Remodeling of synaptic membranes after induction of long-term potentiation.

Several morphological changes of synapses have been reported to be associated with the induction of long-term potentiation (LTP) in the CA1 hippocampus, including an transient increase in the proportion of synapses with perforated postsynaptic densities (PSDs) and a later occurrence of multiple spine boutons (MSBs) in which the two spines arise from the same dendrite. To investigate the functional significance of these modifications, we analyzed single sections and reconstructed 134 synapses labeled via activity using a calcium precipitation approach. Analyses of labeled spine profiles showed changes of the spine head area, PSD length, and proportion of spine profiles containing a coated vesicle that reflected variations in the relative proportion of different types of synapses. Three-dimensional reconstruction indicated that the increase of perforated spine profiles observed 30 min after LTP induction essentially resulted from synapses exhibiting segmented, completely partitioned PSDs. These synapses had spine head and PSD areas approximately three times larger than those of simple synapses. They contained coated vesicles in a much higher proportion than that of any other type of synapse and exhibited large spinules associated with the PSD. Also the MSBs with two spines arising from the same dendrite that were observed 1-2 hr after LTP induction included a spine that was smaller and a PSD that was smaller than those of simple synapses. These results support the idea that LTP induction is associated with an enhanced recycling of synaptic membrane and that this process could underlie the formation of synapses with segmented PSDs and eventually result in the formation of a new, immature spine.

Spine changes associated with long-term potentiation.

High-frequency stimulation of excitatory synapses in many regions of the brain triggers a lasting increase in the efficacy of synaptic transmission referred to as long-term potentiation (LTP) and believed to contribute to learning and memory. One hypothesis proposed to account for the stability and properties of this functional plasticity is a structural remodeling of spine synapses. This possibility has recently received support from several studies. It has been found that spines are highly dynamic structures, that they can be formed very rapidly, and that synaptic activity and calcium modulate changes in spine shape and formation of new spines. Ultrastructural analyses bring additional support to these observations and suggest that LTP is associated with a remodeling of the postsynaptic density (PSD) and a process of spine duplication. This new information is reviewed and interpreted in light of other recent advances concerning the mechanisms of LTP and especially the role of postsynaptic glutamate receptor turnover in this form of plasticity. Taken together, a view is emerging that suggests that morphologic changes of spine synapses are associated with LTP and that they not only correlate with, but probably also contribute to the increase in synaptic transmission.

Infection of organotypic slice cultures from rat central nervous tissue with Trypanosoma brucei brucei.

We recently described a new procedure to grow nervous tissue as organotypic culture. The main feature of these slice cultures is to maintain a well preserved, three-dimensional organisation of the central nervous tissue. As these cultures can be kept for several weeks (up to three months), we have used this in vitro approach to study the complex interactions between host tissue and parasites during late stages of cerebral African trypanosomiasis. Light and electron microscopical studies, as well as electrophysiological recordings demonstrate that the structure and function of the nervous tissue is not severely affected even after several weeks of trypanosome infection. The presence of a large number of parasites does not seem to be deleterious to neuronal survival. Secondly, most of the trypanosomes are located around the periphery of the nervous tissue, but many of them also penetrate into the nervous parenchyma. Thirdly, trypanosomes with well-conserved morphology are found within the cytoplasm of glial cells, which in some cases were identified as astrocytes. These "intracellular parasites" seem to actively invade the target cells. Our study demonstrates that the presence of proliferating trypanosomes does not per se interfere with the neural activity of CNS tissues. Secondly, it provides, to the best of our knowledge, the first in vitro demonstration of intracellular forms of African trypanosomes.

Differential neurotoxic effects of propofol on dissociated cortical cells and organotypic hippocampal cultures.

BACKGROUND: Propofol is a widely used anesthetic agent for adults and children. Although extensive clinical use has demonstrated its safety, neurologic dysfunctions have been described after the use of this agent. A recent study on a model of aggregating cell cultures reported that propofol might cause irreversible lesions of gamma-aminobutyric acid-mediated (GABAergic) neurons when administered at a critical phase of brain development. We investigated this issue by comparing the effects of long-term propofol treatment on two models of brain cultures: dissociated neonatal cortical cell cultures and organotypic slice cultures. METHODS: Survival of GABAergic neurons in dissociated cultures of newborn rat cortex (postnatal age, 1 day) treated for 3 days with different concentrations of propofol was assessed using histologic and cytochemical methods. For hippocampal organotypic slice cultures (postnatal age, 1 and 7 days), cell survival was assessed by measuring functional and morphologic parameters: extracellular and intracellular electrophysiology, propidium staining of dying cells, and light and electron microscopy. RESULTS: In dissociated neonatal cell cultures, propofol induced dose-dependent lesions of GABAergic neurons and of glial cells. In contrast, no evidence for neurotoxic effects of propofol were found after long-term treatment of organotypic slice cultures. Excitatory transmission was not affected by propofol, and inhibitory transmission was still functional. Histologic preparations showed no evidence for cell degeneration or death. CONCLUSION: Although long-term applications of propofol to dissociated cortical cell cultures produced degeneration and death of GABAergic neurons and glial cells, no such lesions were found when using a model of postnatal organotypic slice cultures. This conclusion is based on both functional and morphologic tests.

LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite.

Structural remodelling of synapses and formation of new synaptic contacts has been postulated as a possible mechanism underlying the late phase of long-term potentiation (LTP), a form of plasticity which is involved in learning and memory. Here we use electron microscopy to analyse the morphology of synapses activated by high-frequency stimulation and identified by accumulated calcium in dendritic spines. LTP induction resulted in a sequence of morphological changes consisting of a transient remodelling of the postsynaptic membrane followed by a marked increase in the proportion of axon terminals contacting two or more dendritic spines. Three-dimensional reconstruction revealed that these spines arose from the same dendrite. As pharmacological blockade of LTP prevented these morphological changes, we conclude that LTP is associated with the formation of new, mature and probably functional synapses contacting the same presynaptic terminal and thereby duplicating activated synapses.

Induction of long-term potentiation is associated with major ultrastructural changes of activated synapses.

Long-term potentiation (LTP), an increase in synaptic efficacy believed to underlie learning and memory mechanisms, has been proposed to involve structural modifications of synapses. Precise identification of the morphological changes associated with LTP has however been hindered by the difficulty in distinguishing potentiated or activated from nonstimulated synapses. Here we used a cytochemical method that allowed detection in CA1 hippocampus at the electron microscopy level of a stimulation-specific, D-AP5-sensitive accumulation of calcium in postsynaptic spines and presynaptic terminals following application of high-frequency trains. Morphometric analyses carried out 30-40 min after LTP induction revealed dramatic ultrastructural differences between labeled and nonlabeled synapses. The majority of labeled synapses (60%) exhibited perforated postsynaptic densities, whereas this proportion was only 20% in nonlabeled synaptic contacts. Labeled synaptic profiles were also characterized by a larger apposition zone between pre- and postsynaptic structures, longer postsynaptic densities, and enlarged spine profiles. These results add strong support to the idea that ultrastructural modifications and specifically an increase in perforated synapses are associated with LTP induction in field CA1 of hippocampus and they suggest that a majority of activated contacts may exhibit such changes.

A new cytochemical method for the ultrastructural localization of calcium in the central nervous system.

We have developed a new cytochemical method for the localization of calcium at the ultrastructural level in the central nervous system (CNS). The method is based on the use of phosphate buffer in the primary fixation followed by a mixture of a complex of chromium(III)-trisoxalate and osmium tetroxide (OsO4) which precipitates calcium and results in the formation of a high electron-dense reaction product. Calcium selectivity was verified by reactions made in test tube, by EGTA treatment of the tissue, by electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS). The technique was found to be reproducible, yielding similar results in acutely prepared hippocampal slices or organotypic cultures fixed by immersion and in brain areas fixed by perfusion. In hippocampal slices, calcium deposits were found to accumulate in different subcellular compartments such as endoplasmic reticulum, mitochondria and synaptic vesicles. Interestingly, electron-dense reaction products were also visualized in smooth endoplasmic reticulum structures localized in presynaptic terminals or post-synaptic spines as well as in synaptic clefts and active zones. This new method may thus be of interest for studying the metabolism of calcium, specifically with regard to synaptic activity, in the CNS.

Lesion-induced neurite sprouting and synapse formation in hippocampal organotypic cultures.

By sectioning, using a razor blade, one- and three-week-old rat hippocampal organotypic cultures, we have tested the possibility that neurite outgrowth and reactive synaptogenesis would take place even after several weeks in culture in this in vitro model. At the light-microscopic level, recovery from the section and formation of a thin scar were observed within six days following the lesion. Immunostainings using neurofilament antibodies showed the presence of numerous degenerative and regenerative images one day after the cut and many fibres crossing the section six days after the lesion. Electrophysiological recordings of synaptic responses elicited across the section indicated the formation of new functional synaptic contacts and complete recovery of transmission within three to six days. Interestingly, functional recovery in three-week-old cultures was found to be significantly slower than in one-week-old tissue. These findings were confirmed at the electron-microscopic level. Evidence was obtained for an effective cleaning of the lesion site by macrophages and astroglial cells, the existence of many degenerative and regenerative images one day after the cut and the presence of new dendrites, axonal fibres and synapses in the area of the section six days after the lesion. All these changes were slower in three- than in one-week-old cultures. These results indicate that organotypic cultures can be used as an interesting model for studies of reactive synaptogenesis.

Structural modifications associated with synaptic development in area CA1 of rat hippocampal organotypic cultures.

Using morphological techniques, we characterized the developmental reorganization that takes place during the first weeks after explanation in area CA1 of organotypic hippocampal cultures maintained at the interface between medium and a CO2-enriched atmosphere. Pyramidal neurones redistributed from a vertical into an horizontal cell layer in the middle of a three-dimensional culture, with apical dendrites running above the pyramidal layer. Glial cells redistributed into a thin layer at the bottom of the culture, forming an interface between tissue and culture medium. Astrocytes were identified as the most numerous non neuronal cells. No sign of glial proliferation could be observed, except for a transient increase during the first days after explanation. The density of synaptic contacts in the stratum radiatum decreased immediately after explanation and then increased by about 20-fold to reach values in the proximal part of the apical layer after 4 weeks in culture which were only slightly smaller than those measured in 1-month-old rats. The synaptic density in the most distal part of the dendritic layer which receives connections extrinsic to the hippocampus remained significantly lower than in vivo. The ratio of spine to shaft contacts was comparable to that found in vivo. These results indicate that interface type of organotypic cultures can be used as an interesting model for studies of synaptic development in vitro.

Time course of synaptic development in hippocampal organotypic cultures.

Using electrophysiological recordings of field potentials, we investigated the time course of synapse formation and maturation in organotypic cultures prepared from neonate animals of different ages. Following explanation, the size of the maximal synaptic responses elicited in area CA1 by stimulation of a small group of CA3 neurons increased progressively during the first three weeks in culture in a way that corresponded to the changes observed in synaptic contact density. Growth of synaptic responses was found to occur much more rapidly in cultures prepared from 8-day-old as compared with 2-day-old rats. Development of synaptic connections between CA3 and CA1 neurones was also faster than between granule cells and CA3 neurones. Acquisition of mature synaptic properties occurred in vitro as indicated by changes in degree of paired-pulse facilitation and the onset of long-term potentiation (LTP) after a few days in culture. The onset of LTP was much faster in cultures prepared from 8-day-old as compared with 2-day-old neonates and corresponded approximately to the 12-14th postnatal day. It is concluded that development proceeds in the cultures with a time course that resembles the in situ situation.

A simple method for organotypic cultures of nervous tissue.

Hippocampal slices prepared from 2-23-day-old neonates were maintained in culture at the interface between air and a culture medium. They were placed on a sterile, transparent and porous membrane and kept in petri dishes in an incubator. No plasma clot or roller drum were used. This method yields thin slices which remain 1-4 cell layers thick and are characterized by a well preserved organotypic organization. Pyramidal neurons labelled by extra- and intracellular application of horse radish peroxidase resemble by the organization and complexity of their dendritic processes those observed in situ at a comparable developmental stage. Excitatory and inhibitory synaptic potentials can easily be analysed using extra- or intracellular recording techniques. After a few days in culture, long-term potentiation of synaptic responses can reproducibly be induced. Evidence for a sprouting response during the first days in culture or following sections is illustrated. This technique may represent an interesting alternative to roller tube cultures for studies of the developmental changes occurring during the first days or weeks in culture.

Long-term potentiation, protein kinase C, and glutamate receptors.

Among the various molecular events that have been proposed to contribute to the mechanisms of long-term potentiation (LTP), one of the most cited possibilities has been the activation of protein kinase C (PKC). Here we review various aspects of the cellular actions of PKC activation and inhibition, with special emphasis on the effects of the kinase on synaptic transmission and the N-methyl-D-aspartate (NMDA) and non-NMDA receptor-mediated components of synaptic responses. We discuss the implications of these effects for interpretations of the role of PKC in the mechanisms of LTP induction and maintenance.

Protein kinase C activity is not responsible for the expression of long-term potentiation in hippocampus.

Long-term potentiation (LTP) in hippocampus has been proposed to result from a tonic activation of protein kinase C. This hypothesis predicts that stimulation of the kinase would produce a smaller change in response size on potentiated versus control pathways and, conversely, that inhibition of the kinase would reduce potentiated inputs to a greater degree than control responses. We tested these predictions using phorbol esters to activate and using the antagonist H-7 to inhibit protein kinase C; we found that the actions of these drugs on synaptic transmission were not affected by prior induction of LTP. Both compounds, however, significantly decreased the contribution of N-methyl-D-aspartate receptors to synaptic potentials, a result that accounts for the suppressive effects of these compounds on LTP formation. Thus protein kinase C is probably not involved in the expression of LTP but may play a role in the receptor-mediated events participating in its induction.