Formation of spontaneous and evoked unit activity in organotypical hippocampal tissue culture

Spontaneous and evoked single unit activity of Ammon's horn and the dentate fascia was investigated by extracellular recording in organotypical explants of neonatal mouse hippocampus during the first 2 weeks in culture. Three main types of spontaneous activity were observed: single, group, and volley. After the fifth to sixth day in culture the neurons were found to respond to stimulation of the entorhinal cortex and dentate fascia. Both short-latency (evidently monosynaptic) and long-latency responses were observed, and they usually varied if the stimulus was repeated. The appearance of evoked unit activity in the explants is considered to be due to maturation of synaptic contacts at axon endings of granule cells and perforant path, the formation of which continues during culture.Brain Institute, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 9, No. 3, pp. 257–266, May–June, 1977.     

Intercellular space and nonsynaptic interneuronal connections of the mammalian brain

Electron-microscopic investigations of various parts of the CNS in laboratory mammals and man have demonstrated that a single cerebral intercellular space, presenting an immediate internal medium for the nervous tissue elements, has different structure at the border separating the media. Here is a complex of structures, included into composition of the hemato-encephalic and liquor-encephalic barrier. Data of the literature on chemical composition of the intercellular contents and on specific membranous receptors make it possible to suppose a communication role of the intercellular spaces, ensuring nonsynaptic intercellular connections and modulation of the synaptic transmission. At the level of membranes, that make walls of the intercellular clefts by means of certain phylogenetically ancient humoral mechanisms, interaction of systems of synaptic and nonsynaptic interneuronal connections is secured.     

Transplacental effect of urethane on DNA synthesis in mouse embryonal lung tissue during organotypical cultivation

Kinetics of DNA synthesis was estimated by autoradiography in the organ cultures of murine embryonic lung tissue. Strain A mice were taken for this experiment (intact and exposed to transplacental action of urethane--30 mg/g to a pregnant mouse, subcutaneously, once). The explants were examined 1,7, 15 and 21 days after the cultivation. Transplacental urethane inhibited DNA synthesis the first 24 hours. The label index became approximately 3 times less. The next two days it was restored and then stimulated. The maximal label index was noted in the experimental explants on the 7th cultivation day, i.e. on the 8th-10th day after the transplacental action of urethane on the lung tissue in utero. By the 15th cultivation day the label index dropped but was higher than in the intact explants; by the 21st day DNA synthesis in the intact cultures ceased completely, whereas in the experimental animals--it continued.     

Cell types and neuronal connections in cultures of mammalian central nervous tissue

Summary Various neuronal cell types surviving in cultures of cerebellum, brain stem and cerebral cortex of new-born rats and kittens were described. The regenerative power of neurons in these explants was expressed by the growth of new processes that reached a length of several millimeters. However, nerve fiber regeneration and growth followed an entirely erratic pattern as evidenced by sometimes extensive fiber convolutions.Feed-back collaterals of axonic processes that returned fibers to their own cell bodies or their dendrites were a common feature in many neurons. Short chains of interconnected neurons were detected in a number of cultures. The connections between these neurons were established by apposition of fine axon terminals to presumable postsynaptic cell bodies or dendrites but not by true synaptic boutons. In numerous instances, synapses of the bouton type that appeared morphologically normal could be shown to represent remnants of severed connections since the presynaptic fibers proved to be isolated fiber fragments. Under the sterile conditions of the culture environment these structures apparently persisted without considerable morphological alteration. Evidently, most neurons in our cultures were reduced to the status of isolated cells due to extensive de-afferentiation at the time of explantation. Moreover, there was no evidence for the establishment of new synaptic connections between regenerating neurons.This work was supported by Research Grant NB 03114-05 from the National Institute of Neurological Diseases and Blindness, United States Public Health Service.Dedicated to my esteemed teacher, Prof. Dr. W. Bargmann, on the occasion of his 60th birthday.Sincere appreciation is expressed to Mrs. Eleanor Morris for her assistance in the management of the cultures, and to Mr. E. E. Pitsinger, Jr. for his photographic assistance.     

Formation of extracellular structures in conjugated cultures of agrobacteria

Electron microscopy of noncentrifugated agrobacterial cells on a nitrocellulose membrane labeled with colloid gold-conjugated antibodies to VirB1 showed that the labeled complex bound to acetosyringone (AS)-induced cells but failed to form red-colored stains during incubation with Ti aplasmid cells. Supramembrane structures of AS-treated A. tumefaciens cells were for the first time visualized by transmission electron microscopy. Colloid gold labeling of VirB2-specific antibodies showed that VirB2 proteins produce long thin pilus structures emerging at the poles of AS-induced agrobacterial cells but never on the surface untreated with AS and Ti-plasmid-free agrobacterial cells. As a rule, one (or rarely two) thread-like connections and bridges were observed between the cells at the primary contact stage. The bridges were not destroyed by SDS, did not react with VirB2-specific antibodies, and remained visible at 30 degrees C. Visible close contacts between mating bacteria did not cease after SDS treatment. SDS pretreatment of donor cells or a mating cell suspension significantly modified the efficiency of pTd33 plasmid transfer from donor to recipient agrobacterial cells. In the presence of AS the optimal temperature for transfer was 25 degrees C. The frequency of plasmid pTd33 transfer from A. tumefaciens via vir-dependent pathway decreased 2-4-fold due to increase of temperature from 19.25 to 31 degrees C.     

Conditioned reflex analysis of functional connections between the hippocampus and limbic system structures

Instrumental defensive conditioned reflex elaborated in dogs to stimulation of the dorsal and ventral hippocampus, also appeared, due to generalization, in response to stimulation of a number of limbic structures. Two types of changes in the generalization effects (estimated by parameters of motor conditioned reaction) were observed in the course of conditioned reflex stabilization: enhancement (in response to stimulation of the lateral hypothalamus, lateral nucleus of septum, limbic cortex) and weakening toward complete disappearance (in response to stimulation of the medial nucleus of amygdala and medial hypothalamus). Manifestation of the generalization phenomenon from the brain structures, which are not involved initially into conditioned activity, suggests the existence of close functional connections between these structures and the hippocampus.     

Formation of bone tissue by mouse bone marrow cell suspensions in organ culture

Adult mouse bone marrow cell suspensions prepared by trypsinization were cultivated in gelatin sponges on millipore filters. When HAWP filters were used, multilayer bone structure was formed. It contained mineralized ground substance, incorporated bone cells and osteoblast layer. With the use of AUFS filters, bone tissue developed not only on the top surface, but also inside the filter.     

Ultrastructure of interneuronal connections in the superior cervical sympathetic ganglion in cats

The structure of interneuronal synapses in the superior cervical sympathetic ganglion was studied in cats under normal conditions and after division of the cervical sympathetic nerves and removal of spinal ganglia T₁₂–L₂. A definite number of dendro-dendritic and dendro-somatic junctions is observed in the ganglion and most of them remained intact after operations of both types; they are probably synapses formed by dendrites of neurons located in the ganglion. Synapses of this sort participate in the formation of nest-like complexes, consisting of consecutive junctions of one neuron with several dendrites. The formation of such complexes may provide the anatomical basis for synchronization of rhythmic neuronal activity in the cellular glomeruli of the ganglion. The results of an ultrastructural study of dendro-dendritic junctions suggests that they are synaptic in nature. Some dendro-dendritic junctions underwent degeneration after both types of operation and are probably endings of neurons in spinal ganglia. Wide club-like structures, probably receptor endings, formed by dendrites of afferent neurons of spinal ganglia, also are found in the ganglion. These structures lie freely in the stoma of the ganglion or form contacts with axon terminals and dendrites of neurons located in the ganglion; some of them degenerate after removal of spinal ganglia T₁₂–L₂.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 13, No. 3, pp. 299–306, May–June, 1981.     

Microbial hazards in plant tissue and cell cultures

Summary A wide range of microorganisms (filamentous fungi, yeasts, bacteria, viruses and viroids) and micro-arthropods (mites and thrips) have been identified as contaminants in plant tissue cultures. Contaminant may be introduced with the explant, during manipulations in the laboratory or by micro-arthropod vectors. Contaminants may express themselves immediately or can remain latent for long periods of time. This often makes it difficult to identify the source of contamination. Disinfection protocols have now been developed for a wide range of plant species including those infected with viruses/viroids or endophytic bacteria. They may include the selection of pathogen-free donor plants or donor plant treatments such as thermotherapy. Also microbiological quality assurance systems (e.g. Hazard Analysis Critical Control Point; HACCP procedures) have been adapted to the needs of commercial plant tissue culture laboratories. These are aimed at, preventing the introduction of pathogens, into tissue cultures at establishment and in the laboratory. In established in vitro cultures preventative strategies have proved to be essential, since it is extremely difficult to eliminate environmental bacterial and fungal contaminants using, antibiotics and fungicides. In many cases anti-microbial treatments only inhibit contaminants and low levels of contamination persist. In particular, the use of antibiotics against Gram-negative bacteria (including plant pathogenic bacteria and Agrobacterium tumefaciens vector systems used in genetic engineering) has been shown frequently to be extremely difficult or unsuccessful. Detection of latent contamination may involve the use of general and semi-selective microbial growth media or serological and PCR-based molecular techniques for specific pathogens. However, it is often difficult to detect low numbers of latent bacterial contaminants (e.g. levels present following antibiotic treatment or when acidified plant media are used). This poses a particular risk in the production of transgenic plants where the elimination or detection of Agrobacterium tumefaciens-based vector systems cannot be guaranteed with the currently available methodologies. Recent research has also shown that there is a risk of the transmission of human pathogens in plant tissue cultures.     

Analysis of axonal growth and cell migration in 3D hydrogel cultures of embryonic mouse CNS tissue

This protocol uses rat tail-derived type I collagen hydrogels to analyze key processes in developmental neurobiology, such as chemorepulsion and chemoattraction. The method is based on culturing small pieces of brain tissue from embryonic or early perinatal mice inside a 3D hydrogel formed by rat tail-derived type I collagen or, alternatively, by commercial Matrigel. The neural tissue is placed in the hydrogel with other brain tissue pieces or cell aggregates genetically modified to secrete a particular molecule that can generate a gradient inside the hydrogel. The present method is uncomplicated and generally reproducible, and only a few specific details need to be considered during its preparation. Moreover, the degree and behavior of axonal growth or neural migration can be observed directly using phase-contrast, fluorescence microscopy or immunocytochemical methods. This protocol can be carried out in 4 weeks.     

Glycosaminoglycans of disaggregated foetal mouse brain tissue cultures

Synopsis Disaggregated foetal mouse brain tissue cultures were examined for glycosaminoglycans using Alcian Blue and periodic acid-Schiff staining techniques. It was found that spongioblasts (neuron and glial cell precursors) were rich in sulphated glycosaminoglycans, while astrocytes contained little or no sulphated polymers. The chief acid glycosaminoglycans of the brain reportedin vivo, hyaluronic acid, chondroitin sulphate and sialic acid-bearing polymers, were not demonstrated in the mouse brain cultures. There was a decline in glycosaminoglycan content over two weeks in culture, but during the corresponding periodin vivo an increase has been reported. These deficiencies are possibly correlated with the failure of the cultures to myelinate.     

An analysis of interneuronal connections in the auditory cortex of awake cats]

A statistical analysis has been made of the interaction of the auditory cortex units in alert cats with chronically implanted electrodes. Three neurones with an amplitude ratio of 4:2:1 were singled out from the multineuronal activity. The dependence between the firing of two neurones was determined by the cross interval histograms. The relationships between 78 pairs of units were studied in 26 three units microsystems. About a third of the studied pairs functioned independently. The number of pairs with one-way and two-way connections was about equal (26 and 30 respectively). The neurones which generated spikes of high and medium amplitude, had the largest number of two-way connections. One-way connections were equally represented in all the three neurones, though with regard to direction they depended on the amplitude characteristics of the spikes. In neurones with large and medium spikes, output connections predominated, while in neurones with small spikes input connections predominated considerably. The connection could be of inhibitory, excitatory or mixed type. The inhibitory type of connections was the most frequent occurrence (57 out of 86). At prolonged recording (6 to 16 min) of spike activity, most of the functional connections persisted.