Agerelated Alterations of Synaptic Structural Dynamics

It is well demonstrated that the nervous system is capable of consistent structural adaptive changes throughout the individual's lifespan. Specifically, the synaptic terminal regions are in a very dynamic condition responsible for continuous remodeling interventions to optimize the critical role of the synaptic junctional areas in signal transduction and information processing. Synaptic plasticity is the commonly used term defining such a function-driven adaptive response of the synaptic contact zones, and its meaning includes the many different morphological, biochemical, molecular, and genetic changes occurring at synapses as a consequence of environmental stimulation.

During the last decades, the significant advancement in the accuracy and reliability of the investigative procedures has contributed to a better identification and to an improved quantitative analysis of function-related synaptic parameters, thus furthering our understanding of the basic mechanisms contributing to the fine tuning of synaptic transmission. From a morphological point of view, synaptic junctional areas undergo significant rearrangements of their ultrastructural features that mirror the functional status of the neural network where they are located, thus enabling an estimation of the adaptive capacities (i.e., plasticity) of selected CNS zones. In performing any quantitative morphological, or morphometric, study in a given biological tissue, collection of the data is a laborious time-consuming procedure since several histological sections, and perhaps zones within the tissue samples, must be analyzed and measured in order to obtain statistically comparable pools of findings between the experimental groups. Thus, until the late 1980s, analysis and measurement of tissue sections was a significant limiting step in performing these investigations.

The subsequent introduction in laboratory routine procedures of modern computer-assisted image analyzers, though facilitating and objectifying the morpho-metric procedures, resulted in a marked acceleration of data collection. The application of these procedures to the study of the aging nervous system has provided novel information on the modulation of synaptic architecture to respond to the changing functional needs

(Bertoni-Freddari et al., 1996). It is consistently documented that the staining procedures used in conventional electron microscopy to prepare the tissue samples for qualitative and quantitative analyses enable a better visualization of the whole cellular architecture. This is due to the fact that the traditionally used staining reagents bind ubiquitously to the biological molecules (namely, proteins and lipids), leaving the background completely unstained or faintly stained. The more or less dark and sharp contrast of the cellular structures depends on various factors and, besides the concentration and incubation time of the contrasting reagents (Miquel and Bertoni-Freddari, 2000), also includes the chemical composition of the cellular structure where the staining reaction occurs. In this context, the synaptic contact zones are functionally differentiated areas of the neuronal membrane where polyunsaturated fatty acids are abundant because of the presence of several double bonds between carbon atoms. This very specific feature, although constituting the chemical basis of their plastic condition for adequate functional tasks, is also the reason why pre- and postsynaptic membranes abundantly bind osmium tetroxide and, subsequently other contrasting compounds (namely, uranyl acetate and lead citrate), resulting in being markedly darker than any other zone of the neuronal membrane. These specific properties have favored the conduction of several morphometric studies of synaptic ultrastructure on tissue samples conventionally stained by osmium tetroxide, uranyl acetate, and lead citrate, but they have also prompted the development of various preferential cytochemical techniques with the aim of evidencing selectively fine structural details reporting on specific functional aspects of synaptic morphology. Among the different staining techniques to evidence synaptic junctions, the ethanol phospho-tungstic acid (E-PTA) procedure originally was developed to analyze synapse formation and maturation in cultured neurons, and then it was applied to tissue sections. The E-PTA staining enables visualization of the pre- and postsynaptic membranes against a very faint background that is almost completely unstained (see Figure 40.3). This is due to the fact that the fixed tissue pieces are stained in a 1% ethanolic solution of PTA at 60°C for 1 hour, and the nonpositive structures are degraded substantially during this step.

Although the reasons for the specificity of the synapses to this staining technique are still under investigation, it has been clearly demonstrated that PTA binds to the basic amino acid residues of the proteins as also supported by the E-PTA positivity of the chromatin due to the presence of histones. Whatever the mechanisms or reasons of the E-PTA preferential staining of the synapses, the big molecule of the PTA does not penetrate easily into the tissue. Thus in order to obtain good samples for quantitative analysis it is important that very thin sections must be used for incubation in the E-PTA solution. Because of the sharp precipitate due to the

Figure 40.3 Perforated E-PTA stained synaptic junction from the human cerebral cortex. The postsynaptic density appears as a continuous dark line. The presynaptic apposition can be seen as a dotted line. Arrows indicate the perforations. Bar: 0.5 ^m.

E-PTA cytochemical staining, no further contrasting procedure is needed, thus avoiding the risk of staining artifacts. Accordingly, the synapses may be easily identified as two parallel black lines or as a range of dark peaks and a black sharp line representing the pre-and postsynaptic membrane appositions, respectively. At the electron microscope, in the tissue samples stained by the E-PTA procedure, the identification of the synapses is greatly facilitated and the semiautomatic measurement of ultrastructural parameters with a defined functional meaning may be reliably carried out by applying morphometric formulas with the use of computerized image analysis systems.

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