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Kindling results from intermittent electrical stimulation of a local brain region and leads to a virtually permanent augmentation of synaptic responsiveness in the stimulated circuit. It has been hypothesized that an increase in the number of synapses may represent a structural basis for the enduring expression of synaptic plasticity following kindling, but such an alteration has not been demonstrated unequivocally. The present report provides evidence that hippocampal kindling is indeed accompanied by an increase in synaptic numbers. Young adult rats were kindled via medial perforant path stimulation and sacrificed 4 weeks after reaching a criterion of 5 generalized seizures. Stimulated but not kindled and implanted but not stimulated rats served as controls. Synapses were analyzed in the middle (MML) and inner (IML) molecular layer of the hippocampal dentate gyrus. Using the stereological disector technique, unbiased estimates of the number of synapses per neuron were differentially obtained for 3 morphological subtypes of perforated axospinous synapses characterized by a fenestrated, horseshoe-shaped or segmented postsynaptic density (PSD). A significant increase in synaptic numbers was found to selectively involve only those perforated synapses which are distinguished by a segmented PSD consisting of 2-5 discrete plates. This structural modification was restricted to the terminal synaptic field of stimulated axons (MML), but was not observed in an immediately adjacent synaptic field (IML) which was not directly stimulated during kindling. Since synapses distinguished by a segmented PSD may represent specialized synaptic contacts of an unusually high efficacy, a selective increase in their numbers is likely to provide a structural substrate of the augmented synaptic gain associated with kindling.

Long-term potentiation (LTP) is characterized by a long-lasting enhancement of synaptic efficacy which may be due to an increase in synaptic numbers. The present study was designed to verify the validity of this suggestion using recently developed unbiased methods for synapse quantitation. LTP was elicited in young adult rats by high-frequency stimulation of the medial perforant path carried out on each of 4 consecutive days. Potentiated animals were sacrificed 1 h after the fourth stimulation. Stimulated but not potentiated and implanted but not stimulated rats served as controls. Synapses were examined in the middle (MML) and inner (IML) molecular layer of the hippocampal dentate gyrus. Using the stereological disector technique, unbiased estimates of the number of synapses per neuron were differentially obtained for the following morphological synaptic types: axodendritic synapses involving dendritic shafts, non-perforated axospinous synapses exhibiting a continuous postsynaptic density (PSD) and perforated axospinous synapses distinguished by a fenestrated, horseshoe-shaped or segmented PSD. A major finding of this study is that the induction of LTP is accompanied by a selective increase in the number of synapses with segmented PSDs. This change was detected only in the potentiated synaptic field (MML), but not in an immediately adjacent one (IML) which was not directly stimulated during the induction of LTP. It is strongly suggested by the latter finding that the increase in the number of axospinous synapses exhibiting segmented PSDs is associated with LTP. Such a highly selective modification of connectivity, which involves only one particular subtype of synapses in the potentiated synaptic field, is likely to represent a structural substrate of the enduring augmentation of synaptic efficacy typical of LTP.

Kindling is a consequence of intermittent electrical stimulation of a local forebrain area leading to a durable augmentation of synaptic responsiveness in the stimulated circuit. The basis for this functional change is unknown, but there is evidence suggesting that it entails a structural modification of synapses. The present report demonstrates that hippocampal kindling induces a selective enlargement of active zones in perforated axospinous synapses formed by stimulated axons. Since the active zone is the site of intracellular transmission, its enlargement involving only a certain subpopulation of synapses provides a likely structural substrate of synaptic plasticity associated with kindling.

A comparative analysis of axospinous synapses was performed in the middle (MML) and inner (IML) molecular layer of the hippocampal dentate gyrus of rats kindled via medial perforant path stimulation and sacrificed 4 weeks after reaching a criterion of 5 generalized seizures. The MML was a directly stimulated structure, while the IML was not. Both are immediately adjacent synaptic fields likely to be equally susceptible to any generalized effects of convulsions and hypoxia. In these two subdivisions of the molecular layer, the so-called perforated and non-perforated synapses, distinguished respectively by a discontinuous or continuous postsynaptic density, were differentially quantified. In the MML, the ratio of perforated to non-perforated synapses was found to be markedly increased in kindled rats relative to controls. In the IML, however, no change in this ratio was detected following kindling. Thus, the shift in the relative preponderance of perforated synapses over non-perforated ones is not a consequence of generalized phenomena accompanying the kindling process.

Examination of axospinous synapses in serial sections obtained from the middle molecular layer of the rat dentate gyrus has revealed that some of them involve double-headed dendritic spines. Each spine head is apposed by a separate axon terminal with which it always forms a perforated synaptic contact distinguished by a discontinuous postsynaptic density. The number of perforated synapses on double-headed spines was estimated as a synapse-to-neuron ratio with the aid of the disector technique and found to be significantly increased in rats kindled via medial perforant path stimulation. These results support the notion that perforated synapses involving double-headed dendritic spines represent a structural modification related to enhanced synaptic efficacy.

Synapses on dendritic shafts were examined in electron micrographs of serial sections obtained from the molecular layer of the rat dentate gyrus. Some of these synapses have been found to exhibit profiles of a discontinuous postsynaptic density (PSD). PSD reconstructions from serial sections were performed in a plane perpendicular to that of the sections. The results obtained indicate that profiles of discontinuous PSDs observed in random sections of dendritic shaft synapses are generated by sectioning of PSD plates that contain 1-3 holes or perforations. Earlier serial section studies of osmicated material have demonstrated that a proportion of axospinous synapses also exhibit a perforated PSD. It appears, therefore, that the presence of PSD perforations is a general phenomenon shared by subpopulations of different types of synapses, both those involving dendritic shafts and those involving dendritic spines.

Spatial working memory, which crucially depends on the structural integrity of the hippocampal formation and its afferent connections, is impaired in the most, but not all, of aged rats. This study was designed to verify whether aged animals that do not exhibit the spatial memory deficit are the ones in which the hippocampal synaptic connectivity remains preserved with advancing chronological age. Young adult rats with good spatial memory, aged rats with impaired spatial memory and equally aged rats with intact spatial memory were compared. The number of synapses per neuron was estimated in the hippocampal dentate gyrus. The most important results were obtained when axospinous synapses were divided into perforated and non-perforated ones according to the appearance of their postsynaptic density. A significant decrease in the number of perforated synapses was found in memory-impaired aged rats as compared to either young adults or aged animals without memory deficits. The number of non-perforated synapses per neuron was diminished in memory-deficient aged rats relative to young adults, but not to memory-intact aged rats. However, it was only the loss of perforated synapses which correlated with the degree of spatial memory impairment. Thus, aged rats need a preserved complement of hippocampal perforated synapses to maintain good spatial memory.

Axospinous synapses were examined in the molecular layer of the rat dentate gyrus. Serial section analysis of synapses, which exhibited a discontinuity of the postsynaptic density (PSD) in at least one consecutive section, was performed. Reconstruction of each discontinuous PSD was made in a plane perpendicular to that of serial sections. The results obtained confirm earlier observations that profiles of 'perforated' synapses visualized in random sections of osmicated material are produced by sectioning of synapses with perforated and horseshoe-shaped PSDs. Additionally, it has been found that two other synaptic subtypes, namely synapses with notched and segmented PSD, contribute to the number of profiles of 'perforated' synapses. Synaptic contacts with notched PSD are characterized by an indentation of an otherwise continuous PSD, relatively small dimensions and simple shape. They appear to be unrelated to the category of synapses with discontinuous PSD. Synaptic contacts with segmented PSD are distinguished by the presence of 2-5 discrete PSD segments at the interface between a presynaptic axon terminal and a postsynaptic dendritic spine. Some PSD segments exhibit 1-3 perforations, while others are horseshoe-shaped. It is postulated that the segmented PSD may evolve through the stages of perforated and horseshoe-shaped PSD to form a specialized synaptic contact of an unusually high efficacy. Every PSD segment is a component of a separate synaptic complex, each one comparable to that of a small, simple-shaped synapse. A concerted activation of several synaptic complexes belonging to a single synaptic junction may provide a mechanism for an amplification of synaptic transmission.