Pierre-Alain Buchs

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.

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.

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.

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.

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. Hippocampus 2000;10:596–604.

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.

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 &ggr;-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.

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 activationa and inhibition, with special emphasis on the effects of the kinase on synaptic transmission and theN-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.

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.

Role of protein phosphorylation in long-term potentiation in the hippocampus

The N-methyl-D-aspartate (NMDA) receptor is essential for multiple functions of glutamate neurotransmission that include learning, memory, and brain development. The NMDA receptor subunits NR2A and NR2B are tyrosine phosphorylated in the brain. We found that tyrosine phosphorylation of NR2A was severely reduced in fyn-deficient mice, suggesting that NR2A is a substrate of the Fyn kinase. Indeed, NR2A was tyrosine phosphorylated in 293T cells when coexpressed with Fyn. Furthermore, PSD-95, which binds to and co-clusters with NMDA receptors, promoted Fyn-mediated tyrosine phosphorylation of NR2A. Different regions of PSD-95 associated with NR2A and Fyn, respectively. These suggest that PSD-95 is critical for regulation of the NMDA receptor activity, serving as a molecular scaffold for anchoring Fyn to NR2A. Fyn is also important for tyrosine phosphorylation of NR2B. We showed that tyrosine phosphorylated NR2B bound the ~85 subunit. Biological significance of tyrosine phosphorylation of NRZA and NR2B will be discussed.