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 ...

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