Effects of protein synthesis inhibitors on the sensitization of a ...

Neuroscience and Behavioral Physiology, Vol. 37, No. 5, 2007

Effects of Protein Synthesis Inhibitors on the Sensitization of a Defensive Response in Common Snails and Potentiation of the Cholinosensitivity of Command Neurons M. S. Abramova, A. A. Moskvitin, and A. S. Pivovarov

UDC 612.822.2+594.382

Translated from Zhurnal Vysshei Nervnoi Deyatel’nosti imeni I. P. Pavlova, Vol. 56, No. 3, pp. 355–362, May–June, 2006. Original article submitted September 22, 2005, accepted December 12, 2005. The effects of protein synthesis inhibitors on short-term sensitization of a defensive reaction in common snails and the potentiation of the cholinosensitivity of command neurons were studied. The protein synthesis inhibitor anisomycin did not prevent behavioral sensitization. Anisomycin and the irreversible protein synthesis inhibitor saporin changed the dynamics of potentiation of command neuron cholinosensitivity. We suggest that the sensitization of the defensive response of the common snail studied here does not require the synthesis of new proteins. KEY WORDS: anisomycin, saporin, acetylcholine, potentiation of cholinosensitivity, command neurons, behavioral sensitization, common snail.

mal’s foot is associated with protein synthesis [6]. Another type of long-term sensitization, induced by application of a chemical stimulus (quinine) to the snail’s head, also depends on protein synthesis [11]. Experimental studies on the formation of a contextual conditioned reflex in the common snail demonstrated that reactivation of long-term contextual memory was dependent on protein synthesis [5]. Protein synthesis is not always needed for long-term sensitization. Thus, studies in Aplysia neuron cultures showed that the combination of application of serotonin and activation of the presynaptic input increases its efficiency for a period of more than 24 h, but does not require the synthesis of new proteins or the formation of new synapses [17]. Rhythmic electrical stimulation of the foot in the common snail evokes short-term sensitization of the animal’s defensive reflex in response to tactile stimulation [2]. The enhancement of the defensive response after the sensitizing stimulus has similar dynamics to potentiation of the cholinosensitivity of mollusk defensive behavior command neurons LPa3 and RPa3; this potentiation is induced by rhythmic stimulation of the intestinal nerve with the same parameters as those used in behavioral experiments [1, 2, 13, 15]. It is therefore possible that increases in the cholinosensitivity of the somatic membranes of defensive behav-

Behavioral sensitization (facilitation) is an elementary type of non-associative learning involving enhancement of the animal’s reflex response to a stimulus after presentation of another (usually a strong or harmful) stimulus [7]. The duration of sensitization of a defensive reflex in the marine mollusk Aplysia and the terrestrial common snail is 2–4 weeks [3, 4, 7]. Three forms of sensitization induced by the application of serotonin are distinguished in Aplysia on the basis of the duration of the effect: short-term, lasting 15–30 min, intermediate, lasting up to 1.5 h, and long-term, lasting more than 1.5 h [22, 23, 25]. The significant difference in the mechanisms of the short-term and intermediate types of sensitization on the one hand and long-term sensitization on the other is their dependence on protein synthesis [22]. Short-term and intermediate sensitization of interneuron connections does not require the synthesis of new proteins. Long-term sensitization is accompanied by local synthesis of new proteins in the pre- and postsynaptic neurons in Aplysia [16, 27]. Long-term sensitization of a defensive reflex in the common snail evoked by electrical stimulation of the aniDepartment of Higher Nervous Activity, M. V. Lomonosov Moscow State University; e-mail: [email protected].

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444 ior command neurons may be involved in the neuronal mechanism of sensitization of the animal’s defensive response. The sensitization of the defensive reaction in snails and the potentiation of the cholinosensitivity of their command neurons are not long-lived, so these processes are probably not dependent on the synthesis of new proteins. However, this suggestion requires experimental verification. The aim of the present work was to study the involvement of protein synthesis in the sensitization of a defensive reaction in the common snail and in the potentiation of the cholinosensitivity of command neurons.

METHODS Experiments were performed on Helix lucorum common snails collected in the Crimea close to Sebastopol. Studies included behavioral and electrophysiological parts. The behavioral part of the study used an original apparatus for contact-free recording of the integral defensive reaction of the snail [9]. The animal’s defensive reaction (withdrawal of the antennae, head, and foot into the shell) was evoked by tactile stimulation of the skin of the head using a special apparatus. The behavioral apparatus consisted of a ring rotating around a horizontal axis and a system monitoring rotations of the ring on which the snail was crawling in such a way that the snail’s position remained constant relative to the photodiode sensor used for recording the snail’s reaction. The snail crawled upwards around the ring, which imitates its movement on a vertical surface in natural conditions. The snail was illuminated from the side by a diffuse light source which was detected on the other side by a photodiode sensor. Use of a diffuse light source gave a fuzzy shadow, such that, within limits, deviations in the snail’s position relative to the photodiode were not critical. The monitoring system stabilized the snail’s position in the stationary coordinate system with a precision of 1 mm. The apparatus for tactile stimulation was based on the magnetic components of a loudspeaker and allowed blows of defined energy to be applied, in response to which the snail retracted the tentacles, head, and foot. This decreased the area of the snail’s shadow – changes in shadow area characterized the magnitude of the integral defensive reaction recorded by the photodiode. The signal from the photodiode was amplified and passed to the recording apparatus, i.e., a KSP-4 electronic potentiometer. The strength of the tactile stimulus was determined by the energy of the blow applied by the apparatus (133–241 µJ), which was kept constant throughout the experiment. Substances. Protein synthesis was blocked with anisomycin (a translational inhibitor), which efficiently blocks protein synthesis in Helix pomatia neurons [21]. Anisomycin (Sigma, USA) was injected into the midpart of the foot and into the mantle cavity of the body 50 min before

Abramova, Moskvitin, and Pivovarov series started. The anisomycin dose was 8 mg/kg and injections of 0.1 ml of 7.5 mM anisomycin solution in physiological saline were given (0.2 mg/0.1 ml per animal). This anisomycin dose injected into the mantle cavity of the common snail leads to blockade of protein synthesis [12]. Rhythmic electrical stimulation was applied to the skin of the midpart of the snail’s foot. The stimulating bipolar electrodes were made of nichrome wire of diameter 0.1 mm. Square-wave current impulses (amplitude 900 µA, duration 0.1 sec, frequency 2 impulses/sec, stimulus duration 2 min) were delivered from an ÉSL-2 electrostimulator. Experimental protocol. Several experimental protocols were used. All schemes involved application of tactile stimulation with identical intervals of 10 min. Scheme 1. Spontaneous changes in the amplitude of the snail’s defensive reaction were measured after injection of physiological saline. 1) Three tactile stimuli, 2) injection of physiological saline (0.1 ml into the mantle cavity), 3) 12 tactile stimuli. Scheme 2. Changes in the snail’s defensive reaction after rhythmic electrical stimulation of the skin of the foot on the background of physiological saline (injection of 0.1 ml into the mantle cavity). 1) Three tactile stimuli, 2) injection of physiological saline (0.1 ml into the mantle cavity), 3) five tactile stimuli, 4) rhythmic electrical stimulation of the snail’s foot; 5) seven tactile stimuli. Scheme 3. Spontaneous changes in the amplitude of the snail’s defensive reaction were measured after injection of anisomycin solution (0.1 ml of physiological saline into the mantle cavity). 1) Three tactile stimuli, 2) injection of anisomycin, 3) 12 tactile stimuli. Scheme 4. Changes in the snail’s defensive reaction were measured after rhythmic electrical stimulation of the skin of the foot on the background of treatment with anisomycin (injection of anisomycin in 0.1 ml of physiological saline into the mantle cavity). 1) Three tactile stimuli; 2) injection of anisomycin, 3) five tactile stimuli; 4) rhythmic electrical stimulation of the skin of the snail’s foot, 5) seven tactile stimuli. The amplitude of the defensive reaction after rhythmic stimulation of the snail’s skin was normalized in percentage terms relative to the mean amplitude of the three last defensive reactions before rhythmic stimulation, which decreased the effects of spontaneous changes in the magnitude of the of the defensive reaction during the experiment. In experimental schemes 1 and 3 (without rhythmic electrical stimulation), the mean amplitude of three sequential defensive reactions (6, 7, and 8) after injection of the protein synthesis blocker was taken as 100%. Results were obtained from 31 animals. Results for schemes 1 and 2 were taken from the previous study [2]. The electrophysiological part of the study consisted of experiments performed on identified neurons LPa3, RPa2, RPa3, and RPa2 of the common snail Helix lucorum in semi-intact CNS-visceral sac preparations. These neu-

Effects of Protein Synthesis Inhibitors on the Sensitization of a Defensive Response rons are involved in mediating the defensive behavior of the common snail [8]. The membranes of these cells contain extrasynaptic cholinoreceptors [14]. Semi-intact preparations. Before making semi-intact preparations, animals were anesthetized by cooling in an ice:water mix for 30 min. The periglottal neural ring and its associated nerves (anal, intestinal, right pallial, and left pallial nerves) and the visceral sac were attached to plastic supports with steel microneedles in adjacent chambers connected together by a Vaseline bridge (1.5 × 5 mm) and filled with physiological saline containing 100 mM NaCl, 4 mM KCl, 10 mM CaCl2, 4 mM MgCl2, and 10 mM tris-HCl, pH 7.5–7.7. The periglottal neural ring was placed in a 1-ml flow chamber and the visceral sac was placed in a 41-ml chamber. After treating ganglia with digestase (0.5%, Seatec, Russia, Luxembourg) for 20–60 min at room temperature, the connective tissue layers covering the ganglia were removed. Recording of currents. Transmembrane currents were recorded from neurons using a two-electrode membrane voltage clamping method with virtual earthing. Intracellular glass microelectrodes were filled with 2 M potassium acetate (resistance 42.49 ± 3.14 MΩ). Application of ACh. Acetylcholine (ACh) was applied by local ionotophoretic application from a glass micropipette brought to the neuron body. The micropipette was filled with 1 M ACh chloride (Sigma, USA). Pipette resistance was 7–35 MΩ. Iontophoresis was performed using cationic currents (500–600 nA, 0.2–2.3 sec, 0.54 ± 0.12 sec). The indifferent pipette in the microiontophoresis circuit was filled with physiological saline (resistance 10–25 MΩ). Iontophoretic application parameters in each experiment were kept constant. Electrical stimulation. Changes in neuron cholinosensitivity were analyzed after rhythmic electrical stimulation of the intestinal nerve (current amplitude 0.5 µA, current duration 0.1 sec, stimulation frequency 2 impulses/sec, duration of stimulation 2 min). Square-wave current impulses were delivered from an ÉSL-2 electrostimulator via bipolar metal electrodes made of nichrome wire of diameter 0.1 mm (electrode resistance 80 MΩ). A 302-T electrostimulator was used as the control electrostimulator. The parameters of rhythmic electrical stimulation (impulse duration, frequency and duration of stimulation) in electrophysiological experiments were as in the behavioral part of the study. The extent of postactivation changes in neuron cholinosensitivity was assessed by comparing changes in the amplitude of the acetylcholine-evoked influx current (the acetylcholine current) after stimulation (experimental) and without stimulation (control). Substances. Protein synthesis was blocked using anisomycin (a translational inhibitor) and saporin (an irreversible ribosome inactivator). Anisomycin, which penetrates the cell membrane, was given extracellularly. The chamber containing the periglottal neural ring was supplemented with

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anisomycin (30–50 µl, 1 mM anisomycin solution in physiological saline) using a microsyringe. Exposure to the blocker lasted 60 min. The calculated anisomycin concentration in the flow chamber was 30–50 µM (30 µM in 11 experiments, 40 µM in three experiments, and 50 µM in 21 experiments). Published data indicate that the extracellular anisomycin concentration leading to blockade of protein synthesis is 10–100 µM [10, 18, 21, 29]. Since saporin does not penetrate cell membranes, it was injected intracellularly by spontaneous (passive) diffusion (90 min before testing). One of the two intracellular microelectrodes used for recording the potential was filled with 1 µM saporin in 2 Mm potassium acetate (electrode resistance was 46.4 ± 5.3 MΩ). Passive diffusion is used in experimental neurophysiology for intracellular injection into neurons of compounds unable to penetrate the cell membrane [20, 24, 28, 29]. The last of these studies reported experiments on Aplysia neurons subjected to intracellular injection of the irreversible ribosome inhibitor gelonin (a saporin analog). Injections were performed 60 min before tests were started. The presence of injected gelonin and its concentration in cells bodies were assessed by measuring the volume of fast green released from electrodes after addition to the injection solution. The calculated gelonin concentration in the neuron body 60 min after injection was 100 times less than that in the injection electrode. The similar sizes of neurons in Aplysia and common snails, the similar resistances of the injection microelectrodes, and the longer duration of the spontaneous saporin injection procedure in our experiments (90 min) suggest that the saporin concentration in the bodies of command neurons was also at least two orders of magnitude less than that in the injection electrode, i.e., less than 10 nM. This magnitude is within the range of saporin concentrations (4–3000 nM) at which it induces irreversible inhibition of ribosomes and protein synthesis [19]. Experimental protocols. All protocols included periodic application of ACh to the bodies of the neurons being recorded, with identical intervals of 10 min throughout the experiment. The amplitude of the ACh current in response to the first application of ACh generally showed a marked reduction. Experiments were therefore started after stabilization of the amplitude of the ACh current in the common snail neurons. Scheme I. Spontaneous changes in the amplitude of neuron ACh currents without rhythmic electrical stimuli – 10 applications of ACh. Scheme II. Changes in the amplitude of neuron ACh currents after rhythmic electrical stimulation of the intestinal nerve. 1) Three applications of ACh, 2) rhythmic electrical stimulation of the intestinal nerve, 3) seven applications of ACh. Scheme III. Spontaneous changes in the amplitude of neuron ACh currents without rhythmic electrical stimulation on the background of treatment with anisomycin. 1) Three

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Fig. 1. Effects of anisomycin on the sensitization of the defensive reaction of the common snail. A) Sensitization of the defensive reaction after injection of physiological saline (from [2]); B) after injection of anisomycin. Columns show the amplitudes of the defensive reaction of the common snail to tactile stimulation (mean ± S.E.M.): white columns show amplitudes without rhythmic electrical stimulation; dark column show amplitudes after stimulation. The arrows show the moment at which rhythmic electrical stimulation was applied. The interval between tactile test stimuli was 10 min. Overall results from all experiments are presented.

applications of ACh, 2) intracellular application of anisomycin, 3) 14 applications of ACh. Scheme IV. Changes in the amplitude of neuron ACh currents after rhythmic electrical stimulation of the intestinal nerve on the background of treatment with anisomycin. 1) Three applications of ACh, 2) extracellular application of anisomycin, 3) seven applications of ACh, 4) rhythmic electrical stimulation of the intestinal nerve, 5) seven applications of ACh. Scheme V. Spontaneous changes in the amplitude of neuron ACh currents without rhythmic electrical stimulation on the background of treatment with saporin. 1) Insertion into the neuron of a microelectrode filled with saporin solution, 2) 17 applications of ACh. Scheme VI. Changes in the amplitudes of neuron ACh currents after rhythmic electrical stimulation of the intestinal nerve on the background of treatment with saporin. 1) Insertion into the neuron of a microelectrode filled with saporin, 2) 10 applications of ACh, 3) rhythmic electrical stimulation of the intestinal nerve, 4) seven applications of ACh. Control series (schemes I, III, and V) were performed without rhythmic nerve stimulation to exclude the masking effect of spontaneous changes in ACh current amplitudes during prolonged recording of neuron activity. The amplitudes of evoked ACh currents were normalized in relation to the amplitude of the last ACh current prior to presentation of rhythmic stimulation. Results were obtained from 55 neurons (20 LPa3, 29 RPa2, five LPa2, and one RPa2) in 55 preparations. The cell membrane potential was –45.33 ± 1.47 mV. Neuron input resistance was 3.51 ± 0.45 MΩ. Results for schemes I and II were taken from our previous study [15].

Statistical methods. Results were analyed statistically using MS Excel 2000 and Stadia 6.2. Arithmetic means and standard errors of the mean were calculated (Stadia 6.2). All data sets were initially checked for normal distributions. If the set distribution differed from the normal, even if in only one of the two sets, they were compared using nonparametric tests. The effects of substances on the amplitude of the ACh current were assessed using the non-parametric Wilcoxon and van der Waerden tests for differences in positions (Stadia 6.2). The effects of substances on the dynamics of changes in defensive responses and the ACh current after rhythmic electrical stimulation were assessed using the Wilcoxon test and the signs test for paired data (Stadia 6.2).

RESULTS 1. Effects of Anisomycin on Sensitization of the Defensive Reaction of the Common Snail Anisomycin had no significant effect on the amplitude of the snails’ defensive reaction (number of animals N = 31). This is shown by comparison of the mean amplitudes of the snails’ defensive reactions 50 min after injection of anisomycin and 50 min after injection of physiological saline (N = 20). The magnitudes of the snails’ defensive reaction after injection of anisomycin (86.49 ± 8.15%) and physiological saline (111.45 ± 15.15%) were not different (p > 0.05, Wilcoxon test; p > 0.05, van der Waerden test). Anisomycin did not block sensitization of the defensive reaction and had no effect on its latency (Fig. 1). Rhythmic electrical stimulation of the midpart of the snails’ feet after injection of anisomycin induced sensitization of the snails’ defensive reaction (p < 0.05, Wilcoxon test). The mean dif-

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Fig. 2. Effects of anisomycin and saporin on potentiation of the amplitude of the ACh current in command neurons after rhythmic orthodromic stimulation of the intestinal nerve. A) No pharmacological treatment (from [15]); B) after application of anisomycin; C) after saporin. Columns show the amplitudes of the ACh current (mean ± S.E.M.): white columns show amplitudes without rhythmic orthodromic electrical stimulation; dark columns show amplitudes after rhythmic stimulation. Arrows show the moment at which rhythmic electrical stimulation was applied. The intervals between test applications of ACh were 10 min. Overall results for all experiments.

ference in the amplitudes of the defensive reactions in experimental and control preparations was 21.73 ± 6.04%. The extents of sensitization of the defensive reaction after injection of anisomycin and after injection of physiological saline (data from [2]) were not different (p > 0.05, Wilcoxon test; p > 0.05, signs test). 2. Effects of Anisomycin and Saporin on Potentiation of the ACh-Evoked Influx Current Anisomycin (30–50 µM) decreased the amplitude of the ACh current at 60 min by 22.90 ± 5.75% (number of neurons n = 35, p < 0.01, Wilcoxon test; p < 0.001, van der Waerden test). Anisomycin altered the dynamics of the potentiation of the acetylcholine current, increasing its latency (Fig. 2, B). The mean difference in the medians of the sets of mean ACh current amplitudes in the control (n = 18) and experimental (n = 17) series was 20.41 ± 8.13% (p < 0.05, Wilcoxon test). There was no difference in the extent of potentiation of the ACh current in conditions of exposure to anisomycin and without any pharmacological treatment (p > 0.05, Wilcoxon test; p > 0.05, signs test). Saporin, like anisomycin, altered the dynamics of potentiation of the ACh current, increasing its latency (Fig. 2, C). The medians of the sets of mean ACh current amplitudes in the control (n = 10) and experimental (n = 10) series were significantly different (p < 0.01, Wilcoxon test; p < 0.05, signs test). The mean difference was 15.50 ± 2.92%.

There was no difference in the extent of potentiation of the ACh current after exposure to saporin and without pharmacological treatment (p > 0.05, Wilcoxon test; p > 0.05, signs test).

DISCUSSION The studies reported here show that the protein synthesis inhibitor anisomycin did not prevent the short-term (up to 40 min) sensitization of the defensive reaction in the common snail and had no effect on its latency. This suggests that the types of behavioral sensitization studied here are independent of protein synthesis. This result is in agreement with published data. It is likely that only long-term changes in behavior in the common snail occurring during learning require the synthesis of new proteins [5, 6, 11]. Increases in the cholinosensitivity of common snail defensive behavior command neurons may play a role in the mechanism of short-term sensitization of the animals’ defensive reactions [2]. This conclusion was based on the similarity of the dynamics of sensitization of the defensive reaction and potentiation of the cholinosensitivity of snail defensive behavior command neurons. Both protein synthesis blockers, anisomycin and saporin, act on the potentiation of command neuron potentiation in a similar manner – they change the dynamics of

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potentiation of the ACh current, increasing its latency. We can suggest two possible explanations for this result. We believe that the first is the more likely: protein synthesis blockers induce increases in the latency of that potentiation of cholinosensitivity which is recorded in their absence. This may occur as a result of the effects of anisomycin and saporin not only on protein synthesis, but also on other cellular targets. In particular, anisomycin has been shown to activate c-Jun N-terminal kinase (JNKs) and p38 mitogen-activated kinase (MAPK) and to increase arachidonic acid levels [26, 30]. The second explanation is that protein synthesis inhibitors suppress short-latency potentiation and evoke the appearance of other, long-term forms of potentiation. Our results do not yet allow either of these causes of these changes in the dynamics of potentiation of cholinosensitivity to be verified. What postsynaptic cellular mechanisms could mediate the sensitization of the defensive reaction in the common snail? Two mechanisms are theoretically possible: 1) insertion of newly synthesized receptors into the membrane and 2) recycling of internalized cholinoreceptors from the reserve cytoplasmic pool. The fact that the short-term behavioral sensitization in the common snail is independent of the protein synthesis inhibitor anisomycin as demonstrated in the present study probably allows the first of these cellular mechanisms to be excluded, i.e., the synthesis of new cholinoreceptors. The second cellular mechanism would appear to underlie short-term behavioral sensitization (up-regulation of internalized cholinoreceptors). We suggest that this mechanism may mediate increases in the efficiency of interneuronal connections during sensitization of the defensive reaction for a short period by increasing the transmitter sensitivity of the subsynaptic and extrasynaptic zones of the neuron membrane without the synthesis of new transmitter receptors.

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CONCLUSIONS 1. The protein synthesis inhibitor anisomycin does not prevent the short-term sensitization of the defensive reaction of the common snail. 2. Anisomycin and the irreversible protein synthesis inhibitor saporin altered the dynamics of the potentiation of the cholinosensitivity of defensive behavior command neurons in the common snail. This study was supported by the Russian Foundation for Basic Research (Grant No. 05-04-48400a).

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