Structural stabilization of CNS synapses during postnatal development in rat cortex.

CNS synapses are produced rapidly upon pre- and post-synaptic recruitment. However, their composition is known to change during development and we reasoned that this may be reflected in the gross biochemical properties of synapses. We found synaptic structure in adult cortical synaptosomes to be resistant to digestion with trypsin in the presence and absence of calcium ions, contrasting with previous observations. We evaluated the divalent cation dependence and trypsin sensitivities of synapses using synaptosomes from different developmental stages. In contrast to adult synapses, at postnatal day (P) 10 EDTA treatment eliminated approximately 60% of the synapses, and trypsin and EDTA, together, eliminated all junctions. Trypsinization in the presence of calcium eliminated approximately 60% of the junctions at P10. By P35, all synapses were calcium independent, whereas full trypsin resistance was not attained until P49. To compare the calcium dependence and trypsin sensitivity of synapses in another region of the adult brain, we examined synapses from adult (P50) hippocampus. Adult hippocampus maintained a population of synapses that resembled that of P35 cortex. Our results show that synapses are modified over a long time period in the developing cortex. We propose a model in which the addition of synergistic calcium-dependent and -independent adhesive systems stabilize synapses.

Proteomic comparison of two fractions derived from the transsynaptic scaffold.

A fraction derived from presynaptic specializations (presynaptic particle fraction; PPF) can be separated from postsynaptic densities (PSD) by adjusting the pH of Triton X-100 (TX-100) extraction of isolated transsynaptic scaffolds. Solubilization of the PPF corresponds to disruption of the presynaptic specialization. We show that the PPF is insoluble to repeated TX-100 extraction at pH 6.0 but becomes soluble in detergent at pH 8.0. By immunolocalization, we find that the major proteins of the PPF, clathrin and dynamin, are concentrated in the presynaptic compartment. By using multidimensional protein identification technology, we compared the protein compositions of the PPF and the PSD fraction. We identified a total of 341 proteins, 50 of which were uniquely found in the PPF, 231 in the PSD fraction, and 60 in both fractions. Comparison of the two fractions revealed a relatively low proportion of actin and associated proteins and a high proportion of vesicle or intracellular compartment proteins in the PPF. We conclude that the PPF consists of presynaptic proteins not connected to the actin-based synaptic framework; its insolubility in pH 6 and solubility in pH 8 buffered detergent suggests that clathrin might be an anchorage scaffold for many proteins in the PPF.

Gamma-protocadherins are targeted to subsets of synapses and intracellular organelles in neurons.

The clustered protocadherins (Pcdhs) comprise >50 putative synaptic recognition molecules that are related to classical cadherins and highly expressed in the nervous system. Pcdhs are organized into three gene clusters (alpha, beta, and gamma). Within the alpha and gamma clusters, three exons encode the cytoplasmic domain for each Pcdh, making these domains identical within a cluster. Using an antibody to the Pcdh-gamma constant cytoplasmic domain, we find that all interneurons in cultured hippocampal neurons express high levels of Pcdh-gamma(s) in a nonsynaptic distribution. In contrast, only 48% of pyramidal-like cells expressed appreciable levels of these molecules. In these cells, Pcdh-gamma(s) were associated with a subset of excitatory synapses in which they may mediate presynaptic to postsynaptic recognition in concert with classical cadherins. Immunogold localization in hippocampal tissue showed Pcdh-gamma(s) at some synapses, in nonsynaptic plasma membranes, and in axonal and dendritic tubulovesicular structures, indicating that they may be exchanged among synapses and intracellular compartments. Our results show that although Pcdh-gamma(s) can be synaptic molecules, synapses form lacking Pcdh-gamma(s). Thus, Pcdh-gamma(s) and their relatives may be late additions to the classical cadherin-based synaptic adhesive scaffold; their presence in intracellular compartments suggests a role in modifying synaptic physiology or stability.