D. A. Moshkov

In vitro long-term potentiation of electrotonic responses of goldfish Mauthner cells is accompanied by ultrastructural changes at afferent mixed synapses

The potentiated afferent mixed synapses of the Mauthner cells of fry and adult goldfish in stumps of the medulla oblongata incubated long-term in vitro were studied by electrophysiological and electron microscopic methods. It was shown that brief high-frequency stimulation of posterior branches of the eighth nerve induced a long-term potentiation of electrotonic transmission at large and small mixed club endings. It was about 135% upon subthreshold stimulation and about 200% upon suprathreshold stimulation. The ultrastructural analysis of ultrathin sections of potentiated mixed synaptic endings revealed an increase in the dimensions of desmosome-like contacts which was proportional to the degree of potentiation, about 135% or 200%, depending on the type of stimulation. The dimensions of gap junctions remained unchanged. The dimensions of active zones at potentiated synapses were reduced two-fold as compared with their unpotentiated counterparts, irrespective of the type of stimulation. Considering that desmosome-like contacts consist predominantly of F-actin, a molecule which possesses electroconductivity, it can be assumed that this cytoskeletal protein is involved in the process of potentiation. The increase in the synapse electrical conductivity can be mediated either directly, by shunting the synaptic junction with polymer actin filaments in the region of desmosome-like contacts, or indirectly, via the interaction of actin with gap junction connections situated nearby.

Smooth endoplasmic reticulum in fish Mauthner cells at different functional states

The ultrastructure of Mauthner cells of goldfish fry and adult xenotoca in intact state and after prolonged natural stimulation has been studied qualitatively and quantitatively. Additionally, Mauthner cells of intact adult goldfish and adult rotan Percottus glehni were investigated. In all adult fish the dendroplasm of the two major dendrites was shown to contain a regular network of smooth endoplasmic reticulum, with cisterns and tubules arranged transversally to the dendrite stem. In the Mauthner cells of intact goldfish fry, the reticulum was not clearly expressed, the transversal cisterns occurred occasionally. After stimulation, however, it became more developed probably due to proliferation of additional transversal cisterns. The periodicity of transversally oriented cisterns in the dendrites of Mauthner cells in each fish species studied was nearly the same. However, the number of transversal cisterns per unit of dendrite length, and the total length of cisterns and tubules per unit of cross-section area varied both within and among the species. These parameters increased after stimulation. It is suggested that the proliferation of the transversal cisterns in the endoplasmic reticulum and the extent of their development depend on the functional state of the afferent synapses and the plasticity of the smooth reticulum reflects the involvement of postsynaptic mechanisms in regulation of Mauthner cell stability presumably via the regulation of calcium homeostasis under varying conditions of functioning.

Ultrastructural Changes in the Mixed Synapses of Mauthner Neurons Related to Long-Term Potentiation and Natural Modification of the Motor Function

We examined changes in the ultrastructure of afferent mixed synapses on the membrane of Mauthner neurons (M cells) of the goldfish, which were related to two functional states, long-term potentiation (LTP) of the electrotonic response (a model form of the memory trace) and adaptation (resistivity to fatigue resulting from long-lasting motor training and considered a natural form of the memory trace manifested on the neuronal level). LTP was induced in medullary slices using high-frequency electrical stimulation of the afferent input. Adaptation was produced using natural vestibular stimulation (everyday motor training, which modified motor behavior of the fish and function of the M cell). It was supposed that if the LTP phenomenon is involved in the formation of natural memory, both the adaptation and the LTP states should be accompanied by similar specific structural modifications. Indeed, it was found that in both cases the number of fibrillar bridges in the gaps of desmosome-like contacts (DLC) in the mixed synapses on the M cell surface demonstrated an about twofold increase. These bridges are known to include actin filaments, which function as conductors of cationic signals; thus, the LTP-related increase in the density of bridges corresponds to increased efficacy of electrotonic coupling via mixed synapses. Such a structural correlate of LTP, which probably has the same functional significance in mixed synapses of the “adapted” M cells, allows us to suppose that LTP is a natural property of the nervous system. The LTP-type intensification of the relay function of mixed synapses, which corresponds to adaptation, is probably a compensatory rearrangement allowing M cells to maintain some balance of the synaptic influences and, at the same time, to remain in a stable and plastic state; this is necessary for stable functioning under changing environmental conditions.

Cytokinin-binding protein (70 kDa) from etioplasts and amyloplasts of etiolated maize seedlings and chloroplasts of green plants and its putative function

Cytokinins regulate chloroplast differentiation and functioning, but their targets in plastids are not known. In this connection, the plastid localization of the 70 kDa cytokinin-binding protein (CBP70) was studied immunocytochemically in 4-d-old etiolated maize seedlings (Zea mays L., cv. Elbrus) using monoclonal antibodies (mAbs) against CBP70 recognizing this protein not only in nuclei and cytoplasm, but also in plastids. CBP70 was detected in the amyloplasts of the root cap and etioplasts of the mesocotyl, stem apex, and leaves encircling the stem axis in the node. Immunogold electron microscopy demonstrated CBP70 localization in amyloplasts outside starch grains and revealed a dependence of CBP70 content in etioplasts on the degree of their inner membrane differentiation: the low CBP70 amount in etioplasts at the early stages of membrane development, the high content in etioplasts with actively developing membranes, and a considerable decrease in plastids with the formed prolamellar body. This suggests that CBP70 is involved in etioplast structure development. CBP70 was also observed in chloroplasts of the bundle sheath of green maize leaves. CBP70 purified from etioplasts mediated trans-zeatin-dependent activation of transcription elongation in vitro in the transcription systems of maize etioplasts and barley chloroplasts, suggesting that CBP70 is a plastid transcription elongation factor or a modulator of plastid elongation factor activity. CBP70 involvement in the cytokinin-dependent regulation of plastid transcription elongation could be essential for the cytokinin control of the biogenesis of this organelle.

Effect of the inhibitory neurotransmitter glycine on slow destructive processes in brain cortex slices under anoxic conditions.

Slow destructive processes in brain cortex were studied under deep hypoxia (anoxia). Study of the character and dynamics of DNA destruction showed that apoptosis and necrosis run in parallel under the experimental conditions. These processes typically develop in tens of hours. A similar conclusion was reached from electron microscopic study of the tissue ultrastructure. More detailed study revealed that a relatively rare type of apoptosis not involving cytochrome c release from the intermembrane space of mitochondria and not associated with opening of the mitochondrial nonspecific pore occurs under the experimental conditions. As this is occurring, the process can be slowed by high concentrations of glycine, an inhibitory neurotransmitter. The study of DNA destruction demonstrated that high concentrations of glycine selectively slow apoptosis but have almost no effect on necrosis. Glycine also drastically decreases changes in the tissue ultrastructure, particularly of mitochondria, arising under anoxia. Glycine does not notably influence the mitochondrial oxidative phosphorylation system. Study of impairment of mitochondrial function demonstrated that the oxidative phosphorylation system is not disturbed for 1 h, which is several times longer than the inhibition time of brain function under deep hypoxia. The mitochondrial respiratory system is preserved for a relatively long time (24 h). Malate oxidase activity is deactivated after 48 h. The succinate oxidase fragment of the mitochondrial respiratory chain proved especially resistant; it retains activity under anoxia for more than 72 h. A possible mechanism of the effect of high glycine concentrations is discussed.

Morphological parameters of mauthner neurons of goldfishes with modified asymmetry of motor behavior

We examined the morphological peculiarities of Mauthner neurons, MNs, in goldfishes with a phenotypically different or an experimentally modified preference to perform rightward vs leftward turnings in the course of motor behavior; this preference was characterized by values of the motor asymmetry coefficient (MAC). 3D reconstruction of MNs was performed based on several histological sections; volumes of the soma, lateral and ventral dendrites (LD and VD, respectively), initial segment of the axon, as well as full volumes of the right and left neurons, were calculated. Differences between the above parameters were expressed as structural asymmetry coefficients (SACs). It was shown that clear orientation asymmetry of motor behavior of the fish is accompanied by differences in the dimensions of MNs and their compartments; MNs localized contralaterally with respect to the preferred turning side were considerably bigger than ipsilateral neurons. Experimental influences inducing inversion of the motor asymmetry of fishes inverted structural asymmetry of their MNs. In fishes with no phenotypical preference of the turning side and in individuals whose motor asymmetry was smoothed due to experimental influences (rotational stimulations), structural asymmetry of the MNs was also smoothed. Changes of the structural proportions developed, as a rule, due to decreases in the dimensions of one or both MNs and their compartments. The MAC value was in direct correlation with the value of SAC of the MNs and with values of this coefficient for the soma and the sum soma + LD. At the same time, reciprocal relations were found for the MAC and structural asymmetry of the VD; the decrease in the volume of VD was related to an increase in the preference of the contralateral turning side by the fish, and vice versa. In general, the results of our study demonstrate that both morphological and functional peculiarities of MNs correlate to a significant extent with such a form of motor behavior of fishes as realization of spontaneous turnings.

Asymmetry of Motor Behavior of the Goldfish in a Narrow Channel

We studied swimming of goldfish fries about 3 cm long in a narrow channel by calculating the numbers of spontaneous turns on different sides. The ratio of fishes preferring to turn to the right vs to the left was 1.5:1.0, respectively; two-thirds of the fishes demonstrated an ambilateral behavior. Experiments with compulsory 10-min-long rotation of the fishes (clockwise around the longitudinal body axis for fishes preferring right-side turns and anticlockwise for fishes preferring left-side turns) showed that the behavioral asymmetry smoothed somewhat after such a procedure, and a greater number of the fishes became ambilateral in their preference to turn to one side or another. After a one- or two-day-long test, the initial asymmetry of motor behavior completely recovered. Compulsory rotation of similar fishes in the opposite direction exerted no influence on the asymmetry in the choice of the turning direction. Adaptation-induced training of the fishes (using fatiguing long-lasting vestibular stimulation) resulted in some smoothing of motor asymmetry but did not change its general pattern. Thus, our findings allow us to believe that a noticeable proportion of the goldfish individuals (similarly to other animals and humans) is characterized by an innate asymmetry of the motor function with a clear preference for either right- or left-side turnings. These relations can be smoothed under experimental influences but are recovered later on, i.e., they are stable and are not fundamentally transformed. We assume that the asymmetry of motor behavior of fishes in a narrow channel can be an adequate pre-requisite for further examination of the asymmetry of the brain and motor centers controlling changes in locomotion (body turnings)

ROLE OF DIFFERENT DENDRITES IN THE FUNCTIONAL ACTIVITY OF THE CENTRAL NEURON CONTROLLING GOLDFISH BEHAVIOR

The structural mechanisms that control the neuronal functional activity maintaining the brain functional asymmetry were studied using the relationship between the function and structure of goldfish Mauthner neurons (MNs) responsible for fish motor asymmetry as a model. It was shown for the first time that the dominant activity in one of the two counter neurons symmetrically situated in the medulla oblongata directly correlates with changes in its integral volume and is inversely regulated by the size of its ventral dendrite. It is known that the variability of the neuron dimensions is due to changes in the actin component of the cytoskeleton. The experimental data presented are discussed in terms of the involvement of cytosol actin in the control of the volume of somata and the main dendrites of MNs with the participation of dopamine and glutamate, two major neurotransmitters that are known to regulate the function of MNs.

Effects of optokinetic stimulation on motor asymmetry in the goldfish

In goldfish fries, we examined the effect of the optomotor reaction (drive to swim toward moving images of vertical dark bars) on the behavioral motor asymmetry. Contralateral optokinetic stimulation of fishes (rotation of the bars against the direction preferred by fishes in their turnings) gradually smoothed and, later on, inverted the motor asymmetry, while the asymmetry underwent no modifications in the case of ipsilateral optokinetic stimulation (rotation of the bars in the direction similar to that preferred for turnings). Contralateral optokinetic stimulation also induced long-lasting inversion of the motor asymmetry of immobilized fishes deprived of the possibility to follow the movement of bar images. Ipsilateral optokinetic stimulation of fishes with the enucleation of the ipsilateral eye enhanced their motor asymmetry, while contralateral stimulation either did not modify the motor asymmetry of such individuals or inverted this feature. These data agree with the concept that, in fishes, one eye dominates and more actively provides tracking of the movement of bars, while another eye is a subdominant one. In general, we first found that the use of specific visual stimulation allows one to modify for a long time the behavioral motor asymmetry of the fishes, which, as is known, correlates with the morphofunctional asymmetry of Mauthner neurons (MNs). Visual information that activates MNs influences mostly the ventral dendrites of these neurons; thus, our findings allow us to believe that stimulations, which initiate the optomotor reaction, can serve as an adequate physiological model of natural visual stimulation of MNs (with projection of the respective influences on the ventral dendrites of the above cells). The use of such an experimental paradigm opens up new possibilities for studies of the role of these dendrites in the functions of MNs and of the plasticity of morphofunctional organization of these cells.

Correlation between the sizes of Mauthner neurons and the preference of goldfish to turn to the right or left.

Three-dimensional computer reconstruction working from serial histological sections was used to study the morphology of the right and left Mauthner neurons (MN) in goldfish fry showing marked preferences to turn stably to the right or left in a narrow water channel or showing no asymmetry in their choice of side during turns. Visually, fish with left-sided motor asymmetry had larger MN on the right side, while fish with right-sided motor asymmetry had larger MN on the left side. Fish with symmetrical turns to the right and left showed no differences in MN size. Quantitative assessment of the MN of fish with preferences for turns to one side or the other revealed significant differences in the sizes of the somatic part, the axon hillock, and the axons of neurons located on the contralateral side of the medulla oblongata. Analysis of the statistical relationships between the functional (motor) asymmetry of fish and the morphological asymmetry of the somatic parts of MN in the same fish revealed a stable correlation (0.69) between these measures. Given that MN initiate unilateral turns of the body in free movement, the data obtained here lead to the conclusion that the larger neuron is more frequently activated in natural conditions as compared with the smaller, contralateral, neuron.