Formation of functional synaptic connections between cultured cortical neurons from agrin-deficient mice.

Numerous studies suggest that the extracellular matrix protein agrin directs the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). Strong support for this hypothesis comes from the observation that the high density of acetylcholine receptors (AChR) normally present at the neuromuscular junction fails to form in muscle of embryonic agrin mutant mice. Agrin is expressed by many populations of neurons in the central nervous system (CNS), suggesting that this molecule may also play a role in neuron-neuron synapse formation. To test this hypothesis, we examined synapse formation between cultured cortical neurons isolated from agrin-deficient mouse embryos. Our data show that glutamate receptors accumulate at synaptic sites on agrin-deficient neurons. Moreover, electrophysiological analysis demonstrates that functional glutamatergic and gamma-aminobutyric acid (GABA)ergic synapses form between mutant neurons. The frequency and amplitude of miniature postsynaptic glutamatergic and GABAergic currents are similar in mutant and age-matched wild-type neurons during the first 3 weeks in culture. These results demonstrate that neuron-specific agrin is not required for formation and early development of functional synaptic contacts between CNS neurons, and suggest that mechanisms of interneuronal synaptogenesis are distinct from those regulating synapse formation at the neuromuscular junction.     

Formation of functional synaptic connections between cultured cortical neurons from agrin‐deficient mice

Numerous studies suggest that the extracellular matrix protein agrin directs the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). Strong support for this hypothesis comes from the observation that the high density of acetylcholine receptors (AChR) normally present at the neuromuscular junction fails to form in muscle of embryonic agrin mutant mice. Agrin is expressed by many populations of neurons in the central nervous system (CNS), suggesting that this molecule may also play a role in neuron–neuron synapse formation. To test this hypothesis, we examined synapse formation between cultured cortical neurons isolated from agrin‐deficient mouse embryos. Our data show that glutamate receptors accumulate at synaptic sites on agrin‐deficient neurons. Moreover, electrophysiological analysis demonstrates that functional glutamatergic and gamma‐aminobutyric acid (GABA)ergic synapses form between mutant neurons. The frequency and amplitude of miniature postsynaptic glutamatergic and GABAergic currents are similar in mutant and age‐matched wild‐type neurons during the first 3 weeks in culture. These results demonstrate that neuron‐specific agrin is not required for formation and early development of functional synaptic contacts between CNS neurons, and suggest that mechanisms of interneuronal synaptogenesis are distinct from those regulating synapse formation at the neuromuscular junction. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 547–557, 1999     

Formation of lamina-specific synaptic connections

In many parts of the vertebrate central nervous system, inputs of distinct types confine their synapses to individual laminae. Such laminar specificity is a major determinant of synaptic specificity. Recent studies of several laminated structures have begun to identify some of the cells (such as guidepost neurons in hippocampus), molecules (such as N-cadherin in optic tectum, semaphorin/collapsin in spinal cord, and ephrins in cerebral cortex), and mechanisms (such as activity-dependent refinement in lateral geniculate) that combine to generate laminar specificity.     

Synaptic formations and modulations of synaptic transmissions between identified cerebellar neurons in culture.

1. Synaptic formations between a rat cerebellar granule cell and a Purkinje cell, and also between an inferior-olivary neuron and a Purkinje cell have been accomplished in culture. 2. The synaptic transmission between an inferior-olivary neuron and a Purkinje cell was far much more potent than that between a granule cell and a Purkinje cell in the culture, and the former always induced in a Purkinje cell an action potential followed by prolonged depolarization, which resembled a climbing fiber response in vivo. 3. Synaptic potentiation was induced by repetitive stimulation (2 Hz, 20 sec) of a granule cell, and the synaptic depression was induced by repetitive conjunctive stimulation of both a granule cell and an inferior-olivary neuron as in a slice preparation. 4. When repetitive stimulation of both neurons were given while the postsynaptic Purkinje cell was voltage-clamped at -80 mV, not the depression but the potentiation took place. When repetitive stimulation of a granule cell was coupled with the postsynaptic strong depolarization induced by direct outward current injection, the depression took place. These two experiments indicate that the postsynaptic depolarization during activation of a presynaptic granule cell is both necessary and sufficient to induce the depression, and that the potentiation is induced without the postsynaptic depolarization. 5. The quantal analysis on the synaptic transmission, where fluctuations of amplitudes of synaptic currents in a Purkinje cell induced by a single granule cell were measured, indicated that the synaptic potentiation involves the enhancement of transmitter release from a presynaptic granule cell and that the depression involves changes of postsynaptic receptors on a Purkinje cell.     

Regeneration of functional synaptic connections between widely separated neurons in the adult mammalian central nervous system

The responses to light of retinal ganglion cells with regenerated axons can be recorded from axons teased from peripheral nerve grafts replacing the optic nerve of the adult rat or hamster. These responses resemble those of normal retinal ganglion cells but can no longer be observed several months after grafting, concomitant with ongoing loss of the population of axotomized retinal ganglion cells. Synapses formed with neurons in the superior colliculus by retinal ganglion cell axons regenerated through peripheral nerve grafts mediate both excitatory and inhibitory responses. These experiments demonstrate that when provided with an appropriate milieu for elongation, neurons indigenous to the adult mammalian central nervous system can make functional reconnections with distant targets within the nervous system.     

An assessment of the manifestation of the functional connections between brain structures]

Responses of hypothalamic neurons to single (1/s, 20 impulses) stimulation of the prefrontal (area 8), cingulum (area 24), periamygdaloideus (RPA) cortex and hippocampus (field CA3) were studied on experimental cats anesthetized with ketamine. The routine elaborated for IBM PC/AT 386 provided: 1) selection of such neurone reaction with the latent period variation less than its length per the set value (5-20%); 2) selection of repeated neurone reactions with the same variability of latent periods from 20 cortical stimuli; 3) ranging of neurone reactions according to these indices. Quantitative estimation of the priority ranges made it possible to determine the profundity of the functional relations between the cerebral structures.     

Seasonal plasticity of synaptic connections between identified neurones in Lymnaea

Here we investigate the synaptic connectivity of the giant dopamine containing neurone (RPeDI) of Lymnaea stagnalis during the winter months, in wild and laboratory bred animals. RPeD1 is one of the three neurones forming the respiratory central pattern generator (CPG) in Lymnaea and initiates ventilation under normal circumstances. Many of the follower cells of RPeD1 are ventilatory motor neurones. The connections of RPeD1 to its follower cells were investigated using standard intracellular recording techniques and dopamine was applied to the follower cells using a puffer pipette. During February and early March, RPeD1 was functionally disconnected from its follower cells, but connections reappeared towards the end of March. Most functionally disconnected cells failed to respond to applied dopamine, consistent with the hypothesis that there is down regulation of dopamine receptors in the follower cells of RPeD1 in the winter months. Behaviourally, Lymnaea that survive the winter, are not active at this time and do not indulge in lung ventilation, but stay quiescent. Thus functional disconnection of neurones from the CPG may be either a cause or a consequence of this change in behaviour.     

Formation, maintenance, and functional uncoupling of connections between identified Helisoma neurons in situ

Previous work with identified Helisoma neurons has characterized an array of neuroplastic responses to axotomy that include the generation of new neuritic outgrowth, the reinnervation of target organs, and the formation of new electrical synapses. These responses are not random, but rather occur in a precise, predictable manner under a variety of culture conditions. The present investigation demonstrates that specific identified neurons display similar neuroplastic "behavior" within the living animal. In response to in situ nerve crushes, neurons B4 and B5 generate new neuritic outgrowth, neuron B4 functionally reinnervates the salivary glands, and new electrical synapses form between the left and right neurons B5. The in situ paradigm employed in the present experiments made it possible to examine responses to axotomy over longer periods than in earlier studies with organ cultures. New B5R-B5L connections, previously found to be stable over the short term in culture, gradually decreased in strength in situ, and the cells effectively uncoupled by 8 weeks after axotomy. This uncoupling did not depend upon target reinnervation and occurred in the continued presence of neurites in the buccal commissure. It is suggested that the stability of new connections is related to whether the connection previously existed in the unperturbed nervous system. The similarities between the ability of identified neurons to grow and to form synaptic connections in situ and in culture suggests that neurons are endowed with a specific program of regenerative responses that can be expressed reliably in a wide variety of environmental conditions.     

Formation of nerve fibers between organotypic explants of the hippocampus and mammillary bodies of newborn mice]

Nervous fiber bundles formed between the explants during the organotypic cocultivation of the hippocampus and mamillary nuclei of the newborn mouse. Some of the fibers within these bundles were the continuation of axons localized in the alveus of the Ammon's horn. It is suggested that under conditions of cultivation the hippocampal pyramidal neurons deprived of the afferent leads-in from other brain structures are capable of forming the projection efferent connections typical of the intact hippocampus neurons.     

Molecules involved in the formation of synaptic connections in muscle and brain.

Synapses are highly specialized structures designed to guarantee precise and efficient communication between neurons and their target cells. Molecules of the extracellular matrix have an instructive role in the formation of the neuromuscular junction, the best-characterized synapse. In this review, the molecular mechanisms underlying these instructive signals will be discussed with particular emphasis on the receptors involved. Additionally, recent evidence for the involvement of specific adhesion complexes in the formation and modulation of synapses in the central nervous system will be reviewed. Synapses are specialized junctions between neurons and their target cells where information is transferred from the pre- to the postsynaptic cell. At most vertebrate synapses, this transfer is accomplished by the release of a specific neurotransmitter from the presynaptic nerve terminal. The release of neurotransmitter is initiated by the action potential and the subsequent influx of Ca(2+) into the presynaptic nerve terminal. This results in the rapid fusion of vesicles with the nerve membrane and the release of the neurotransmitter into the synaptic cleft. The neurotransmitter then diffuses across the cleft and binds to specific postsynaptic receptors, resulting in a change in the membrane potential of the postsynaptic cell. This can result in the generation of an action potential. The high precision of synaptic transmission requires that pre- and postsynaptic structures are both highly organized and in juxtaposition to each other. In addition, alterations in synaptic transmission are the basis of learning and memory and are likely to be accompanied by the remodeling of synaptic structures (Toni et al., 1999). Thus, the study of how synapses are formed during development is also of relevance for the understanding of the cellular and molecular processes involved in learning and memory. This review focuses on the molecular mechanisms involved in the formation and the function of synapses.     

Survival of bone tissue in organotypic culture under intermittent mechanical loading. Preliminary results

With the aim to study the mechanism of transduction of mechanical stimuli in biological ones we have realized an experimental device for the application of intermittent mechanical forces on bone specimens in vitro. The scheme of the device is reported in Fig. 1. It is constituted by a drive shaft which rotates on eccentric axis (1) supporting a longitudinal bar (2) with the load (3). The latter rests on a piston (4) only during a limited period of every shaft revolution, so that the load becomes intermittent. The bone specimen (5) is placed under the piston and the two are placed in a tube containing the culture medium. This latter is BGJ mod. Fitton-Jackson (Gibco), enriched with fetal calf serum (10%) and ascorbic acid (70 microliters/ml). Right metatarsi from 18-day-old rats were removed aseptically and placed under the piston for 2-6 days after resection of both ends. The homotypic ones, unloaded, were placed in 30 mm Petri dishes, and used as a control. The incubator environment was 5% CO2 in air (A group), or enriched with O2 (25-35%) (B group). At the end of the experimental period the bone specimens were fixed in 4% formalin buffered and treated for conventional histologic methods. In the A group most of the osteocytic lacunae were empty. The osteoblasts disappeared already at the 2nd day; the periosteal fibroblast dedifferentiated and multiplied. The deposition or calcification of osteoid were completely lacking. The application of mechanical load promoted deposition of granular degenerative material around the bone, and the periosteal cells, well differentiated, were surrounded by metachromatic material, which resembles cartilage matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

The modulating effect of amino acids on an organotypic culture of lymphoid tissue

The effect of 20 essential and nonessential L-amino acids on the dynamics of development of spleen explants from 1- and 21-day-old rats on an organotypic tissue culture was studied. The hydrophilic amino acids with a higher molecular mass (asparagine, lysine, arginine, and glutamic acid) induced an inhibitory effect on the growth zone of explants of immature tissue from 1-day-old animals and an opposite, stimulating effect on the mature spleen tissue of 21-day-old rats. An immunohistochemical analysis revealed a reciprocal correlation between the expression of the proapoptotic protein p53 and the cell proliferation upon the action of lysine, asparagine, and glutamic acid. The role of polar amino acids in the modulation of cell proliferation and apoptosis in dependence on the period of ontogenesis was determined. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.     

The modulating effect of amino acids on an organotypic culture of lymphoid tissue

The effect of 20 essential and nonessential L-amino acids on the dynamics of development of spleen explants from 1-and 21-day-old rats on an organotypic tissue culture was studied. The hydrophilic amino acids with a higher molecular mass (asparagine, lysine, arginine, and glutamic acid) induced an inhibitory effect on the growth zone of explants of immature tissue from 1-day-old animals and an opposite, stimulating effect on the mature spleen tissue of 21-day-old rats. An immunohistochemical analysis revealed a reciprocal correlation between the expression of the proapoptotic protein p53 and the cell proliferation upon the action of lysine, asparagine, and glutamic acid. The role of polar amino acids in the modulation of cell proliferation and apoptosis in dependence on the period of ontogenesis was determined.