cathode cover. Next, slide the new filament over the gauge, until the pressure laminae clamp the ends of the filament.Then, remove the setting gauge and insert the carbon rod. Fix it, by tightening the collet chuck of the evaporator rod holder. Ensuring that the height of the end of the rod is at the middle of the second coil from the bottom.After that, replace the deflector plate, and insert the electron-beam gun into the freeze-fracture unit. In this procedure, adjust the current and voltage for evaporation. Next, insert the frozen carrier sandwich into the double-replica table in liquid nitrogen.Then, transfer the double-replica table to Dewar vessel, and fix it to the specimen stage receiver at an angle of 45 degrees. Pick up the double-replica table with a table manipulator. Insert it into the freeze-fracture unit onto the cold stage, and wait approximately 20 minutes to allow the temperature of the double-replica table to adjust to negative 115 degrees Celsius.Afterward, fracture the tissue by rotating the wheel counterclockwise manually, which is connected to the shroud above the double-replica table. When the shroud turns, it forces the double-replica table to open, which results in fracturing the tissue. A layer of carbon from a 90 degree angle is applied to the fractured faces.Subsequently, remove the replicated specimens from the double-replica table, and transfer them to a ceramic 12-well plate filled with TBS. Using a platinum loop-wire rod, remove the replicated tissue from the specimen carrier. For SDS digestion, transfer a replica to a four milliliter glass vial filled with one milliliter of SDS-digestion buffer.Allow it to digest for 18 hours at 80 degrees Celsius with shaking. For immunolabeling, wash the replica for 10 minutes in fresh SDS-digestion buffer. Then, incubate it with primary and secondary antibodies, diluted in 2%BSA-TBS, in a humid chamber at 15 degrees Celsius for 24 to 72 hours.After that, mount the replica on form-far coated, 100-line parallel bar grid. Image the replica with a transmission-electron microscope at 80 or 100 kilo-volts. Then, acquire the digital images through a CCD camera.When it is offline, find the corresponding regions on the image from the replica, using different landmarks. Four weeks after AAV injection, to express Channelrhodopsin-2 in the posterior thalamic nuclear group, Channelrhodopsin-2 is effectively transported anterogradely along the thalamic axons to reach the amygdala intercalated cell masses. The post-synaptic specialization of glutamatergic synapses can be recognized in a replica as a cluster of intramembrane particles on the E-face of the plasma membrane, and is often accompanied by the P-face of its presynaptic element.Here, Channelrhodopsin-2 and glutamate receptors were visualized using gold particles of different sizes conjugated to the secondary antibodies. Because of the lack of structural or molecular tools to detect on the same replica whether the postsynaptic membrane belonged to the intercalated neurons. The corresponding replica was labeled for mu opioid receptors, a marker for these neurons.As an example of the quantitative analysis of glutamate receptor density at these synapses, here are the scatter plots of the number of gold particles for ampa receptors versus the synaptic area on spines and dendrites. Which reveal a positive correlation in both structures. While attempting this procedure, it's important to remember, that the individual steps are highly interdependent.Therefore, a mistake in one of the steps can jeopardize the whole procedure. Viewing large portion of plasma membrane specializations on a two-dimensional surface of the replica, allows the inspection of the spatial distribution and physical continuity of molecules of interest without laborious and time-consuming reconstruction of serial artrophin sections. This approach can be used by other investigators to gain insights into structure-function relationships of specific synapses in neural circuits.Where it is untangling the origin of the inputs and the nature of the postsynaptic elements. Is crucial, but problematic.

Research

JoVE Journal

Neuroscience

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

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

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

Muscle Velocity Recovery Cycles to Examine Muscle Membrane Properties

Although conventional nerve conduction studies (NCS) and electromyography (EMG) are suitable for the diagnosis of neuromuscular disorders, they provide ...