Correlative Microscopy of Cerebellar Molecular Layer Postsynaptic Proteins and Postsynaptic Receptors

Correlative microscopy of cerebellar molecular layer allowed us to image the localization of postsynaptic receptors using a comparative and intermicroscopy study applying transmission electron microscopy (TEM), freeze-etching and direct replicas for transmission electron microscopy (FFTEM), field emission scanning electron microscopy (FESEM), and confocal laser scanning microscopy (CLSM) using immunohistochemistry of Synapsin-I and PSD-95, immunohistochemistry of Ca2+/calmodulin dependent protein kinase II alpha and immunohistochemistry GluR1 subunits of AMPA receptors alpha. These techniques showed the images of postsynaptic densities of parallel fibers-Purkinje dendritic ramifications containing postsynaptic proteins and glutamate postsynaptic receptors. The freeze-etching replica method for TEM showed the three-dimensional structure and intramembrane morphology of postsynaptic ellipsoidal and spheroid intramembrane particles. Confocal laser scanning microscopy using Synapsin-I (Syn-I) and PSD-95, GluR1 subunits of ionotropic glutamate receptors, and Ca2+/calmodulin dependent protein kinase II alpha contribute to image the precise localization of postsynaptic receptors. The high resolution FESEM showed 25-50nm globular subunits at the spine postsynaptic density corresponding to the localization of postsynaptic proteins and/or postsynaptic glutamate receptors.


Introduction
Purkinje dendritic spines have been studied for more than one century since Ramon and Cajal pioneering studies with the light microscope. The advent of electron microscopy and the freeze etching technique made possible to visualize spine morphology and its intramembrane morphology [1][2][3][4][5][6]. The development of confocal laser scanning microscopy and the immunohistochemistry techniques [7] allowed us to study the domains of mayor proteins inside the postsynaptic region of Purkinje dendritic spines [8][9][10].
The postsynaptic density (PSD) is a dynamic multi-protein complex attached to the postsynaptic membrane composed of several hundred proteins such as receptors and channels, scaffolding and adaptor proteins, cell-adhesion proteins, cytoskeletal proteins, G-proteins and their modulators and signaling molecules including kinases and phosphatases [11][12][13]. Glutamate receptors are the most abundant excitatory neurotransmitter receptors in the brain, responsible for mediating the vast majority of excitatory transmission in neuronal networks. The AMPA-and NMDAtype ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate the fast-synaptic responses, while metabotropic glutamate receptors (mGluRs) are coupled to downstream signaling cascades that act on much slower timescales [14][15]. Tao et al. [16] have developed an integrative approach combining cryo-electron tomography and correlative fluorescence microscopy to visualize 3D ultrastructural features of intact excitatory and inhibitory synapses in their native state. Yun-Hong et al. [17], by using the immunoabsorption method, reported binding partner proteins of two proteins residing in the interiors, PSD-95 and α-tubulin, and those of two proteins residing in the peripheral regions, elongation factor-1α and calcium, calmodulin-dependent protein kinase II α subunit, of cerebral and cerebellar postsynaptic densities (PSDs). Nanoscopy studies, ranging from quantification of receptors and scaffolding proteins in postsynaptic densities and their dynamic behavior, to imaging of synaptic vesicle proteins and dendritic spines in living neurons or even live animals has been carried out by means of super resolution microscopy [18].
In the present paper we used a combined intermicroscopy study of postsynaptic Purkinje dendritic spines by means of transmission electron microscopy and freeze etching technique, field emission scanning microscopy and confocal laser scanning microscopy immunohistochemistry methods in an attempt to get insight into the molecular composition of postsynaptic density and image postsynaptic receptors.

Transmission electron microscopy (TEM)
For transmission electron microscopy (TEM), slices 1-2mm thick of mouse cerebellar cortex were immediately fixed by immersion in 4% glutaraldehyde in 0.1 M phosphate buffer solution (pH 7.4) for 4-16h at 4°C; they were post fixed for1h in a similarly buffered 1 % osmium tetroxide solution, dehydrated through graded concentrations of ethanol and embedded in Araldite. Thin sections were stained with uranyl and lead salts and observed with a JEOL 100B electron microscope.

Freeze-etching and direct replicas for transmission electron microscopy (FFTEM) [3]
The freeze-etching direct replica technique was applied to the cerebellar cortex of adult Swiss albino mice. The brains were carefully removed, and thin 1-2mm slices of the cerebellar cortex were fixed in 1% ice-cold glutaraldehyde 0.1 M phosphate buffer, pH 7.2-7.4, for 1h. All cut pieces were immersed in three changes of 25% glycerol in a similar buffer for 30min, mounted on gold discs and frozen in Freon at N2 temperature for 3-5sec. They were immediately trans-ferred to a Balzer BAF-301 freeze-fracture unit equipped with an electron beam gun, at -110 °C, in a vacuum of 4 X 106 or better. Fractured surfaces were shadowed with a layer of carbon-platinum about 2.5 nm thick. Replicas were floated off on water, cleaned in Chlorox overnight, rinsed in water, bathed in 50% H2SC>4 and rinsed in multiple changes of water. Cleaned replicas were mounted on grids usually coated with parlodion or Formvar films and examined with a JEOL 100B electron microscope at 80kV.

Field emission scanning electron microscopy (FESEM) [4]
Albino mouse cerebellar cortex was excised, cut into 1-2 mm slices and immersed in 4% glutaraldehyde in 0.1 M phosphate buffer solution, pH 7.4, for 24 h at 4 °C; and post-fixed for 1 h in a similarly buffered 1 % osmium tetroxide solution.
Rapid freezing of packets was performed by plunging into LN2.
First, the packet was transferred from the LN2 storage vessel with LN2 chilled forceps in order to avoid thermal damage. Secondly, the cooled fracture blade was removed from the LN2, the packet was orientated under the blade, and immediately struck with a heavy tool. Fractured tissue fragments were transferred into chilled absolute etlianol (4 °C) and thawed. Tissues were loaded into fresh absolute ethanol filled mesh baskets within the boat of a Polaron E-3000 critical point drier. The boat was then loaded into the Tissues were loaded into fresh absolute ethanol filled mesh baskets within the boat of a Polaron E-3000 critical point drier. The boat was then loaded into the drier, and exchanged with CO 2 gas at a rate of 1.2 1/min. The CPD chamber was then thermally regulated to the critical temperature and decompressed at the same rate. Dried speci-mens (shiny face up), were mounted onto aluminium stubs 9mmX2mmX1mm for the ISI DS-130 SEM with silver paste and degassed at 2X107 torr prior to sputter coating.

Metal coating for FESEM imaging
Specimens were chromium coated with a continuous 1 nm film in a Denton DV-602 turbo pumped sputter deposition system operated in a vacuum of Argon at 5X103 torr.

High resolution field emission scanning electron microscopy [19]
Specimens were staged in-lens of an ISI DS-130F equipped with Schottky field emitter. The instrument was operated at accelerating voltages of 5kV in order to produce a small spot size and minimal specimen penetration while attaining excellent adequate signal to noise ratio at all magnifications. Images were digitally recorded in 116sec with TIF-files size of 4.8 megabytes, contrast processed with Adobe Photoshop.

Synapsin-I and PSD-95 immunohistochemistry [7]
Animals were used in accordance with NIH and institutional guidelines. For synapsin-I and PSD-95 immunohistochemistry, cerebellar cortex slices were derived from 14-21 days old rats.

Immunohistochemistry of Ca 2+ /calmodulin dependent protein kinase II alpha by means of confocal laser scanning microscopy [7]
Animals were used in accordance with NIH and institutional guidelines. For CaMKII alpha labeling, cerebellar cortex slices were derived from 14 -days-old postnatal (P14) rats anesthetized with CO 2 . After decapitation, the lateral lobules of cerebellum Transmission electron microscopy (TEM) shows the Purkinje tertiary dendritic ramifications emitting mushroom-shaped dendritic spines and making synaptic connection with the

Am J Biomed Sci & Res
Copy@ Orlando J Castejón, et al. 56 presynaptic ending of parallel fibers at the outer third cerebellar molecular layer (Figure 1).

The freeze-etching replica method for TEM
The freeze-etching replica method for TEM showed the three-dimensional structure and intramembrane morphology of Purkinje dendritic ramification and their spines. About 90 IMPs were observed in the spine head, and about 65 IMPs at the short neck ( Figure 2).

Synapsin-I (Syn-I) and PSD-95 immunohistochemistry of rat cerebellar cortex Dailey
The

Am J Biomed Sci & Res
Copy@ Orlando J Castejón, et al.

The immunohistochemistry of Ca 2+ /calmodulin dependent protein kinase II alpha (CaMKII)
The immunohistochemistry of Ca 2+ /calmodulin dependent protein kinase II alpha (CaMKII) in rat developing cerebellar cortex depicted the immunopositively reaction as small puncta at the outer third molecular layer corresponding to the localization of parallel fiber-Purkinje spine synapses ( Figure 6).

Am J Biomed Sci & Res
Copy@ Orlando J Castejón, et al.

High resolution field emission scanning electron microscopy
The field emission scanning electron microscopy showed the fractured presynaptic endings of cerebellar parallel fibers or granule cell axons in the cerebellar molecular layer containing the synaptic vesicles aggregated toward the presynaptic membrane, the homogenous dense presynaptic axoplasm and the SEI profile of nonspecialized presynaptic membrane (Figure 8).  The aggregated globular subunits can be observed at the postsynaptic ending as round substructures 8 ( Figure 10).

Transmission electron microscopy and freeze-etching replica technique
In earlier investigations we studied a freeze-fracture scanning electron microscopy and comparative freeze-etching study of parallel fiber-Purkinje spine synapses of vertebrate cerebellar cortex (Castejon,1990). Lately we reported a combined conventional and high resolution field emission scanning electron microscopy of vertebrate cerebellar parallel fiber-Purkinje spine synapses [4][5][6][20][21][22]. These previous studies demonstrated the potentiality and resolution power of correlative and inter microscopy studies and their related technique to approach to the study of synaptic receptors.
The hypothesis that some proteins of the postsynaptic membrane are locally synthesized at postsynaptic sites [23] is supported by our finding of aggregates of ribosomes found at the site of emergency of postsynaptic spines. We have used the TEM and freeze etching technique to study protein membrane morphology of postsynaptic spines and intramembrane particle distribution.
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 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 [24]. Immunogold labeling shows that the presynaptic active zone provides a scaffold for key molecules involved in the release of neurotransmitter, whereas the postsynaptic density contains ligand-gated ionic channels, other receptors, and a complex network of signaling molecules [13]. Freeze etching replicas exposed postsynaptic intramembrane particles that can be correlated with the globular subunits observed at high resolution FESEM.

Synapsin-I and PSD-95, GluR1 and CaMKII alpha immunohistochemistry
In the present study we have found positive immunoreaction for Synapsin-I and PSD-95, GluR1 and CaMKII. The GluR1 protein, a 106KDA glycoprotein, appears predominantly in synaptic plasma membranes, where it is enriched in the postsynaptic density [25]. The GluR1 forms Ca 2+ permeable channels and exhibit inward rectification [26,27]. CaMKII alpha presence in the main excitatory and inhibitory circuits of developing cerebellar cortex is presumably related with its participation in information, motor learning and memory processes. Our findings support the role of CaMKII as a molecular switch that is capable of storing long-term synaptic memory as postulated by Lismann et al. [28]. According to Hansel et al. [29] alpha CaMKII is essential for motor learning and long term potentiation on hippocampus, cerebral cortex and Purkinje cells of cerebellum. PSD 95, a main member of MAGUK family, interacts directly with carboxyl termini of NMDA receptor subunits and clusters them to the postsynaptic membrane. In addition, PSD 95 is involved in binding and organizing proteins connected with NMDAR signaling [12].

High resolution field emission scanning electron microscopy
In an intent to extract relevant information from the outer and Homer are perfectly suited to act as a major scaffold in postsynaptic density [12].
Previous studies by means of biochemical methods, electron microscopy and confocal laser scanning microscopy have localized the CaMKII at the level of postsynaptic density, being central to the regulation of glutamates synapses. According to Lisman et al. [28] previous work indicates that a binding pattern for CaMKII is the NMDA receptor within the postsynaptic density. The AMPA receptor subunit GluR1 is also phosphorylated by CaMKII enhancing channel function. Significant differences were found in protein composition and organization across PSDs from the different brain regions. The signaling protein, βCaMKII, was found to be a major component of each PSD type and was more abundant than αCaMKII in both hippocampal and cerebellar PSDs. The scaffold molecule PSD-95, a major component of cortical PSDs, was found absent in a fraction of cerebellar PSDs and when present was clustered in its distribution. In contrast, immunogold labeling for the proteasome was significantly more abundant in cerebellar and hippocampal PSDs than cortical PSDs. Together, these results indicate that PSDs exhibit remarkable diversity in their composition and morphology, presumably as a reflection of the unique functional demands placed on different synapses Significant differences were found in protein composition and organization across PSDs from the different brain regions. The signaling protein, βCaMKII, was found to be a major component of each PSD type and was more abundant than αCaMKII in both hippocampal and cerebellar PSDs [31].
To address the critical aspect of synaptic organizational dynamics, Li & Blanpied [32] performed single-molecule tracking of transmembrane proteins using universal point accumulation- for-imaging-in-nanoscale-topography (uPAINT) over PSDs whose internal structure was simultaneously resolved using photoactivated localization microscopy (PALM). Their results provide important experimental confirmation that PSD scaffold protein density strongly influences the mobility of transmembrane proteins.

Futures avenues for research on postsynaptic receptors
Because numerous proteins thought to be involved in