Sensitization in - Springer Link

Neurophysiology, VoL 25, No. 2, March-April, 1993

Sensitization in Helix Snail: Morphofunctional Correlates in Command Neurons of Withdrawal Behavior V. D. Goncharuk, S. A. Kozyrev, and V. P. Nikitin

UDC 612.822

Translated from Neirofiziologiya, Vol. 25, No. 2, pp. 150-157, March-April, 1993.

Received October 19, 1992.

Electrophysiological effects have been studied in command neurons of withdrawal behavior inHelix snail. In parallel, correlated changes in the content of bound calcium (Ca-b), as well as changes in DNA condensation, were investigated using a chlortetracycline fluorescent probe and the fluorescent dye bisbenzimide, respectively. Short-term electrophysiological changes (depolarization of the membrane and elevation of its excitability) in sensitized snails have been found to be accompanied by an increase in the Cab level in the cell nucleus and by partial DNA decondensation. Long-term effects were characterized by more pronounced synaptic components of the responses - - slow EPSPs evoked by sensory stimuli, as well as by further DNA decondensation and considerable elevation of the Ca-b content in the nucleus and cytoplasm. The results obtained in in vitro conditions have shown that Ca-binding nonhistone proteins of chromatin are components of the cell nucleus whose content may be measured by chlortetracycline fluorescence.

nounced phasic changes in the Ca-b content in these cells [5]. However, the technique of vital spectrophotometric registration of CTC fluorescence in these experiments allows determination of total Ca-b content only in the whole soma or in a substantial portion. Only morphological methods can reveal distribution of the dye in individual structural components of the cell. Therefore, in the present work we attempted to assess redistribution of calcium-binding components in command neurons at different stages of sensitization with the use of histochemical methods. Neurons were fixed in liquid nitrogen and stained with CTC. It is known [ 1, 6, 7] that elaboration of new skills is closely associated with the functions of the genetic apparatus of nerve cells. In this connection, for staining slices of command neurons, we used, together with CTC, another fluorescent dye, Hoechst 33342 (bisbenzimide-BB), which is thought [8, 9, 10] to reveal functional DNA activity of cells. Since it was found during investigation that CTC stains nuclear structures of nerve cells most intensely, in in vitro experiments we studied possible calcium-binding nuclear components, in particular, nonhistone proteins of chromatin and DNA for which CTC can be regarded as an indicator.

INTRODUCTION The results of investigations performed in the last few years convincingly demonstrate a leading role for intracellular metabolic processes in the realization of specific functions of nerve cells, including the elaboration of new skills [ 1]. Further progress in this area depends to a considerable extent on the development and application of techniques allowing recording of different aspects of neuronal metabolism. In this r e s p e c t , the use of f l u o r e s c e n t p r o b e s is promising. In b i o c h e m i c a l studies, a c h l o r t e t r a c y c l i n e (CTC) probe is widely used to register the dynamics of Ca 2+ binding (Ca-b) to hydrophobic components of ceils [2]. This method was first used in our studies on vital changes in Cab content in intact nerve cells of animals [3, 4]. In particular, the experiments conducted on command neurons of withdrawal behavior in Helix snails showed that elaboration of sensitization (one of the simplest forms of learning) leads not only to rearrangement of electrical activity, but to pro-

P. K. Anokhin Institute of N o r m a l P h y s i o l o g y , R u s s i a n A c a d e m y o f Medical Science, Moscow, Russia.

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Sensitization in Helix Snail

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Fig, 1. Changes in responses of command neurons of withdrawal behavior to application of 0.75% quinine solution following sensitization with 10% quinine solution, a) Initial neuronal response to application of 0.75% quinine solution to molluscan head (right-side inset -- response to rectangular current pulses passage through a microelectrode); b) neuronal response to sensitizing application of 10% quinine solution to molluscan head (arrow denotes the moment of quinine application), c-e) Neuronal responses to application of 0.75% quinine solution 15, 50, and 90 rain following sensitization stimulus, respectively. Lines under records indicate application of 0.75% quinine solution. Upper parts of action potentials are not shown.

METHODS Experiments were c o n d u c t e d on the snail Helix lucorum, L. A semi-intact preparation has been prepared according to conventional technique [11]. Simultaneously, electrical activity of command neurons of withdrawal behavior, LPlt and RPll, was recorded intracellutarly using glass microelectrodes filled with 3 mole/liter KC1 solution [11]. After amplification, the recorded potentials were recorded by means of an oscillograph and an ink-recorder. Sensitization was produced by applying four drops of 10% quinine hydrochloride solution to the skin of the anterior part of the head. Test stimuli (0.75% quinine solution) were applied to the snail head every 15-20 rain for 1,5-2 h until the sensitizing influence was attained and for 2-3 h after its administration. The level of excitability of the cytoplasmic membrane was evaluated by the number of action potentials (AP), evoked by the passing of a depolarizing current (1-2 nA, 10 sec) through the intracellular electrode. In total, 46 neurons were investigated in electrophysiological experiments. Dynamics of the Ca-b content in command neurons of the snail were studied under conditions similar to those of electrophysiological experiments. Spectrophotometry of neurons stained with CTC was performed by means of a luminescent contact microscope according to the earlier described method [3-5]. The fluorescent signal was recorded in a range with a maximum o f 520 nm for an excitation spectrum with a maximum of 4 t 0 nm. The optical signal was transformed into an electrical one by a photoelectric multiplier and recorded with an ink recorder. After record-

ing of the natural fluorescence of the unstained command neuron with the use of optical probe (150 gin), the ganglia were perfused with p h y s i o l o g i c a l saline c o n t a i n i n g 50 gmole/liter CTC ("Sigma," USA). The dynamics of the Cab content were inferred from the change in the level of fluorescence of investigated cells, which was expressed in relative values (the level of fluorescence prior to sensitizing influence was taken as 100%). As is known [2, 4, t2], the method of CTC-fluorescent probing allows evaluation of the dynamics of the content of intracellular calcium bound mainly to hydrophobic components, including calciumbinding proteins. In totaI, 24 neurons were investigated in spectrophotometric experiments. For histochemical investigation the subesophageal rings of the ganglia of semi-intact preparations from six Helix snails were frozen in a liquid n!trogen. Two of these snails were nonsensitized (control), and the rest were frozen 30 (two snails) and 90 rain (two snails) after application of sensitizing stimulus. Serial cryostat sections of 5 gm thick were placed into coverslips and fixed in cold acetate. A portion of the sections were stained for 20 rain in BB solution (8 gg/ ml). The sections adjacent to these were stained in CTC solution (6 gg/ml). After histochemical reaction was stopped, the sections were rinsed and dried out. Visualization and photographing of the sections were done with a fluorescent microscope. Wavelengths of excitation and fluorescence for preparations stained with BB were 365 and 460 nm, respectively. In order to measure the intensity o f fluorescence o f nonhistone proteins of chromatin and purified DNA in the presence of CTC, a spectrophotometer was used. Acid nonhi-

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Fig. 2. Dynamics of bound calcium content in LPll neuron during sensitization, 1) Changes in fluorescence of sensitized snail neurons; 2) the same in intact snails, In every case, data are averaged from 12 neurons, Abscissa) time, hours; ordinate) relative change of the intensity of fluorescence of a neuron, % (the intensity of fluorescence before sensitization is taken as 100%). Arrow denotes the moment of application of 10% quinine solution.

stone proteins [13], as well as purified DNA [14], were obtained by Drs. E. A. Shumova and M. A. Gruden in the laboratory of Molecular Neurophysiology at the P. K. Anokhin Institute of Normal Physiology, Academy of Medical Sciences of Russia. Total nonhistone proteins of chromatin were separated into three fractions by a step-like elution with increasing (graded) concentrations (0.1, 0.3, and 0.5 mole/liter) of NaC1 solution with DEAE-sepharose 4B added. These fractions were distinguishable by the value of negative charge. Calmodulin ("Sigma," USA), a calciumbinding protein, served as a control.

RESULTS Background electrical activity of LP1 t and RPll neurons was either absent or, if present, was characterized by generation of rare APs and fast EPSPs. The resting potential was on the order of 60-64 mV. In response to application of 0.75% quinine solution, the neurons generated a slow wave of depolarization (sEPSP), with an amplitude of 3-5 mV and a duration of 70-100 sec, and several APs (Fig. 1). Application of 10% quinine solution to the snail head elicited a pronounced depolarization shift of neuronal membrane potential by 12-16 mV, AP generation with high frequency for 2-5 min, acceleration of EPSP generation, and an increase of excitability of cytoplasmic membrane by a factor of 1.5-3. The initial level of membrane potential was restored by the 15-20th rain after the sensitizing influence. The excitability of the membrane decreased by the 50-75th min to the level exceeding its initial value by 20-30% and remained unchanged till the end of the experiment. In the 15 min following application of sensitizing stimulus, the expression of responses to application of 0.75% quinine solution was slightly increased. In particular, the amplitude and duration of sEPSP increased by 10-20% as

compared with their initial values. By the 50-60th min following sensitization, the neuronal responses elicited by chemical stimuli displayed 15-20% reductions in duration and amplitude of sEPSP (Fig. 1). Beginning with the 5060th min, the responses to sensory stimuli increased substantially. By the 90-120th rain, the amplitude and duration of sEPSP elicited by 0.75% quinine solution exceeded the initial values by 50-70%; this pattern was maintained till the end of the experiment (Fig. 1). Against the background of maximal sEPSP increase, the number of APs in neuronal responses to sensory stimuli outnumbered the initial number by a factor of 4-8. In intact snails, the intensity of fluorescence of LPll and RPtl neurons stained with CTC was relatively stable over several hours of recording (Fig. 2). Administration of 10% quinine solution led to pronounced changes in fluorescence consisting of several phases. Within 30-100 sec following quinine application, the fluorescent signal increased, reaching 10-14% by the 15-20th rain (Fig. 2). Within the following 30-40 rain, the level of fluorescence gradually increased by 2-6%. By the 50-70th min after sensitizing stimulation, a repeated marked enhancement of the intensity of fluorescence was observed for 30-40 rain. By the 85-100th rain, the fluorescent signal was stabilized at a level exceeding the initial one by 30-40%. Histochemical experiments performed on the slices of command neurons of nonsensitized snails revealed that the sites stained with BB were localized in the cell nuclei. They had the appearance of rather large, brightly luminous islets surrounded by fields with scattered fluorochrome granules. Their fluorescence was diffuse and less bright than that of islets (Fig. 3a). Analysis of adjoining slices stained with CTC showed that the domains with the brightest fluorescence of this fluorochrome often coincided with the domains of diffuse BB fluorescence. It should be noted that sometimes small, weakly fluorescent loci of CTC could be observed in those areas where BB fluorescence was absent (Fig. 3b). By the 30th rain following the sensitizing influence, islets of intense BB fluorescence were encountered very rarely; the area of each islet was much smaller than that observed in nonsensitized snails (Fig. 3c). Domains of CTC fluorescence had the appearance of bright spots or elongate strips, sometimes exceeding the boundaries of the areas stained with BB (Fig. 3d). Analysis of serial sections showed that the area of the CTC fluorescence domains exceeded, on the average, that in nonsensitized animals. By the 90th min after the sensitizing influence, the islets of bright BB fluorescence were practically absent and the stained structures in the slices had the appearance of elongated, weakly fluorescent bundles (Fig. 3e). As a result of CTC staining of adjoining slices, brightly fluorescent domains of this fluorochrome showed significant overlapping with portions stained with BB, which occupied almost all the nucleus and a greater part of the cytoplasm. In this case, it was impossible to identify the boundary of the nucleus be-

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Fig. 3. Staining of LP1t commandneurons of withdrawal behavior with chlortetracycline (b, d, f) and bisbenzimide (a, c, e) at different stages of sensitization, a, b) Control (nonsensitized snails; c, d) in 30 rain and e, f) 90 rain following sensitizing influence, a, c, e) Photographs of sections adjacent to those in b, d, f, Arrow shows a brightly fluorescent islet of densely packed DNA; broken line indicates the boundary of the neuronal soma. cause the stained parts of the nucleus and the cytoplasm come into contact with one another at a considerable distance (Fig. 33'), In in vitro experiments the change in fluorescence of nonhistone proteins of chromatin stained with CTC was studied. When 1.0 mg of each of three fractions of these proteins were added to a solution containing deionized water and CTC (50 gmole), the fluorescence increased by 300500% (a level of 50 gmole CTC dissolved in deionized water was taken as 100%) (Fig. 4). After application of 1.0 mmole EDTA, a calcium chelator (Serva, Germany), to the cuvette, the intensity of fluorescence sharply dropped to 2060%. Adding of 0 2 mg calmodulin to the CTC solution increased the fluorescence by 16% (Fig. 4). Subsequent addition of 100 gmole Ca 2+ produced a pronounced increase of fluorescence by 290% and i n t r o d u c t i o n o f 1 m m o l e of EDTA to this medium reduced the increase to 18%. Addition of 1.0 mg of purified DNA to the initial solution containing 50 gmole CTC, as well as subsequent application of 100 gmole Ca 2+ and 1.0 mmole EGTA to this solution, had almost no effect on the fluorescence.

DISCUSSION The results of our experiments have shown that a single sensitizing influence with 10% quinine solution produces short- and long-term changes in different indices of electrical activity of command neurons for withdrawal behavior as

well as a pronounced restructuring of the Ca-b level in these cells. During the short-term development of stabilization, the following events were detected: a considerable decrease in the level of membrane potential, an increase in excitability of the plasma membrane, some facilitation of responses to sensory stimuli immediately after the sensitizing stimulation and suppression of such responses by the 50-60th rain after the stimulation, as well as an initial intense increase of the Ca-b level. The long-term shift occurring about 1 h after sensitizing stimulation was characterized by pronounced facilitation of synaptic components in neuronal responses (sEPSP) and repeated p r o n o u n c e d elevation of the Ca-b level. The first phase of the increase in neuronal Ca-b content is likely due in part to the entry of Ca 2+ into the cell during membrane depolarization and its binding with near-membrane structures. Earlier, we showed [15] that removal of Ca 2+ from the bathing solution or inhibition of catmodulin by a blocker, W7, prevents the changes in the Ca-b level elicited by sensitizing stimulation in command neurons of the withdrawal reflex. However, these changes in the plasma membrane and the cytoplasm of command neurons are obviously a part of the changes observed in the Ca-b level in the cell during sensitization. Morphological experiments demonstrated that already in intact (nonsensitized) snails, it is precisely the nuclear content of the cell which is the most intensely stained with CTC. It was established in model experiments performed by us earlier in in vitro conditions [12] that a CTC probe is an adequate indicator of Ca 2+ binding

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12 t23 f Z t Z Fig. 4. Changes in fluorescence of chromatin nonhistone proteins stained with chlortetracycline (CTC) in in vitro conditions. In a-c: 1) fluorescence of solutions containing 50 ~tmole CTC and 1.0 mg of chromatin nonhistone protein fractions; 2) the same after adding 1 mmole EGTA. In d: 1) fluorescence of solution containing 50 ~tmole CTC and 0.2 mg calmodulin; 2) the same after adding 100 ~tmole Ca2+; 3) after adding 1.0 mmole EGTA. All data are averaged from two measurements. Vertical line) relative intensity of fluorescence, % (the level of the fluorescent signal of 50 ~tmole CTC dissolved in deionized water is taken as 100%). by calcium-binding proteins. Proceeding from this fact, it can be suggested that Ca 2+ binding with calcium-binding proteins may be one of the direct reasons for fluorescence of the CTC stained neurons. At present, there is little information on the role of calcium-binding proteins in the functioning of nuclear structures. A calcium-dependent isoenzyme of protein kinase C has been found in the nuclei of cerebellar Purkinje cells and in the nuclei of cortical pyramidal cells [16]. This protein kinase phosphorylates a number of nuclear proteins, including histones [17], lamin B, the main component of the nuclear membrane [18], proteins of nuclear matrix [19], and topoisomerase II [20]. The calcium-binding proteins parvalbumin and calbindin D-28k have been detected in the nuclei of most of the cerebellar Purkinje cells, spinal ganglia neurons, in cortical interneurons and in single cells of other regions of rat cortex [21]. A modulatory effect of Ca 2+ on the expression of a gene coding the synthesis of calbindin D28k was detected in experiments on cultured cells from the duodenum [ 10]. Such calcium-binding proteases as calpains were shown to play a leading role in the metabolism of nuclear proteins. In particular, they modulate the activity of protein kinase C phosphorylating histone H 1 in the nuclei of rat hepatic cells [22] as well as activate a leupeptine-sensitive protease which breaks down high molecular proteins of the nuclear matrix [22, 23]. A widely occurring calciumbinding protein, calmodulin, appears to be of vital importance for DNA repair and effects on cell proliferation [24].

Based on the analysis of the data available, some authors suggest a peculiar participation of nuclear calciumbinding proteins in gene expression [20, 21, 25]. It was found in our in vitro experiments that CTC fluorescence greatly increases in the presence of total fractions of chromatin nonhistone proteins and decreases if EDTA, a calcium chelator, is introduced into this medium. The increase of fluorescence of the CTC-nonhistone proteins complex is comparable with both the increases of these proteins in different fractions and with the increase of fluorescence of the CTCcalmodulin complex; this allows one to suggest the presence of a considerable amount of calcium-binding proteins in nonhistone proteins of chromatin. In the 30 min following application of the sensitizing stimulus, the intensity of fluorescence of CTC probe increased somewhat in the cell nucleus. At the same time, the brightness of the fluorescence was reduced and the distribution p a t t e r n o f f l u o r e s c e n t g r a n u l e s stained with BB changed, It is known [8, 9, 26] that the intensity of fluorescence of fluorochrome BB in the cell nucleus reflects the processes of decondensation of chromatin and directly depends on the density of the chromatin packing. Decondensed chromatin (euchromatin) represents the parts of s-chromosomes which are in a diffuse state. It is established [27] that precisely these condensed parts of chromosomes are the locations where transcription of messenger RNA takes place and, on the other hand, that condensation of these parts of chromosomes is accompanied by suppression of gene expression localized in these loci. The above facts imply that as early as the 30th min following sensitizing stimulation, the activation of the genome of c o m m a n d neurons for the withdrawal reflex occurs; moreover, it is likely that calcium-binding nonhistone proteins o f chromatin are involved in this activation. It is known that genome activation can be realized relatively quickly. In particular, it has been shown that within about 10-20 rain after various stimulating influences on cells [28], including obviously sensitization, the so-called early genes or proto-oncogenes are activated. One of the mechanisms for excitation transmission from neuronal synaptic inputs to the cell genome may be intracellular secondary messengers [1, 7]. By 90 rain after the sensitizing stimulation, practically all the nucleus and a considerable part of the cytoplasm were stained with CTC. At the same time, staining with BB was diffuse, which indicates a considerable decondensation of chromatin. The area of the section covered with fluorescent CTC significantly overlapped the area occupied by fluorescent BB. In this connection, one may suppose that calcium-binding proteins interacted not only with heterochromatin but also with euchromatin, which is known to be the most active in relation to gene expression. Thus, it appears that by the 90th min following sensitization, a considerable number of DNA segments for command neurons are activated and that the quantity of Ca 2+-

Sensitization in Helix Snail binding proteins is augmented. The reason for the increase in the content of nuclear calcium-binding proteins might be a massive diffusion of newly synthesized proteins into the nucleus. In favor of this suggestion is the tendency toward contact between labelled regions in the neuronal cytoplasm and nucleus, which may reflect a process of penetration into the nucleus by calcium-binding proteins synthesized at the cytoplasmic near-nuclear region. One more reason for the change in staining of nuclear structures with CTC may be increased synthesis of a relatively small number of regulatory molecules which, when penetrating into the nucleus, induce b i n d i n g o f Ca 2+ with a c o n s i d e r a b l e a m o u n t of calcium-binding protein of the nuclear matrix. It is interesting to note that the pronounced elevation of the activity of the genetic apparatus of command neurons within 90 rain following sensitizing stimulation coincides in time with the end of the so-called temporal window during which the protein-dependent process of consolidation of long-term memory is thought ~6, 7] to take place. Changes in staining with fluorescent dyes within 90 rain after application of sensitizing stimulation correlate with long-term neuronal effects of sensitization ~ considerable increase of sEPSP parameters in response to sensory stimuli. Therefore, it is not ruled out that a portion of the changes in CTC fluorescence in the near-nuclear region are related to the synthesis of proteins involved in the regulation of synaptic transmission. In addition, during this period of time the so-called late genes can be activated; these genes are responsible for the synthesis of proteins maintaining longterm changes in neuronal activity [7, 28]. The data presented here lead us to believe that the sensitizing influence results in an increase of genetic activity o f the nucleus of command neurons of withdrawal behavior in snails which is manifest within 30 min and which is most expressed within 90 rain after application of the sensitizing stimulus, An important role in this process obviously belongs to nuclear calcium-binding proteins, including nonhistone proteins of chromatin. Activation of the genome is likely to induce synthesis of protein molecules which participate in the regulation of the efficiency of synaptic transmission in neurons and which possibly participate in prolonged restructuring of the activity of the genome itself.

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