Journal of Integrative Neuroscience, Vol. 8, No. 4 (2009) 453–469 c Imperial College Press DOI: 10.

1142/S0219635209002319

Research Report PRINCIPLES OF CELLULAR-MOLECULAR MECHANISMS UNDERLYING NEURON FUNCTIONS
ALEXANDER S. RATUSHNYAK∗ and TATIANA A. ZAPARA Design Technological Institute of Digital Techniques SB RAS Novosibirsk, 63009, Russia ∗ ratush@mail.ru Received 27 October 2009 Revised 2 December 2009 In the present work, it was experimentally shown that a neuron in vitro was capable of responding in a manner similar to habituation, Pavlov’s reflex and avoidance of the reinforcements. The locality of plastic property modifications and molecular morphology, as well as the connection between functional activity and cytoskeleton have been revealed. A hypothesis is formulated that the neuron is a molecular system which may exercise the control, forecast, recognition, and classification. The basic principles of the molecular mechanisms of the responses underlying integrative activity, learning and memory at the neuronal level are discussed. Keywords : Neuron information functions; recognition; classification; forecasting; learning.

1. Introduction For the past ten years, vast amount of experimental data have been accumulated in neurobiological and cell molecular studies. The list of pathways of signal transduction of molecules, enzymes and genes taking part in the information processes is enormous. It allows understanding of the main principles of the organization and function of molecular information devices — the neurons. However, unequivocal answers to the main conceptual questions of bioinformatics have remained elusive still. There is an opinion that such “analytic approach has been exhausted, we have another and possibly more difficult problem. We literally sink in data. You can connect all data in the world, but without the model these data will always be insufficient” [23]. The main part of current neurobiological investigations is devoted to analysis of the processes taking place in synapses during learning and memorization and executed with the use of reduced neuronal systems as models, including those of mollusks [1, 2, 5, 8, 10, 15, 16, 18, 22, 33]. Plasticity is a fundamental property of the nervous systems. One of the best characterized forms of synaptic plasticity is long-term potentiation (LTP) [7]. Currently, 1124 proteins have been identified
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in the synaptic terminal and only 466 of them were validated by detection in two or more studies [9]. DNA microarray techniques allowed examination of the characteristics of the activity-regulated genes (ARG) which alter their expression during tetanus-induced LTP. The ARG-associated processes are complicated, including signal transduction, transcription regulation, and modulation of synaptic structure and functions. Structural changes at the synapse and new synapse formation are thought to be critical for longer lasting LTP [25, 32]. A significant number of ARGs are involved in neurite outgrowth and synapse formation. Many ARGs are implicated in the cytoskeleton regulation and cell-to-cell or cell-to-extracellular matrix interactions. Activity-regulated expression of these genes may directly contribute to the synaptic modification or formation of the synapse, because some ARG proteins which regulate cell–cell/extracellular matrix interactions and cytoskeletons are known to be enriched at the synaptic regions [27]. ARGs also reveal the novel molecular processes underlying LTP. For example, CDC25B is an ARG that is a well-known cell cycle regulator [13] but it plays a role in the regulation of synaptic plasticity as well [27]. Unlike an analytical path there is a direction in which the study of a cell and the brain as a whole is directed first of all to creation of imitation models with their subsequent experimental verification mainly under the black box scheme. In this case, for the most part the verifications are restrained by creation of mathematical models of different complexity levels. Until recently, a neuron was thought as a conductor of information and a simple threshold linear adder. Such ideas were used as the basis for most of the neurocomputer metaphors. An attempt to bring the neuron model nearer to the real object was undertaken in the Blue Brain project [23]. The notion of neuronal cells as rather complicated molecular device is the base of this project [14, 17, 20]. However, in this case undertaken attempts are also reduced to creation of a cell model based not on conceptual principles but on rather conflicting and inexhaustible experimental data. The results of such attempts clearly demonstrate the problem of knowledge synthesis from the infinite body of information while the conceptual model is absent. The big set of neurobiological investigations of neurons were undertaken in the Blue Brain project to increase the data volume, specify the data and take away the contradictions of known data. These studies mainly touched on the function of ion channels and membrane mechanisms. Using these data and supercomputer IBM, a model of neocortical columns containing several tens of thousands of model neurons has been created. According to the author’s opinion, the model under development is similar to the rat brain neurons and is exhausted by the limiting/existing possibilities of computer systems. However, it is not clear how fully this model reflects the real possibilities of simulated structure, because the function of this structure of the brain is still not fully revealed. Thus distinction of power consumption and speed on several orders from prototypes can serve by the indirect evidence of considerable mismatches (not only “technological” ones). Unfortunately the real informational neuron properties were not the focus of study in this mega project. Works which showed that the simple neuronal structures and separate cells are capable, in the experimental conditions, to generate enough

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complicated responses were not practically considered [3, 8, 11, 16, 22, 24, 34]. It is necessary to note that the data could not be used because of their incompleteness and inconsistency. The integration of the many known facts and the experimental results obtained in the past years allow us to suggest that the neuronal cell has rather complicated functions. However, on the conceptual level, the questions of the main informational neuron properties are not been yet solved. There are no answers to the most conceptually important questions. Namely, what is a neuron? Are its functions limited in the nervous system by the simple operations of signal processing and signaling? Is it possible to selectively change the effectiveness of separate input of a neuron? Is it possible to receive specific neuronal response after signaling from receptors to effectors by the non-selective intracellular pathways? Without solving these questions, the creation of models for both single cell system and higher level systems will only lead to creation of simulations weakly resembling real neuronal systems. Solving these problems would permit development of the conceptual models of biological information systems. That in turn will bring closer possibility purposefully to solve problems of medical correction pathological and cognitive changes in the nervous systems, and with another to use principles (and also molecular mechanisms) functioning of such biological prototypes of information systems in the field of neurocomputers nano- and molecular electronics. 2. Methods and Experimental Model In this study, we attempt to solve the above questions. It is necessary to carry out direct investigations of isolated neurons with the aim to show the functional and information possibilities of such cells. It was necessary to detect experimentally a change of the weights for separate inputs and fixation of new functional meanings by recording of spatial distribution of neuron activity. It is important on the reduced system (the isolated neurons) to determine experimentally the properties of processes of the functional plasticity and the participation of separate intracellular systems in such processes. To achieve these tasks, the selection of the experimental model is important. The subject must be rather simple, but it must permit organization of several information inputs to control and modify the molecular organization. The methodological possibility of storage of the main properties is necessary. One of the subjects which fits the majority of these requirements is isolated mollusk’s neurons cultivated outside of the organism. The large size of such cells permits organization of rather simple system of informational inputs. The present work was carried out using this model. The functional activity of isolated cultivated mollusk’s neurons was recorded using the imaging, microelectrode and patch-clamp techniques. Several types of the plasticity paradigms to be described below were developed. The formation of external electric inputs and registration of ion channel functioning (molecules forming the output signal) on the micro-areas was performed using the glass coaxial micropipettes with 5 µm end diameter. Supplying the substances was

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Fig. 1. Isolated neuron of Lymnaea stagnalis mollusk with micropipettes positioned for simulating the system of inputs and recording of the biological properties. The chosen areas for recording the imaging signals are marked on the cell soma by number. The bottom part of the figure shows the imaging signal from some chosen areas. On the left, the imaging signal from the chosen areas in another time scale (the scale is 10 minutes is shown). The scale of image is 10 µm. The time-signal scale is 10 seconds.

performed using the microcapillaries located inside these pipettes. Using these methods, several electric and chemical inputs were simulated. The cell response and ionic currents were recorded. Recording and comparison of ionic currents of stimulated and testing areas of a cell were conducted at the various stages of formation of the neuron response. The transformation of molecular morphology was estimated by redistribution of the cell structures decorated by endogenous pigments to be characterized for the chosen mollusk type. The mechanisms of interaction between various cells’ zones were investigated by influencing on the sites of a cell, located on different distances. For this, several micropipettes were put in the immediate vicinity, limited only by the thickness of their wall (3–5 µm) (Fig. 1). 3. Results 3.1. Information characteristics of the isolated neurons 3.1.1. Experimental models of plastic responses Many cellular analogs of learning paradigms to study the membrane mechanisms of the plastic responses were developed using mollusk’s neurons [1, 5, 15, 18, 33].

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However, these experimental models and methods did not reveal the answer to the question: Are there any homogeneous modifications of conductance of the whole membrane or these modifications can be local and specific for different zones of somatic membrane? In particular, the question of whether whole neuron is modified or the selective change of effectiveness of separate input is possible during training remains to be elucidated? The goal of the present study included search for a model which would allow investigation of the local changes of plastic properties of a somatic membrane. Plastic response modifications of isolated neurons occur by electric stimulation of soma’s zones. The modifications of initial response, transition from action potential (AP) to the local responses and transition from local response to AP were investigated. It is known that type, dynamics and maintenance time of plastic modifications of the response depend on individual properties of a neuron and parameters of applied influences (namely, intensity, sequence and frequency of stimuli). In our experiments, among the possible variety of applied stimuli were picked those which caused necessary changes of the response. The new neuron response had to be preserved during the period, which would exceed membrane processes. Several inputs onto the cell were performed. The stimuli were supplied to these inputs according to classical rules of plastic response development. The influences which increase or decrease the effectiveness of such responses to the signal supplied regionally to the selected inputs were used. The paradigms of unassociated, associated and auto-reinforcement stimulation have been chosen. The parameters of the stimuli for the paradigm of unassociated stimulation were selected such that the stimuli evoked the AP of a neuron. The single extracellular impulses of a current (0.1–0.5 nA) were supplied to one or several inputs with an interval of 1–2 seconds. The time course of response changes and AP restoration after the stimulation termination were recorded. The parameters of the stimuli for the paradigm of associated stimulation were selected such that the first in the pair of stimuli (applied to a micropipette) caused the local response, namely, a change of the membrane potential (MP) on 1–3 mV. The second stimulus was supplied on an intracellular electrode with some delay. Its parameters (i.e., amplitude and duration) were selected so that the second stimulus caused AP to every presentation. Thus, the second stimulus caused activation of ionic channels of the whole cell. The experimental model with autostimulation (biofeedback) has been chosen as the third paradigm to produce the neuronal plastic response [28–30]. Such feedback allowed correction of the stimuli presentation by means of the neuron output signal. In the experiments performed on spontaneously active cells, the duration of neuronal interspike (pulse-to-pulse) interval (ISI) was the operating parameter. Dynamics of the ISI averaged values was estimated as the coefficient of reinforcement (Cr). Cr = taisi /tcisi , where taisi is averaged interspike interval of background and tcisi is the current interspike interval.

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3.1.2. Plasticity of somatic areas of isolated neurons under local unassociated influences At the first stage, two or more micropipettes were placed on a somatic membrane and then the microelectrode was inserted into a cell for registration of intracellular activity. The stimuli parameters were selected such that initially every stimulus generate identical number of AP. Hyper- and depolarization stimuli were used. The multiple applications of the same extracellular non-periodic stimulus with stable parameters and the interval accidentally changing from 3 to 15 seconds in many cases evoked transition from the generation of spike responses to local responses. The probability and time course for decreasing the spike responses depended on the intensity of the initial response of a neuron. The time course of the reorganization of the responses to stimulus causing generation of four to seven APs was studied in detail. The multiple applications of local extracellular stimuli on the soma area (limited by the tip of the micropipette) led to the gradual decrease of the number of neuronal APs. The number of APs was reduced to one after the application of 30–50 stimuli. Then the first local responses appeared. At this stage (during 8–10 stimulus presentation), local responses were irregular and the periods of the APs generation were observed. Starting with the 40–60th presentation of the same stimulus, generation of APs completely stopped but APs can be evoked by modification of stimulus parameters such as amplitude, duration and polarity. Such dynamics of the neuron responsibility to the stimulus with stable parameters was developed in 86% of cases. In the remaining 14% of cases, the multiple applications of stimuli on the neuron somatic areas did not lead to the appearance of local responses, although reduction of AP number up to one or two was observed. To define the time of maintenance of the neuron response modification, the stimuli with interval 0.5–1 minute was applied to the soma area. It were revealed that the generation of a single AP in neuronal response was resumed after 1–2 minutes. The restoration of the initial number of APs was observed after 2–5 minutes. In repeated stimulation, the above mentioned stages of modification of neuronal responses could be evoked by lesser number of stimuli presentations. In particular, a reduction of AP number occurred after 8–10 stimuli, spike responses became irregular after 15–20 stimuli and only local responses were only observed after 20–25 stimuli. Test stimuli were supplied to other areas of the membrane in three stages reduction the AP numbers in a response, appearance of the first new (local) responses and stable generation of these responses. Such stimuli did not caused the change of the resting potential (RP) to be more than background variations (1–2 mV).

3.1.3. Plasticity of the soma areas of isolated neurons under associated local and general influences The stimulation of different soma areas by means of local influences was carried out in combination with intracellular stimulation by an algorithm similar to the

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conditioning. The parameters of local extracellular stimulus were chosen such that originally the stimulus applied though each of the micropipettes produces a local response. The amplitude of local responses originally was not more than 1–3 mV (RP of neurons was equal to −55–60 mV). The parameters of stimulus through the intracellular electrode were chosen so that they produced one or two APs. The paired stimuli were used. In each pair, the intracellular stimulus was applied after the extracellular stimulus with interval 50–100 ms. This interval was determined in each concrete case as a period to restore the neuron membrane potential up to RP after AP. The paired stimuli were applied irregularly with 5–15 seconds interval. In 69% of cases, the repeated application of local electric stimulus (coupled with a threshold stimulus) was accompanied by alteration of the neuronal response. The application of the first 15–20 associated stimuli lead to increase of the local response amplitude. When the stimulation was continued, the neuron began to generate AP on initially sub-threshold stimulus. At this stage (from 20–30 stimuli) the spike responses alternated with the subthreshold ones (Fig. 2I, 2C). After 30–35 associated stimuli, the generation of the AP in response to previously ineffective stimulus applied to the micropipettes became regular (Fig. 2I, 2D). The stimulus provoked the generation of AP (in this case response to the second stimulus of the pair did not changed). In our experiments, in 14% of cases, such stimulation did not imply response reconstruction after application of 40–60 coupled stimuli; in this case, further stimulation was ceased. In 17% of cases, the response to the second stimulus of a pair was changed, in this case the further stimulation was ceased. Single and associated stimulation did not changed RP of neuron soma. To define the time of maintenance of the neuron response modification, a single stimuli (with interval of 0.5–1 minute) were applied to the soma area. It was revealed that the response modification (to the stimulus with initially local response) was kept for 5–7 minutes. While the application of the single stimuli has the same frequency as in paired stimulation, the generation of AP was kept in response to 10–18 stimuli. Then the responses to the stimulus applied through the micropipettes became local (as it was before using the pair stimulation) (Fig. 2III). At repeated paired stimulation the change of response (generation of AP to the first stimulus) ensued with the result of application of less number of associated stimuli (6–10). 3.1.4. Response plasticity of the isolated neurons under autostimulation This experiment was carried out on the spontaneously active cells that generated APs without stimulation. The interspike interval of such background activity was varied as a rule from spike to spike. At the initial stage, the background current tcisi was calculated and according to the obtained value, the reinforcement threshold was determined. Two experimental protocols were used to form the external feedback by autostimulation/reinforcement of a particular interval, namely long and short intervals. In the first case, the stimulus was applied to the cell when interspike

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Fig. 2. Examples of plastic response modifications of the isolated neurons: I: responses to stimuli (AP) under associative stimulation (A: at the beginning of the experiment, B: after 10 stimuli, C: after 25 stimuli, D: after 50 stimuli, E: after withdrawal of reinforcing stimulus, F: at the end of experiment); II: unassociative stimulation (A: at the beginning of the experiment, B: after 10 stimuli, C: after 25 stimuli, D: after 40 stimuli, E: after 50 stimuli, F: changing the stimulus amplitude and duration); III and IV: the averaged time course of development and restoration of responses under associative (III) and unassociative (IV) stimulation (↑: beginning of stimulation, ↓: end of stimulation). The ordinate axis corresponds to the percentage of responses to a stimulus. The abscissa axis denotes the time.

interval duration was more than the predetermined threshold. In the second case, the stimulus was applied in the time period equal to 5–10% of the value of tcisi calculated before the stimulation. In such mode of stimulations, the reinforcement of the long intervals switched the cell in the regime of more frequent generation of AP (decrease of ISI, Fig. 3). At reinforcement of the short intervals, the value of ISI was increased. In a number of the cases, the cell was transferred to the regime of the generation of the burst consisting of 3–7 AP with a short ISI (less than 5% from taisi ). Between the bursts, the ISI was higher than the predefined threshold. Such differently directed activity modifications could be generated on the same cell (or on the different areas of a neuron) many times during the experiment. The dynamics of the modification of the reinforcement coefficient is given in Fig. 4. 3.1.5. Local plasticity of somatic membrane of isolated neurons The local character of the response modification was determined by stimulation of several soma areas of the same neuron. The same series of stimuli were applied to the first and second micropipettes. If the stimulation of the first area caused the response

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Fig. 3. The examples of the isolated neuron responses to auto-reinforcement of the intervals (between the spontaneous action potentials) which are shorter than the threshold determined according to the current averaged interspike interval in the background period (↑: the beginning of stimulation). The ordinate axis corresponds to the value of interspike interval. The abscissa axis denotes the time.

Fig. 4. The averaged temporal course of the response of the isolated neuron to auto-reinforcement of intervals (between the spontaneous action potentials). RSI : reinforcement of the short intervals; RLI : reinforcement of long intervals; ↑: the beginning of stimulation; ↓: the end of stimulation. The ordinate axis denotes the value of the coefficient of reinforcement (Cr). Cr = taisi /tcisi , where taisi represents the averaged interspike interval during full recording period and tcisi represents currrent interspike interval. The abscissa axis corresponds to the time.

modifications then, after restoration of initial response to these stimuli, we began to stimulate the area under the second micropipette. During the successive irritation of two somatic areas, it was shown that multiple local influences on these areas could have different effects on the neuron responses. The neurons whose responses were changed under stimulation of two areas or only one chosen area were revealed. In 14% of cases, the multiple stimulations did not cause changes of initial responses to one of the neuron soma areas. The stimulation through one of micropipettes did not change the neuron responses to the stimulus applied to other membrane areas. 3.2. Biophysical properties of areas of local neuron plasticity To investigate the possible cellular mechanisms of the phenomena described above we studied the properties of cell’s areas limited by micropipettes using these areas as model of information inputs.

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3.2.1. Change of the integral ionic currents of the areas of somatic membrane of neuron during development of plastic responses The total ionic currents flowing through the areas of the somatic membrane limited by end of micropipette (5 µm) were registered. The stimuli series were accompanied by alternate registration of currents flowing through the zones of the somatic membrane limited by micropipettes. In this experiment, the current’s registration in these areas was carried out at different stages, namely: (1) before stimulation, (2) at the stage of alternation of spike and local responses, (3) at the stage of stable responses when the changed responses were recorded to 10–15 of stimuli. At the stage of alternation of spike and local responses, the changes of the input and output currents were observed if response changes were recorded. The amplitudes of the input currents were increased to 50–200% in comparison with those registered before stimulation. At the stage of the stable responses, the amplitude of input currents was decreased in comparison with the currents to be registered at the stage of response alteration. The output currents were moderately changed relatively to the initial ones (the changes in 80% of cases were not significant). In other experiments the amplitude of the output currents was decreased to 10–15%. The ionic currents in the control areas were not changed. If the multiple stimulations did not cause the response change then the currents were not changed in those areas of the neuron somatic membrane. The main changes of the integral ionic currents during local plastic modifications for the neurons placed in the solid solution without sodium ions consisted of increase of the amplitude of the input currents; the output currents were decreased insignificantly. 3.2.2. Optical density in the zone of the local influences producing the plastic responses During local stimulation of neurons producing the plastic responses, the change of relative optical density in the zone of influence and the fluorescent label (for actin and tubuline) were registered. The relative optical density was increased from 5–15% to 250%. On the average, the change was equal to 44% (in 15 experiments). The change of optical properties correlated in time with the change of neuron responses and transmembrane ionic currents. The quick restoration (during 1–2 minutes) of initial optical density took place after ceasing the stimulation in 33% of cases, in 54% of cases the decreasing of optical density was observed during 5–40 minutes. 3.3. The influence of selective modification of molecular neuron structures on the plastic changes of responsiveness 3.3.1. The influence of microfilament modification on the neuron plastic properties The influence on the neuronal microfilament system was performed by injection of phalloidin and cytochalasin B into the micropipette solution. The monomeric and

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polymerized (microfilaments) cellular actin were in the dynamic equilibrium state. Phalloidin interacts with the microfilaments by blocking the reaction of depolymerization and stabilizing the cellular microfilaments. Cytochalasin B interacts with monomeric actin, blocking the reaction of polymerization and formation of new microfilaments. To investigate the influence of actin polymerization blockade on the plastic properties of neurons, cytochalasin was injected into the micropipettes solution. In these experiments, the check electric stimulation was carried out to find two neuronal areas with the determined plastic properties. Cytochalasin was injected into one of the pipettes only. The injection of cytochalasin B did not influenced the plastic responses development during the first 30–45 minutes. During the following stages after the injection, the single stimulation did not evoke transformation of spike responses into local ones in 65% of experiments (such effect was revealed in 7% of the control experiments). In 27% of cases, local responses appeared, but the stage of response alternation was maintained even after application of 50–100 stimuli. The associative stimulation while using cytochalasin B evoked AP in 9% of cases. The plastic properties of the check neuronal area were not changed. The neuronal areas at which the local stimulation evoked the response change were influenced by phalloidin. Local stimulation was carried out on those two areas. If the stimulation of each area caused the response change, one of these areas was processed by phalloidin. The substance was injected at the various stages of experiment, namely: after the series of stimuli evoking the response change; after the simulation causing the irregular responses; before stimulation and after spontaneous restoration of the initial response. In the first case, we began to inject phalloidin into a micropipette immediately after the response change and continued stimulation (20–25 minutes). Then to reveal the time of maintenance of the response change, the stimuli were applied to this neuron area at 1–2 minutes interval. The restoration of the initial response to the stimulus did not occur during the time (1–1.5 hours) of observation

Fig. 5. The time course of the plastic response development under associative stimulation after injection of phalloidin. The results of four groups of experiments (Ph1–Ph4) are accumulated. →: the beginning of substance application, ↑: the beginning of stimulation. The ordinate axis corresponds to the percentage of responses (AP) averaged on the group of neurons. The abscissa axis denotes the time.

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(Fig. 5, Ph1). In the control area, the time of response restoration did not changed. If phalloidin was injected before the stimulation or after the restoration of the initial responses and the second series of stimulations was made, then the response change was not observed even after application of 100–150 stimuli (Fig. 5, Ph4). The processing by phalloidin of the neuron areas completely blocked the development of all plastic responses during the 1–1.5 hours. In the process of testing the effects of phalloidin it was revealed that the maintenance time of the new response was increased if the stimulation was carried out not earlier than 45–60 minutes after the substance injection into a micropipette. Reactions with maintenance time of more than 15 minutes were revealed most often. In this case, the cell response was not restored during all period of observation (up to several hours). The averaged dynamics of responses to the associative stimulation is given in Fig. 5, Ph1–Ph4. 3.3.2. Influence of microtubules modification on the plastic properties of neuron The influence on the microtubules system was performed by injection of colchicine or taxol into the micropipette solution. The monomeric and polymerized forms of tubulin protein (microtubules) were in the cell in dynamic equilibrium state. Taxol interacts with microtubules and blocks the reaction of depolymerization, stabilizing the polymeric form of tubulin in a cell. Colchicine in turn interacts with monomeric tubulin blocking the reaction of polymerization and the formation of new microtubules. Colchicine was injected in one of the pipettes and it was impacted on development of the plastic responses for 10–15 minutes. The single stimulation did not evoke local responses or they developed only at the stage of response alteration. In 43% of experiments (in the case when the stimulus initially evoked 1–2 AP), the plastic response to the single stimulation did not develop. In 57% of experiments, dynamic change of the response development took place. The first local response appeared only after 50–100 stimulations. In this case, response alteration occurred even after 300–500 stimuli. Cessation of stimulation led to restoration of the initial response for 20–30 seconds. During associated stimulation, the local responses did not transformed in AP even after 50–100 stimuli. The plastic properties of the check neuron area did not change. Taxol was injected in the micropipette solution and its effects were detected after 20–30 minutes. The dynamics of response change were close to the check ones, independently from the type of stimulation (Fig. 6, T1). The restoration of initial response happened more quickly in 40–60% of cases. The second session of stimulation through the pipette with taxol showed that it was necessary to use more stimuli to change the responses. In the second session, the number of stimuli to develop a new response was increased by 20–25% (Fig. 6, T2). The time of maintaining the new type of response did not change relatively to the first series. During the third and following sessions, the number of stimuli necessary for response change was

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Fig. 6. The time course of the plastic response development during unassociative stimulation after taxol injection. T1–T4: the series of stimulation, ↑: the begin of stimulation. The ordinate axis: the percentage of responses (AP) averaged over the group of neurons. The abscissa axis: the number of stimulus.

being increased further (Fig. 6, T3–T4). The plastic properties of the check neuron area did not change. In the repeated session of stimulation through the check pipette, the response change took place as the result of using lesser number of stimulations. The dynamics of responses, averaged over the neuron group is shown in Fig. 6, T1–T4.

4. Discussion Up to now there is relatively small number of the investigations aimed at the direct experimental analysis of information neuronal functions. It was shown that simple neuronal systems and single cells in vitro were capable of forming complex responses [3, 8, 11, 16, 23, 25]. According to these data, a neuron may be considered as a complicated molecular information system. The neuron response modifications revealed in our study are phenomenologically similar to the behavioral type of habituation and Pavlov’s reflex, but they are generated by a single cell. As the plastic modifications were developed in evolution as the system level response of molecular and cellular complexes to the environment influences, it can then be suggested that the common basic mechanisms are characteristic for both the behavior level of an organism and for neuronal elements [2, 8, 10, 22, 33]. Thus, the results obtained confirm the hypotheses that the isolated neuron is a complex information controlling system. It was shown that every cellular input was modified according to preceding learning. A significant number of electrical or chemical inputs (the size of one cellular region to be activated in our experiments were less 1/1000 of the total surface of a cell) can be organized on a cell. By the influence on each of such local inputs we can change (increase or decrease) the weight of active input. Our results may be considered as indirect confirmation of the idea that the principles of the neuron functions are defined by their morphology [4, 6, 19, 34]. Some structural elements of such molecular “nanocomputer” are defined.

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Besides, the influences on these molecular systems permitting to accelerate and block the weight changing for local inputs are found. The compilation of the results obtained and known modern data in molecular cellular organization allow us to formulate (heuristic to a great extent) a hypothesis about the leading role of regrouping of neuronal receptors and effectors in the process of learning. It was shown that the isolated neuron can control external stimulation by changing output signals to minimize reinforcement. All these permit us to consider the cell as a molecular information system capable of modifying its input and output elements according to a definite program genetically determined but can be influenced by environmental factors. The mechanisms of such system operations have not been sufficiently investigated yet. It is known that ionic channels and receptors are as a rule connected with the cytoskeleton by the specialized protein domains. In the initial concept of signal transmission it was suggested that all main components, namely, receptors, G-proteins and their targets, protein kinases, and ionic channels are freely distributed, and information transfer in the intercellular space takes place during the occasional collisions and interactions of process partners [26]. A huge number of receptors for various neurotransmitters, hormones and sensor stimuli interacting with the system of the secondary mediators are known. Nevertheless, the neurons are capable of distinguishing one stimulus from another within microseconds. A new concept of signal transmission in the eukaryotes based on the results of previous investigations appeared. It implies that the domains of protein–protein recognition allow physical interactions between molecules partners. Various components such as receptors, enzymes and their substrates, and the final targets are structurally combined in the functional complexes — the microdomains. The formation of microdomains takes place on the structure-forming proteins, which due to their genetically conditioned properties, can form ordered connections with receptors, channels, proteins, and can interact with cytoskeleton and molecular movers [12]. Various protein–protein interactions permit formation of clusters of ionic channels and/or receptors on specific subcellular sites of neurons which locate near to the proteins of intracellular signal cascades. Such structure-module protein organization (named as microdomains) permits the input signal to be concentrated and combined with signal transduction and transport pathways of a cell [12, 21]. The specialized receptors and ionic channels of the cellular plasmatic membrane have a unique individually-arranged structure of interactions with their partners (Fig. 7). This microstructure organization permits the receptors and ionic channels of the plasmatic membrane to react differentially on the outward influences using the same systems of the secondary mediators. The data about the mechanisms of the neuronal (non-synaptic) plasticity obtained by us on the neurons of the mollusk — evolutionally older subject than the mammals, allow us to consider the principles of the response of the cell system to the changing environment and the role of the cytoskeleton in the formation and maintenance of the plastic responses. Based

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(A)

(B)

Fig. 7. A possible scheme of the functional domain formation by rearrangement of the receptors and effectors proteins and attaching them to the cytoskeleton. A: before formation of the domain, B: after the structural rearrangements.

on literary evidence and our experimental data, it is possible to assume that the basic element of a neuron while realization of its main information function (record and storage of the information) is the structural microdomain formed during interactions of a neuron with the environment and maintained by the systems of the cellular structure stabilization. The set of such domains being formed by regrouping during the leaning process permits the cell to recognize, classify and use the multidimensional vectors of outward signals for control (on the basis of previous experience). We can suggest that the molecular information neuron system on the basis of previous leaning performs positional coded fixation of the multidimensional vector. It forms a memory matrix consisting of the microdomains. The matrix being formed in such a manner can include all input signals (conditioned and unconditioned stimuli) as elements because neuron has the responses to them. The recognition of the image (even in the absence of some elements of a multidimensional vector) can lead to “predicting” previously connected events. Such identification can be performed as distribution dynamics (defined by the matrix) of a wave of excitation from one to another structure-functional cellular element. The activation of effectors of cell included in the vector of recognition can lead to the formation of response to avoid or minimize the external influences.

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Thus the main function of a neuronal cell is recognition, classification, forecast and control, similar to biological systems of higher level of organization [3, 31]. Using the above conceptual neuron model and taking into account the neuron’s main information properties when creating imitation systems may bring such systems closer to their biological prototypes.

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