The general structure of a chemical synapse is shown schematically in Figure 5.1B. The space between the pre- and postsynaptic neurons is substantially greater at chemical synapses than at electrical synapses and is called the synaptic cleft. However, the key feature of all chemical synapses is the presence of small, membrane-bounded organelles called synaptic vesicles within the presynapticterminal. These spherical organelles are filled with one or more neurotransmitters, the chemical signals secreted from the presynaptic neuron, and it is these chemical agents acting as messengers between the communicating neurons that gives this type of synapse its name. There are many kinds of neurotransmitters (see Chapter 6), the best studied example being acetylcholine, the transmitter employed at peripheral neuromuscular synapses, in autonomic ganglia, and at some central synapses.
Transmission at chemical synapses is based on the elaborate sequence of events depicted in Figure 5.3. The process is initiated when an action potential invades the terminal of the presynapticneuron. The change in membrane potential caused by the arrival of the action potential leads to the opening of voltage-gated calcium channels in the presynaptic membrane. Because of the steep concentration gradient of Ca2+ across the presynaptic membrane (the external Ca2+ concentration is approximately 10–3M, whereas the internal Ca2+ concentration is approximately 10–7M), the opening of these channels causes a rapid influx of Ca2+ into the presynaptic terminal, with the result that the Ca2+ concentration of the cytoplasm in the terminal transiently rises to a much higher value. Elevation of the presynaptic Ca2+ concentration, in turn, allows synaptic vesicles to fuse with the plasma membrane of the presynaptic neuron. The Ca2+-dependent fusion of synaptic vesicles with the terminal membrane causes their contents, most importantly neurotransmitters, to be released into the synaptic cleft.
Sequence of events involved in transmission at a typical chemical synapse.
Following exocytosis, transmitters diffuse across the synaptic cleft and bind to specific receptors on the membrane of the postsynapticneuron (see Chapter 7). The binding of neurotransmitter to the receptors causes channels in the postsynaptic membrane to open (or sometimes to close), thus changing the ability of ions to flow into (or out of) the postsynaptic cells. The resulting neurotransmitter-induced current flow alters the conductance and usually the membrane potential of the postsynaptic neuron, increasing or decreasing the probability that the neuron will fire an action potential. In this way, information is transmitted from one neuron to another.
Electrical synaptic transmission and the ultrastructural correlate of electrical synapses – gap junctions – are now accepted features of neuronal circuitry in many areas of the mammalian central nervous system (CNS; Bennett and Zukin, 2004; Hormuzdi et al., 2004). In the hippocampus, gap junctions and electrical communication between dendrites of interneurons are well-documented ultrastructurally and electrophysiologically (Kosaka, 1983; Fukuda and Kosaka, 2000; Haas et al., 2011). Because gap junction channels also allow direct intercellular passage of small molecules, early observations of cell-to-cell passage of fluorescent dyes (“dye-coupling”) between hippocampal pyramidal cells was taken as evidence of electrical synapses between these cells (MacVicar and Dudek, 1980, 1982). Based on electrical coupling, dye- and tracer-coupling, and computer modeling, electrical synapses were proposed to occur between the axons of hippocampal pyramidal cells, particularly near their somata (Schmitz et al., 2001). Paired recordings from a large number of hippocampal pyramidal cells provided further electrophysiological support for electrical coupling between these cells (Yamamoto, 1972; Mercer et al., 2006). However, proof of electrical coupling via glutamatergic mixed synapses between hippocampal principal cells – dentate granule cells (DG) and pyramidal cells – as well as between glutamatergic interneurons, is lacking. Recently, however, evidence for mixed electrical–chemical synaptic transmission from mossy fibers (MFs) to CA3 pyramidal cells (CA3pyr) has been presented (Vivar et al., 2012). On the other hand, although electrical coupling and connexin-36 (Cx36)-containing gap junctions have been demonstrated between interneuronal dendrites in other regions of neocortex and olfactory bulb (Rash et al., 2005; Fukuda et al., 2006), neither electrical coupling nor the connexin composition of neuronal gap junctions has been adequately examined in hippocampus between MF terminals and CA3pyr, between glutamatergic interneurons, between glutamatergic interneurons and principal cells, or between granule cells and their innervating axons of the perforant path. This pathway, which arises from the entorhinal cortex, forms the major input to the DG, which receives no extrinsic inputs from other cortical structures (Do et al., 2002). In addition to innervating the DG, neurons of the perforant path also innervate CA3pyr on their proximal and distal dendrites (Do et al., 2002), as well as the dendrites of interneurons within the hilus (Acsady et al., 1998), providing additional potential sources of glutamatergic mixed synapses in the hippocampus. In addition, hilar mossy cells (Jackson and Scharfman, 1996) and putative glutamatergic interneurons (Soriano and Frotscher, 1993) synapse on CA3pyr, on granule cells, and on mossy cell dendrites in stratum oriens (pathways summarized in Kondo et al., 2008). Finally, because evidence indicates that CA3pyr synthesize Cx36 (Belluardo et al., 2000), and therefore presumably form gap junctions, local recurrent collaterals of CA3pyr may provide additional sources of glutamatergic mixed synapses.
Four major difficulties in understanding proposed functional contributions of electrical vs. mixed synapses between hippocampal principal cells or between interneurons and other neurons are: (1) the lack of ultrastructural evidence for gap junctions between most of those neurons, (2) the uncertainty regarding the sizes and anatomical locations at which gap junctions might occur, (3) lack of evidence for the connexin composition of the gap junctions, and (4) lack of identification of the neurotransmitter(s) that might be involved at mixed synapses. Despite extensive analysis, thin-section transmission electron microscopic (tsTEM) studies that have described many dozens of large (0.2–0.6 μm diameter) dendrodendritic gap junctions between interneurons in the rat hippocampus (Kosaka, 1983; Kosaka and Hama, 1985; Fukuda and Kosaka, 2000) did not detect gap junctions on principal cells or at axon terminals, potentially because: (a) axodendritic and axosomatic gap junctions between hippocampal neurons may be smaller and more difficult to detect in conventional thin-section images (Rash et al., 1998); (b) the number and distribution of gap junctions may vary considerably in different regions of hippocampus or in different rodent species; or (c) gap junctions may occur at locations other than between the apposed dendrites that were the specific targets of previous studies. With CNS tissues having small intercellular spaces (i.e., ca. 10 nm vs. the 20-nm space found in most other chemically fixed, plastic-embedded, thin-sectioned tissues reviewed in Staehelin, 1974), recognizing small gap junctions in tsTEM images of hippocampus may be especially problematic (Rash et al., 1998). Although electrophysiological recordings from DG and CA3pyr suggest the existence of glutamatergic mixed synapses between MFs and their principal cell targets (Vivar et al., 2012), no ultrastructural evidence has been published for gap junctions between MF terminals and their primary targets on dendritic shafts or spines of CA3pyr. Likewise, neither electrical coupling nor gap junctions have been demonstrated between the glutamatergic axon terminals of the perforant path synapsing on granule cells, CA3pyr, or interneurons, or between interneurons and principal cells or other interneurons. Thus, deciphering the nature of the gap junctions, the types of neurons they connect, and the cellular sites at which these connections are established are critical steps in understanding hippocampal physiology. Indeed, electrical coupling between hippocampal neurons has been proposed for generating gamma (30–80 Hz) and very fast (>80 Hz) oscillations (Traub et al., 2001, 2010) and is also thought to contribute to epileptogenesis (Traub et al., 2001).
The functional importance of neuronal gap junctions at mixed synapses has been extensively documented in lower vertebrates, particularly at the glutamatergic giant club-ending terminals on Mauthner cells in the teleost brain (Pereda et al., 2003). Each of those giant synapses contains 80–200 small to large gap junctions (30–4000 connexons each), intermixed with clusters of 10-nm intramembrane particles (IMPs) on their postsynaptic extraplasmic leaflets (E-faces; Tuttle et al., 1986; Nakajima et al., 1987) that were later shown by freeze-fracture replica immunogold labeling (FRIL) to contain N-methyl-D-aspartate (NMDA) glutamate receptors (Pereda et al., 2003). Intracellular monitoring of those giant mixed synapses documented that a large electrical “spikelet” (or “fast prepotential”) immediately precedes the excitatory postsynaptic potential (Pereda et al., 2003, 2004), thereby revealing the electrophysiological signature of these giant excitatory mixed synapses. However, the smaller and fewer gap junctions found to date on mammalian principal neurons (Rash et al., 2005, 2007) suggest that detection of fast prepotentials will be difficult in hippocampus (Dudek et al., 2010; but see Mercer et al., 2006; Vivar et al., 2012), and indeed, that the function of gap junctions at mammalian mixed synapses may be other than for strong electrical coupling.
This reports presents: (a) tsTEM evidence for gap junctions between glutamatergic MF axon terminals at thorny excrescences of CA3pyr dendrites in adult rat hippocampus, and (b) evidence for limited dye-coupling between CA3pyr somata and dendrites and their closely apposed MF axons. This report also provides: (c) FRIL evidence that connexin-36 (Cx36) is the primary connexin in ultrastructurally identified gap junctions between neurons at diverse locations in hippocampus; (d) that rather than “plaques,” many were unusual “reticular” gap junctions, and (e) that most of the gap junctions occurred immediately adjacent either to glutamate receptor-containing postsynaptic densities (PSDs) and/or to distinctive clusters of 10-nm E-face IMPs within the same axonal contact area, defining most as either glutamatergic mixed synapses or as gap junctions that are closely associated with “extrasynaptic” AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) glutamate receptors. Finally, although one presumptive GABAergic mixed synapse was found by tsTEM on a CA3pyr dendrite, 10 of 20 gap junctions found by FRIL were at ultrastructurally or immunocytochemically identified glutamatergic mixed synapses, and three were at likely dendrodendritic electrical synapses, but none were at ultrastructurally identified GABAergic synapses in FRIL replicas, suggesting that glutamatergic mixed synapses are much more abundant than GABAergic mixed synapses, and possibly more abundant than dendrodendritic electrical synapses. Regardless, the functional significance of “miniature” gap junctions at glutamatergic mixed synapses is yet to be determined.
Materials and Methods
All animals used in this study were treated under the protocols approved by the Institutional Animal Care and Use Committees of the State University of New York, Downstate Medical Center, Mount Sinai School of Medicine; Colorado State University; and the Center for Research and Advanced Studies, Mexico City. All experiments were conducted according to Principles of Laboratory Animal Care (U.S. National Institutes of Health Publication No. 86-23, 1985). Chemical agents were purchased from Sigma (Sigma Aldrich, St. Louis, MO, USA) unless separately indicated.
Fluorescent Dye Injection and Confocal Microscopic Imaging
For morphological reconstructions, 20 Sprague-Dawley rats (3-6 month old) were deeply anesthetized by intraperitoneal injection of a 30% solution of chloral hydrate (400 mg/kg) in distilled water and fixed by whole-body perfusion for 1 min via the ascending aorta using cold 1% formaldehyde dissolved in PBS. Secondary fixation was by perfusion with cold 4% formaldehyde plus 0.125% glutaraldehyde in PBS for 12 min. Brains were removed from the skull and post-fixed for 4–6 h in 4% formaldehyde at 4°C. Brains were transferred to PBS, and transverse slices from temporal hippocampus were immediately cut using a VT1000 vibrating-blade microtome (Leica Microsystems, Bannockburn, IL, USA) and placed in PBS for up to 4 days, pending dye injection. Under visual inspection, Lucifer Yellow (LY; mw = 457; Molecular Probes, Eugene, OR, USA) was iontophoretically injected into the weakly fixed CA3pyr neurons using sharp electrodes, according to our published methods (Rodriguez et al., 2003). Detailed methods for imaging and confocal reconstruction are also described elsewhere (Rodriguez et al., 2006, 2008). These samples for 3-D morphological analysis were secondarily found to be useful for analysis of dye-coupling, as shown in this report.
Thin-Section Transmission Electron Microscopy
Ten adult Sprague-Dawley rats (age 6 weeks to 6 months) were deeply anesthetized by intraperitoneal injection of 0.3–0.5 ml of 30% chloral hydrate in distilled water and perfused with warm 1% formaldehyde prepared from freshly depolymerized paraformaldehyde in 2 mM CaCl2, 4 mM MgSO4, and 0.1 M sodium cacodylate buffer (pH 7.25, 38°C). After 1 min, the fixative was switched to 38°C 2.5% formaldehyde and 3% glutaraldehyde in the same buffer for 12 min. The CA3 region was cut from six brain slices, post-fixed with 2% OsO4 in phosphate buffered saline (PBS, 4°C) for 2 h in the dark, washed in sodium acetate buffer, and stained en bloc with aqueous unbuffered 0.5% uranyl acetate at 10°C for 2 h in the dark (modified from Rash and Fambrough, 1973). After washing, tissue slices were dehydrated in ethanol, then propylene oxide, infiltrated with Araldite-Epon plus 1.5% DMP-30 catalyst (Araldite 502/EMbed 812 Kit, Electron Microscopy Sciences, Hatfield, PA, USA), and polymerized at 60°C for 48 h. A series of 50 ultrathin sections (50–90 nm thickness) were cut using a Reichert Ultracut E ultramicrotome (Reichert-Jung, Nussloch, Germany), mounted on Formvar-coated slot grids (Electron Microscopy Sciences, Hatfield, PA, USA) and stained for 45 min with aqueous unbuffered 1% uranyl acetate and 3 min with Reynolds’ lead citrate (procedure modified from Friedrich and Mugnaini, 1981). Approximately 45,000 μm2 of stratum lucidum were examined at 20,000–30,000× magnification at 80 kV in a JEOL 1200EX electron microscope (JEOL USA, Peabody, MA, USA) and photographed using a 2000 × 2000-pixel Advantage CCD camera (Advanced Microscopy Techniques, Danvers, MA, USA). Images were processed using Adobe Photoshop CS2 (Adobe Systems, San Jose, CA, USA), with “Levels” used for maximal contrast expansion, and both “Levels” and “Brightness/Contrast” used to optimize image contrast and definition over large areas within individual images (i.e., equivalent to wide area “dodging”). In montages, composite photographs were matched using contrast and brightness over each entire image component.
FRIL Transmission Electron Microscopy
Six male Sprague-Dawley rats (147–479 g) were deeply anesthetized by intraperitoneal injections of ketamine and xylazine (120–160 and 12–16 mg/kg, respectively) and fixed by whole-body vascular perfusion with 2 or 4% formaldehyde in Sørensen’s phosphate buffer according to our published methods (Hudson et al., 1981; Rash et al., 2005). Coronal and horizontal slices of hippocampus were cut at 100 μm thickness using a refrigerated Lancer Vibratome 3000 (now sold by Leica Microsystems, Inc., Buffalo Grove, IL, USA) that maintained samples at 4°C during slicing (to minimize lipid leaching and the formation of IMP-free lipid blebs; Shelton and Mowczko, 1979). Unfixed hippocampal samples for labeling NR1 and AMPA glutamate receptors (but not gap junctions) were obtained from one adult male Sprague-Dawley rat (585 g) that was anesthetized and decapitated. The brain was quickly removed, the hippocampus was dissected free, chilled to 4°C, 200 μm-thick slices were obtained using a McIlwain Tissue Chopper (Stoelting Co, Wood Dale, IL, USA), the slices were placed in 34°C tissue culture medium, placed on aluminum freezing supports, and ultrarapidly frozen using a Gentleman Jim metal-mirror freezer (Phillips and Boyne, 1984).
Freeze-fracture replicas were made according to our published methods (Rash and Yasumura, 1999). Immediately after fracturing, the samples were pre-coated with a nominal 1–1.5-nm of carbon (i.e., 6–10 atoms thick), which acts as a “wetting agent” for the subsequent coat of ca. 1.5 nm of platinum (Furman et al., 2003). This carbon “tinning” pre-coat provides for improved resolution. However, if the carbon pre-coat is too thin and discontinuous, immunogold labeling efficiency (LE) is reduced, as described and illustrated below, whereas if too thick, replica resolution is reduced, also as described below. After platinum shadowing, a second, 10–20 nm thick coat of “backing” carbon was applied to complete the replica film. The replicated but still frozen samples were bonded to a gold “index” grid using 2.5% Lexan (polycarbonate plastic) dissolved in dichloroethane; the solvent was evaporated at −30°C; and the Lexan-stabilized replicas were thawed and photomapped with a Zeiss LSM510 META laser scanning confocal microscope (Rash et al., 1995, 1997), then washed 29 h at 48.5°C in 2.5% SDS detergent in 0.16% Tris–HCl buffer (pH 8.9; Rash and Yasumura, 1999, as modified in Kamasawa et al., 2006). To minimize re-deposition of glutamate receptors that are dissolved from the bulk tissue slice, with consequent detection as a major source of background “noise” (Rash and Yasumura, 1999), samples were washed in multiple successive wells of SDS detergent.
After rinsing, one replica was single-labeled with rabbit polyclonal antibody to Cx36 (Ab298; characterized in Rash et al., 2000, and further characterized in Pereda et al., 2003; seven gap junctions found; H1–H7; see Table 1); and one replica was triple-labeled for Cx36 (Ab298) and mouse monoclonal antibodies to Cx45 (courtesy of Steven Coppen; as characterized in Coppen et al., 2003) and NMDA receptor subunit NR1 (NR1; Pharmingen #60021a; now BDBiosciences, San Jose, CA, USA; catalog # #556308; nine neuronal gap junctions found; H8–14, H21, and H22). (No gap junctions labeled for Cx45 were detected, but the apparent absence of Cx45 labeling using this one antibody was not considered sufficiently definitive to conclude absence vs. relatively low abundance of that connexin in hippocampal tissues.) To address then-current controversies regarding whether Cx32 might represent a neuronal connexin (Alvarez-Maubecin et al., 2000; Colwell, 2000) vs. a glial connexin (Rash et al., 2001), one replica was double-labeled for Cx36 + Cx32 [rabbit polyclonal Cx36 ab 36-4600, Invitrogen/Zymed Laboratories; now Life Technologies, Grand Island, NY, USA; and anti-Cx32 monoclonal antibody MAB3069, Chemicon; now EMD Millipore, Temecula, CA, USA; two Cx36-labeled neuronal gap junctions found (H17 and H18), plus >20 Cx32-labeled oligodendrocyte gap junctions (not shown here, but relevant data and images were previously shown in Kamasawa et al., 2005), thereby demonstrating the efficacy of the anti-Cx32 labels and providing further evidence that Cx32 is present solely in oligodendrocyte gap junctions and is not present in neuronal gap junctions (Rash et al., 2001; Nagy et al., 2004)]. One sample was double-labeled or Cx36 (#51-6300; Invitrogen; now Life Technologies) plus GluR2 AMPA glutamate receptors (MAB397, Chemicon; now EMD Millipore; gap junction H20). Three samples were double-labeled for both NMDA-R1 and for AMPA glutamate receptors (“pan-AMPA” anti-GluR1-4; courtesy of Elek Molnar, University of Bristol, Bristol, UK), to identify the fraction of E-face PSD clusters that contain glutamate receptors and to determine if NMDA and AMPA subunits are co-localized within individual PSDs. Finally, one replica was double-labeled for Cx36 (Invitrogen/Life Technologies #37-4600) plus “Cx35/Cx36” (“phospho 276” antibody; courtesy of John O’Brien; University of Texas Houston Medical School; H19). (Cx35 is the fish ortholog of Cx36, and these two antibodies were made to two different shared but non-overlapping amino acid sequences of Cx35 and Cx36; O’Brien et al., 2004, 2006). Double-labeling for Cx35 and for Cx36 was used as an internal control to document the specificity of FRIL labeling of connexins within the same gap junction hemiplaque. Two gap junctions on unidentified membrane fragments (probable astrocyte fingers) that had partially overlapping clumps of gold beads for Cx36 were designated as “false positive labeling” or “noise” (Rash and Yasumura, 1999; H15, H16) and, hence, were excluded from this analysis. All primary antibodies were diluted to 10 μg/ml.
Table 1. Twenty numbered anti-Cx36-immunogold-labeled and grid-mapped gap junctions in adult rat hippocampus, with their anatomical locations, size ranges (number of connexons, if countable), and their associations with labeled and unlabeled glutamate receptor PSDs (as tight patches of 10-nm E-face IMPs) vs. dispersed 10-nm E-face IMPs that are thought to represent extrasynaptic or “reserve” receptors.
After primary labeling, replicas were rinsed and counter-labeled for 17 h using various combinations of goat anti-rabbit immunoglobulin-G (IgG) conjugated to 6-nm and/or 18-nm gold beads (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and goat anti-mouse IgG, conjugated to 12-nm gold beads (Jackson ImmunoResearch), according to our detailed methods (Kamasawa et al., 2006). Samples double-labeled for NR1 and pan-AMPA were labeled with 10 and 20-nm gold, respectively (both from Chemicon International Inc., as reported in Rash et al., 2005; Chemicon antibodies now available from EMD Millipore).
After labeling and rinsing but before TEM viewing, the immunogold-labeled samples were coated a third time with 20–30 nm of carbon on the labeled side to: (a) surround and immobilize gold beads, (b) anneal thermal-expansion cracks in the replica prior to removal of the Lexan support film, thereby helping to maintain replica integrity, and (c) prevent displacement of the replicas with respect to the grid during subsequent removal of the Lexan support film. The Lexan support film was then removed by immersing the grids in 60°C dichloroethane for 10–18 h. FRIL samples were examined at 100 kV in a JEOL 2000 EX-II TEM (JEOL, USA). Stereoscopic images obtained with an 8° included angle were used for: (a) assessing complex 3-D membrane topography, (b) confirming that each immunogold bead was on the tissue-side of the replica (Rash and Yasumura, 1999), and (c) discriminating the smaller (6-nm) gold beads from the similarly electron-opaque platinum caps on 5- to 10-nm IMPs (Pereda et al., 2003; Rash et al., 2004). Every immunogold-labeled gap junction found by FRIL was examined at multiple tilts, and where needed, at multiple rotations (i.e., to obtain optimum views of important features, such as PSDs and interiors of spines). Freeze-fractured neuronal and glial processes were identified according to our published criteria (Rash et al., 1997, 2004). FRIL TEM negatives were digitized by an ArtixScan 2500f digital scanner (Microtek; Santa Fe Springs, CA, USA) and processed using Adobe Photoshop CS2 (Adobe Systems, San Jose, CA, USA), with “Levels” used for maximal contrast expansion, and “Brightness/Contrast” used to optimize image contrast and definition.
Labeling Efficiency, Signal-to-Noise Ratio, and Confidence in IMP Labeling
Factors affecting LE
As noted in our previous reports (Pereda et al., 2003; Nagy et al., 2004; Ciolofan et al., 2006; Kamasawa et al., 2006), two or even three sizes of gold labels for a single connexin provide internal controls for assessing labeling specificity of the primary antibody, as well as for assessing LE of each size of immunogold-conjugated secondary antibody. Likewise, using two different primary connexin antibodies (each with a different size of gold-tagged secondary antibody) allows determination of different LEs for different primary antibodies, but more importantly, provides great confidence in assigning a particular connexin to an individual, ultrastructurally identified gap junction hemiplaque (in this case, only neuronal gap junctions) but not to any other ultrastructural feature.
Labeling efficiency in FRIL is defined, not as the number of gold beads vs. number of copies of a specific membrane protein, but instead, LE is defined as the number of gold beads vs. number of IMPs in a target array (Kamasawa et al., 2006), regardless of whether or not other transmembrane or scaffolding proteins may also be present. For example, astrocyte gap junctions have three different connexins (Cx43, Cx30, and Cx26; Rash et al., 2001; Nagy et al., 2003), each with a different LE. The sum of the three connexin LEs provides a relative measure of the overall LE for those gap junctions.
Many factors affect LE, including whether or not a very thin (1–5 nm) “pre-coat” of carbon is applied before platinum replication (Masugi-Tokita and Shigemoto, 2007; Kasugai et al., 2010) or in areas of high replica contour, where carbon is present without platinum (Rash and Yasumura, 1999; Kamasawa et al., 2005; Schlörmann et al., 2007), as illustrated below to document that glutamate receptor IMPs far outnumber their immunogold labels. Conventionally shadowed freeze-fracture replicas [i.e., those shadowed with 1–2 nm of platinum (Pt), followed by 10–30 nm of carbon (C)] typically have LEs between 1:10 and 1:100 (i.e., one gold bead per 10 connexons vs. one per 100 connexons, respectively). However, in samples “pre-coated” with carbon, or in areas where local contour prevented deposition of platinum, LE can be as high as 1:1 (Masugi-Tokita and Shigemoto, 2007; Kasugai et al., 2010), although it should be noted that those authors defined LE as the number of gold beads vs. the number of electrophysiologically determined receptor ion channels, and not with respect to number of IMPs. For tightly packed multi-subunit IMPs, such as connexons (which have center-to-center spacings of ca. 10-nm) and aquaporin-4 arrays (in which the subunit particles have center-to-center spacings of 6.5 nm), this theoretical upper limit for LE of 1:1–1:4 is reached because each primary antibody molecule (usually bivalent IgG; 8 nm × 8 nm × 12 nm; Valentine and Green, 1967) can be conceived of as a barrel that occupies a planar area equal to or greater than the entire surface of the target IMPs in closely packed arrays, precluding binding of multiple primary antibodies per IMP. Moreover, secondary antibodies, whether fluorescently tagged or gold-tagged, further enlarge the labeling complex, with resulting steric hindrance making it impossible to have LEs for tightly clustered IMPs (e.g., gap junctions, AQP4 arrays) greater than ca. 1:1, either by fluorescence microscopy or by FRIL. However, for loosely packed IMPs, such as glutamate receptors, which are separated by 20–30 nm, several antibody “barrels” can be fitted radially in the spaces around each IMP, allowing the potential for LEs ≥1:1.
Factors affecting detectability of different sizes of gold beads
Small gold beads are difficult to discriminate against Pt-coated 8–10 nm IMPs, which may be of greater electron density than the 6-nm gold labels. Thus, stereoscopic viewing is required for positive recognition of small gold beads beneath the replica, as illustrated below. (Recognition of small gold beads is not a problem for thin-sections because there are few if any 6-nm electron-dense granules, except in defective samples having precipitated staining reagents.) Nevertheless, in FRIL, secondary antibodies that are attached to smaller gold beads (i.e., 5–6 nm) have 8–16 times greater LE than 18- to 30-nm larger gold beads (Nagy et al., 2004). On the other hand, larger gold beads (≥18-nm) have such high image contrast and are so large that they are readily detected in low magnification (10,000×) “scans” of the replica (i.e., they act as “flags” or “beacons”). Thus, in FRIL, the use of a combination of large and small gold beads as labels for the same primary antibody gives the dual advantages of increased LE of the small gold beads and increased detectability of the large gold beads. However, a disadvantage of large gold beads, especially at high LE, is that the gold beads often completely obscure the labeled IMPs. Thus, for finding and quantifying target IMP clusters (i.e., gap junctions and glutamate receptor PSDs), we strive for what we consider an “optimum LE” of ca. 1:10, usually by adjusting antibody concentrations and labeling times.
Disadvantages of a carbon “pre-coat”
Vapor-deposited carbon is extremely adsorptive (Dinchuk et al., 1987), and this adsorptivity is the basis for the SDS-FRL technique, which permits labeling of the membrane proteins that remain adsorbed to the C- or Pt/C-replica following detergent washing (Fujimoto, 1995). However, the highly adsorptive carbon pre-coat greatly increases both “signal” (immunogold labeling) and the non-specific adsorption of both primary and secondary antibodies and of any proteins displaced and readsorbed during washing, all of which provide major sources of labeling “noise.” Indeed, we have found that along with increased labeling times, increased thickness of the carbon pre-coat, greater volume of samples/increased release of proteins, inadequate number or volume of detergent or water rinses, or by the use of ineffective blocking buffers, noise often increases faster than signal. Thus, even with a lower LE, manipulation of these factors creates the potential for improving signal-to-noise ratio (SNR). Often, the highest SNR occurs when LE is ca. 1:10 (Rash and Yasumura, 1999; Kamasawa et al., 2005). Failure to optimize LE and SNR results in excessively high “background” and, in the absence of stereoscopic analysis, the inability to assign individual gold beads as either label (signal) or noise.
A separate issue arising for samples rotary-coated with a 5-nm-thick layer of carbon prior to platinum deposition (Masugi-Tokita and Shigemoto, 2007; Kasugai et al., 2010) is that the sizes of all IMPs are increased by 10 nm (i.e., 5 nm of carbon on both sides of an IMP, prior to platinum shadowing), thereby converting nominal 5-nm IMPs to 15-nm IMPs, and 10-nm IMPs to 20-nm IMPs, completely disguising factors that allow approximate molecular weight assignment (Eskandari et al., 1998; Rash et al., 2004). Equally important, membrane pits are often partially or completely obliterated by carbon pre-coating, making it impossible to demonstrate “complementarity” of replica faces (Steere and Moseley, 1969; Challcroft and Bullivant, 1970), an essential element for determining whether a layer of pure “pre-carbon” is present vs. a layer of water vapor contamination. When water vapor contamination is present, adsorptivity of membrane proteins to the carbon layer is reduced, but in addition, the platinum and carbon layers often separate, resulting in fragmentation of some replicas. Also important, when a thick layer of pre-carbon is present, the variably increased IMP sizes makes it difficult to compare data regarding IMP identifications made by different laboratories, and even makes it difficult to discriminate between nearby IMPs differing by as much as 5 nm (Masugi-Tokita et al., 2007), but which in conventional freeze-fracture replicas are easily distinguished (Rash et al., 1979; Rash and Giddings, 1989). Based on the above, we typically use a nominal 1–1.5 nm thick carbon pre-coat (thereby not significantly increasing IMP diameter or significantly decreasing the width or depth of membrane pits), realizing that the replicas will have a slightly reduced LE but improved SNR. For clarity, we illustrate in this report how each of these factors affects replica quality and LE.
Recognition of “noise” and determination of SNR
In our previous reports, we defined SNR as the number of gold beads per unit area of target structure (e.g., gap junction or PSD) vs. number of gold beads on a representative area of non-target structures (typically nucleoplasm, extracellular space, and plasma membranes of different cell types). In “acceptable” FRIL replicas, there are few if any “background” gold beads, yielding SNR = 5000:1–50,000:1 (Meier et al., 2004). Using stereoscopic viewing, we also identified “definitive noise” as any gold bead above the Pt/C-replica, on the side formerly coated with Lexan, where no specific labeling is possible (Rash and Yasumura, 1999). In samples whose non-specific labeling was minimized due to use of adequate “labeling blocking buffers” (Dinchuk et al., 1987), >90% of gold bead “noise” was on the (formerly) Lexan-coated side of the replica, rather than on the tissue-side. When present in our images, gold beads as definitive noise are designated by a white circle with anoblique cross bar (⃠) stereoscopically superimposed over the offending gold bead.
Advantages and disadvantages of FRIL vs. SDS-FRL
“Freeze-fracture replica immunogold labeling” was named by Gruijters et al., 1987, almost a decade before Fujimoto’s landmark report describing sodium dodecylsulfate-digested freeze-fracture replica labeling (SDS-FRL; Fujimoto, 1995), which allows visualization and high-resolution immunogold labeling of diverse membrane proteins in broad expanses of biological membranes. However, SDS-FRL utilized vigorous immersion-washing of unsupported replicas, which resulted in severe fragmentation that precluded histological-scale mapping of complex CNS tissue, which is the object of our research. With its defining additional step of Lexan-stabilization for high-magnification confocal “grid-mapped freeze-fracture” (GMFF) of samples prior to washing and immunogold labeling (Rash et al., 1995; Rash and Yasumura, 1999), we designated the combined technique as FRIL, in deference to the original FRIL method (Gruijters et al., 1987).
Although progress has been made in developing labels for a few types of neurons for SDS-FRL (Masugi-Tokita et al., 2007), most classes of neurons in hippocampus currently cannot be positively identified by any freeze-fracture technique. An additional disadvantage is that, unlike methodical serial-section reconstruction afforded by tsTEM, conventional freeze fracturing results in a single, essentially random fracture, potentially at any depth within the tissue slice. This near-randomness of the fracture plane, combined with searching for immunogold “flags” rather than for specific cell appositions, reduces the potential for investigator bias in the semiquantitative analysis of gap junctions along different types of cell processes (e.g., CA3pyr dendrites vs. interneuronal dendrites for example). Nevertheless, because flatter membrane expanses are preferentially cleaved over highly convoluted membranes (e.g., thorny excrescences), FRIL may result in overestimates of gap junctions on the flatter, elongate dendritic shafts, as well as underestimates of gap junctions within convoluted membranes, such as thorny excrescences. Indeed, by FRIL, we have as yet found no Cx36-labeled gap junctions in the small portions of ca. 10 positively identified thorny excrescences. However, FRIL analysis using highly visible 18-nm gold beads allows sampling over large areas and relatively unbiased quantification of the labeled structures encountered. Finally, additional sources of sampling bias for both tsTEM and for FRIL include: (a) reduced detection of the smallest gap junctions (particularly those smaller than 30 connexons and therefore having few or no large gold beads to act as “flags”), and conversely, (b) a relative overestimation of the proportion of the large gap junctions that were the subjects of previous reports (Kosaka, 1983; Kosaka and Hama, 1985; Katsumaru et al., 1988; Fukuda and Kosaka, 2000).
Pyramidal Cell-Mossy Fiber Dye-Coupling
Based on the need for high-resolution 3-D reconstructions of CA3pyr for our computer modeling, we injected the strongly fluorescent dye Lucifer Yellow (LY) under direct microscopic visualization into lightly fixed CA3pyr slices from adult rat ventral hippocampus (see Materials and Methods). Serendipitously during image analysis, LY was occasionally noted to reveal apparent CA3pyr-to-MF dye-coupling, apparently at MF axon contact areas within thorny excrescences (Figure 1), as we later confirmed by tsTEM of conventionally fixed samples (Figure 2). However, dye-coupling in these weakly fixed slices occurred at a much lower frequency than that demonstrated between adjacent CA3pyr in living tissue slices injected with LY (MacVicar and Dudek, 1980; Andrew et al., 1981). Using maximum intensity projection of confocal laser scanning microscopy image stacks, we examined ca. 500 dye-loaded CA3pyr, four of which (∼1%) had dye-loaded smooth processes or thin processes with varicosities, but in each case, clearly contacting thorny excrescences in their proximal apical dendrites (Figures 1A,D). Most of the smooth dye-labeled processes were oriented approximately perpendicular to the spiny apical dendrites of CA3pyr, and all four were in the stratum lucidum, with none in the infra-pyramidal bundles in stratum oriens. The smooth processes were either single (n = 1, Figures 1B,C) or appeared in bundles (n = 3, Figure 1E). Based on the morphology and location, we identified these fine-diameter smooth processes as MF axons with varicosities. Their identity as MF axons was further confirmed by high-resolution 3-D morphological reconstruction (Figures 1B,C,E) of local dilatations that had the shape and large size of MF boutons (Figures 1B,C; double arrows). Moreover, the dye-coupled MF axons established direct contacts with dye-injected CA3pyr dendrites (Figure 1B) or with their smaller spine-covered dendrites (Figure 1E, lower left arrowhead), potentially revealing the sites for gap junctional dye transfer. [Note: As in CA1pyr (Megías et al., 2001), the proximal 10–20 μm of CA3pyr dendrites are essentially devoid of spines (Figure 1A inset, yellow brackets), providing a potential confounding factor in the FRIL identification of mixed synapses on proximal dendritic shafts of CA3pyr vs. aspiny interneurons.] Overall, the relatively low frequency of dye transfer detected (ca. 1%) may reflect the use of the classical dye, LY (457 mw), which is slightly above or very near the cutoff molecular weight for gap junction-permanent dyes in vertebrate species (ca. 450 mw; Hu and Dahl, 1999). We are also aware that the rate of permeation of LY though open vs. closed connexon channels may be significantly reduced by even brief, weak formaldehyde-fixation. However, the improvement in structural detail, as well as the reduced likelihood of simultaneously impaling and injecting two cells, gives this approach certain added advantages. Thus, we suggest that future experiments by others should simultaneously inject LY and Neurobiotin (286 mw, Kita and Armstrong, 1991) to better assess the rate and extent of dye transfer for both dyes though gap junctions in living vs. briefly fixed CA3pyr neurons.
Figure 1. Dye-coupling between lightly fixed CA3pyr neurons and MF axons. (A) Maximum intensity projection of confocal z-stack showing a LY-injected CA3pyr and dye-coupled MF axons (arrows). Arrowheads, CA3pyr dendrites. Double arrow, CA3pyr axon. Inset, Higher magnification view of the bases of several dendrites, showing that few if any spines occur within 10–20 μm of the soma (yellow brackets). (B,C) High-resolution 3-D reconstructions (in two opposite z directions) of the boxed area in A showing CA3pyr dendrites dye-coupled to MFs. Arrows, MFs. Double arrow, MF boutons. Arrowheads, CA3pyr dendrites. (D) Maximum intensity projection of a confocal z-stack showing two LY-injected CA3pyr. (E) High-resolution 3-D reconstruction of the boxed area in (D) showing CA3pyr dendrite dye-coupled to a bundle of MF axons (single right arrow). The left two arrows mark MF axons that appear to be dye-coupled to CA3pyr dendritic spines at a different coupling site than the coupling site of the MF bundle. Arrowhead, CA3pyr dendrite. Scale bars are 10 μm.
Figure 2. Thin-section TEM evidence for MF-CA3pyr mixed synapses. (A1) Image showing part of a CA3pyr dendrite (Dend) and adjacent MF boutons (mfb) and MF axons (mfa), with multiple dendritic spines (asterisks). (A2) Higher magnification of boxed area in (A1). A typical large-diameter MF bouton containing numerous synaptic vesicles surrounds a CA3pyr dendritic spine at a synaptic contact containing both close membrane appositions characteristic of gap junctions (whitearrows) and wider membrane appositions with asymmetric dense cytoplasmic material (arrowheads) characteristic of postsynaptic densities (PSDs). Often these PSDs were opposite clustered 50-nm-diameter round synaptic vesicles that characterize glutamatergic chemical synapse. Black arrow, spine neck. (A3) Higher magnification of boxed area in (A2) showing the gap junction contact (white arrow) between the MF bouton and the CA3pyr dendritic spine. Synaptic vesicle (black arrowhead). (B1) Image showing a CA3pyr dendrite (Dend) and adjacent MF boutons (mfb) and CA3pyr dendritic spines (asterisks) within a complex thorny excrescence. (B2) Higher magnification of boxed area in B1. At this MF synapse with a CA3 dendritic spine, a five-layered gap junction contact [arrow in (B2) boxed area, enlarged as (B3)], as well as PSDs (arrowheads) of the closely associated chemical synapse. (B3) Higher magnification of boxed area in (B2) showing a putative gap junction contact (arrow) between MF bouton and CA3pyr dendritic spine, adjacent to the PSD of a chemical synapse (arrowhead). (C1) Image showing part of a CA3pyr dendrite thorny excrescence, with several CA3pyr dendritic spines (asterisks) and adjacent MF boutons (mfb) and MF axons (mfa). (C2) Higher magnification of boxed area in (C1). MF bouton containing numerous 50-nm round synaptic vesicles surrounding a CA3pyr dendritic spine. The synaptic contact contains a five-layered membrane apposition, identified as a gap junction (arrow), and several PSDs of a chemical synapse (arrowheads). (C3) Higher magnification of boxed area in (C2) showing a presumptive gap junction (arrow) between a MF bouton and a CA3pyr dendritic spine. Scale bars in all tsTEM and FRIL micrographs are 0.1 μm, unless otherwise indicated.
Gap Junctions between Mossy Fiber Terminals and Pyramidal Cell Dendritic Spines
Using tsTEM as the preferred method for detecting gap junctions in thorny excrescences, we examined an estimated 45,000 μm2 of CA3b stratum lucidum from adult rat ventral hippocampus in ca. 50 ultrathin sections (ca. 30 μm × 300 μm in each section). Five synapses with close membrane appositions that we designated as putative gap junctions were found between the glutamatergic MF terminal boutons and branched CA3pyr dendritic spines within classical thorny excrescences (Figure 2; dendritic spines indicated by asterisks). In four of these five glutamatergic MF terminals, components of chemical synapses [i.e., PSDs opposite clusters of 50-nm synaptic vesicles at presynaptic electron densities of presumptive active zones (Figures 2A2,B2,C2, arrowheads point to PSDs)] were observed in close proximity to putative gap junctions (Figures 2A3,B3,C3; arrows), thereby defining these glutamatergic synapses as probable mixed synapses. In the fifth example (not shown), the presumptive gap junction was on the neck of a spine of a CA3pyr dendrite, with the spine surrounded by and enclosed within a MF terminal, also consistent with its classification as a glutamatergic mixed synapse on a spine within a thorny excrescence. Within these putative gap junctions, the plasma membranes showed a pentalaminar configuration, with a total thickness of 18–20 nm, characteristic of gap junctions in many other tissues prepared by similar fixation and staining methods (Staehelin, 1974, also Kosaka, 1983; Kosaka and Hama, 1985; Fukuda and Kosaka, 2000). However, the small gap junctions described above lack the “semi-dense cytoplasmic matrix” found in those larger neuronal gap junctions (Sotelo and Korn, 1978; Kosaka, 1983; Kosaka and Hama, 1985; Fukuda and Kosaka, 2000). These presumptive gap junctions had profiles ranging from 35 to 70 nm (mean = 53 nm) in width, suggesting that they contain only a few dozen connexons, as compared to the 200–600 nm-diameter dendrodendritic gap junctions reported in hippocampal interneurons (Kosaka and Hama, 1985), that due to their larger sizes, we estimate to contain 400 to >3000 connexons.
FRIL Analysis of Mixed Synapses in Adult Rat Hippocampus: Proximity of Cx36-Containing Gap Junctions and Glutamate Receptor-Containing PSDs
In freeze-fracture replicas of adult rat hippocampus, neurons, and glia were identified according to established criteria (Rash et al., 1997). Using FRIL, we identified 20 neuronal gap junctions scattered throughout adult rat hippocampus (Table 1), all of which were found because they were labeled for Cx36 by the high-visibility 18-nm gold “flags.” Although additional gap junctions were found on glial cells, no unlabeled neuronal gap junctions were found, suggesting that Cx36-labeled most if not all neuronal gap junctions in these samples.
In a sample that was triple-labeled for Cx36, Cx45, and NMDA-R1 glutamate receptor subunits (6 and 18-nm gold beads for Cx36 and 12-nm gold beads for both Cx45 and NMDA-R1; Figures 3A–C), no gap junctions labeled for Cx45 were found. However, nine Cx36-labeled gap junctions were found, including one large axodendritic gap junction (Figure 3B, blue overlay; from stratum oriens). Its ∼360 connexons were labeled by 21 6-nm and six 18-nm gold beads, the latter of which are easily detected even at low magnification (Figure 3A). (See Materials and Methods for multiple rationales for using two sizes of gold beads for Cx36.) This large crystalline plaque gap junction was on a dendrite (red overlay) of either a spiny interneuron or of a CA3pyr, the latter possibility suggested by its location within stratum oriens, abundance of attached larger-diameter axon terminals covering most of its surface (purple overlays), and by its continuity with a branched spine (Figure 3C, arrow) that may represent the base of a thorny excrescence. In stratum oriens, thorny excrescences are found only on CA3pyr cells, at contacts between the infra-pyramidal MF axons and CA3pyr basal dendrites (Gonzales et al., 2001), but not on interneurons (Amaral, 1978; Scharfman, 1993). The base of a second probable thorny excrescence (Figure 3A, branched red overlay at bottom) may also represent a portion of the same dendrite, but a 20-nm break in membrane continuity (narrow break between red overlays) prevented its certain identification. Thus, these images are consistent with the identification of this medium-size dendrite as a portion of either a spiny interneuron or a CA3pyr. However, because the abundant spines of CA3pyr distal dendrites are much more numerous and are often club-shaped, whereas the spines of spiny interneurons are often branched tubes (as seen here), and most of their synapses are onto their dendrite shafts (as illustrated next), we interpret this glutamatergic mixed synapse as on a spiny interneuron in stratum oriens.
Figure 3. Freeze-fracture replica immunogold labeling images of a spiny dendrite in stratum oriens, with gap junction double-labeled for Cx36 (6 and 18-nm gold beads