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Indian red scorpion venom depresses spinal synaptic transmission without involving NMDA receptors
Neuroscience Letters 475 (2010) 85–88
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Indian red scorpion venom depresses spinal synaptic transmission without involving NMDA receptors Amar N. Maurya, Shripad B. Deshpande ∗ Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, UP 221005, India
a r t i c l e
i n f o
Article history: Received 8 January 2010 Received in revised form 17 March 2010 Accepted 20 March 2010 Keywords: Spinal cord Synaptic transmission MSR PSR NMDA receptor
a b s t r a c t Stings of Indian red scorpion (Mesobuthus tamulus, MBT) produce neurological abnormalities such as convulsions and paralysis. These parameters indicate the activity at ␣-motoneuron. The present study was therefore, undertaken to evaluate the effect of MBT-venom on spinal reflexes and the involvement of N-methyl-d-aspartate (NMDA) receptors. The experiments were performed on isolated hemisected spinal cords from 4 to 6 days old rats. Stimulation of a dorsal root with supramaximal strength at 0.1 Hz evoked monosynaptic (MSR) and polysynaptic reflex (PSR) potentials in the corresponding segmental ventral root. Superfusion of MBT-venom depressed the spinal reflexes in a time- and a concentrationdependent (0.1–1 g/ml) manner. MBT-venom at 0.1, 0.3 and 1.0 g/ml produced maximal depression of 55, 75 and 90% at 30, 10 and 7 min, respectively. The time required to produce 50% depression (T-50) of MSR was 19.0, 8.0, and 3.6 min and for PSR was 15.0, 5.6, and 2.9 min at 0.1, 0.3 and 1 g/ml of venom, respectively. Pre-treatment with DL-␣-2-amino-5-phosphonovaleric acid (APV) decreased MSR by 26% and abolished PSR. In the presence of APV, the MBT-venom-induced depression of MSR was not different from the venom only group. The results indicate that venom-induced depression of spinal reflexes did not involve NMDA receptors. © 2010 Elsevier Ireland Ltd. All rights reserved.
The Indian red scorpion (Mesobuthus tamulus, MBT) stings produce fatal manifestations with abnormalities in nervous, cardiopulmonary, endocrine and haematological systems [1,6,15,16,20]. The neural manifestations are characterized by local pain, paralysis, autonomic storm, alteration in neuronal excitability, convulsions, etc. [1,2,9,17,18,22]. The convulsions or paralysis represent the increased or decreased motoneuron activity, respectively. These motor alterations involve Ia-␣-motoneuron synapse in the spinal cord. The increased or decreased activity at this synapse can be produced either by the inhibition of excitatory system or by the excitation of inhibitory system [3,5,13,21]. In our previous studies, the Ptychodiscus brevis toxin (PbTx), aglycemia/ischemia and 3-nitropropionic acid (3-NPA) depressed the spinal reflexes. Further, NMDA receptors are shown to be involved in the depression induced by PbTx or aglycemia/ischemia [13,21]. It is also shown that the venom increases the excitability of peripheral axons [4]. However, the effect of venom on spinal reflexes is not known. Therefore, this study was undertaken to examine the effect of venom on spinal reflexes in isolated spinal cord. Since glutamate is a putative transmitter at Ia-␣ motoneuron synapse [12], the role of N-methyld-aspartate (NMDA) receptors in mediating these responses was also ascertained.
∗ Corresponding author. Tel.: +91 542 2309551/6703274; fax: +91 542 2367568. E-mail address: [email protected] (S.B. Deshpande). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.03.052
Methods for isolation and preparation of spinal cord have been described earlier [8,21]. Briefly, the neonatal rats (4–6 days) of Charles-Foster strain were anesthetized with diethyl ether and the vertebral column was quickly removed and placed in a sylgard plated petridish containing physiological solution bubbled with 100% O2 (pH 7.3). By dorsal laminectomy, the spinal cord was carefully removed from mid thoracic to mid sacral region along with the roots. Care was taken to keep the corresponding dorsal and ventral roots intact during dissection. The spinal cord was then placed ventral surface upwards, pinned and hemisected sagitally. The hemisected cord was then transferred to a small Plexiglas bath (total volume about 1 ml) which was continuously superfused with oxygenated physiological solution (3–5 ml/min) at 25.0 ± 0.5 ◦ C. All the experiments were performed according to the guidelines of the Institute of Medical Sciences, Banaras Hindu University, Varanasi for conducting the animal experiments. Suction electrodes were prepared by using borosilicate glass capillary tube (1.2 mm OD; 0.9 mm ID). By fire-polishing, the diameter of the capillary opening was reduced to the size slightly lesser than the dorsal or ventral root. The cut ends of the corresponding dorsal/ventral roots between L3–5 segments were sucked gently in the capillary tubes prefilled with physiological solution. The physiological solution had the following composition (mM) (NaCl, 124.0; KCl, 5.0; KH2 PO4 , 1.2; CaCl2 , 2.5; NaHCO3 , 4.5 and glucose-11.0; pH, 7.3). The temperature of the physiological solution was maintained at 25 ± 0.5 ◦ C. The tissue was
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Fig. 1. Time–response effect of various concentration of MBT-venom (0.1–1.0 g/ml) on spinal reflexes in vitro. In the left panel, actual reflex recordings at different time intervals after exposure to three concentrations of venom (0.1–1.0 g/ml) are shown. Horizontal calibration is 5 ms and vertical calibration is 1 mV. The preparation was washed (W) with normal physiological solution for 30 min. Right panel from B to D shows, mean ± SEM values after exposing to 0.1 g/ml (n = 9), 0.3 g/ml (n = 10) and 1.0 g/ml (n = 5) of MBT-venom. MSR, monosynaptic reflex, PSR, polysynaptic reflex. ‘*’ indicates P < 0.05 (Student–Newman–Keul test for multiple comparisons). Note the time- and concentration-dependent depression of MSR and PSR.
allowed to stabilize in the experimental chamber for 2 h for recovery. The stimulation of a dorsal root with rectangular pulses of 0.5 ms at 0.1 Hz evoked monosynaptic (MSR) and polysynaptic reflex (PSR) potentials in the segmental ventral root. The voltage was adjusted to get the maximal response (40–50 V). The potentials were amplified by Harvard AC–DC amplifier and digitized by DMS 708A AD converter (Dynalog Microsystems, Bombay) and stored in a personal computer for on-line or off-line analysis. The averaged potential of five consecutive reflexes was recorded and analyzed using UnkelScope software (MIT, MA, USA). The experiments were divided into two groups. In group-I, the concentration response of venom was determined. In this group, control reflex recordings were obtained after stabilization, then the cord was exposed to venom for 60 min (for 0.1 g/ml) or 30 min (for 0.3 or 1.0 g/ml) and the recordings were performed at every 5 min. In addition, the recordings at 3 and 7 min were also performed for 1.0 g/ml of venom. At the end, the cord was washed with normal physiological solution for 30 min and then the reflex potentials were recorded. In a given experiment the cord was exposed to only one concentration of venom. In time-matched control experiments, the recordings of reflexes were made up to 60 min with normal physiological solution. In group-II, effect of DL-␣-2-amino-5-phosphonovaleric acid (APV) on MBT-venom-induced alterations on the spinal reflexes was examined. After the control recordings, the cord was exposed to APV (10 M) for 30 min and then the reflex potentials were recorded. Subsequently, the preparation was exposed to MBTvenom (0.3 g/ml) in the presence of APV and the recordings were made at every 5 min till 30 min. The cord was washed with normal physiological solution for 30 min and the recordings were made. MBT-venom was obtained from Haffkine Institute, Bombay, India. APV was obtained from Sigma Chemicals, St. Louis, MO, USA.
Stock solutions were prepared in distilled water and stored in a freezer. Reflex activity was quantified by measuring the peak amplitude of the reflex potentials. The responses after stabilization were taken as the initial response for normalisation. The data were pooled to obtain mean ± SEM. Differences between the groups were compared by using one-way/two-way analysis of variance (ANOVA). Student’s t-test was used whenever required. A P < 0.05 was considered significant. Superfusion of physiological solution containing MBT-venom (0.1 g/ml) produced depression of spinal reflexes in a timedependent manner (Fig. 1A and B). The depression of MSR began immediately and reached maximum (about 55%) by 30 min and remained at that level up to 60 min. On the other hand, the depression of PSR began after 5 min, the maximum depression (about 54%) was seen at 30 min. Time to produce 50% depression of MSR and PSR was 19.0 ± 2.7 and 15.0 ± 2.4 min, respectively (Fig. 2). The effect of venom was not reversed by washing the cord with normal physiological solution for 30 min (Fig. 1A and B). In time-matched control experiments (n = 4), the MSR and PSR magnitude were not altered even up to 60 min. Superfusion of 0.3 g/ml of MBT-venom depressed both MSR and PSR in a time-dependent manner (Fig. 1A and C). The maximum depression for MSR (about 60%) and PSR (about 75%) was seen at 10 min and remained at that level up to 30 min. The depression of PSR was greater than MSR between 10 and 20 min (Fig. 1C). The duration of MSR before and after venom was 3.46 ± 0.44 and 4.51 ± 0.37 ms, respectively. In case of PSR, the values were 4.11 ± 0.4 and 3.11 ± 1.0 ms, respectively for before and after exposure to venom. The durations of reflexes after venom (0.3 g/ml) were not significantly different from before values (P > 0.1; Student’s t-test for paired observations). The time to produce 50% depression of MSR was 8.0 ± 0.8 min and PSR was 5.6 ± 0.4 min (Fig. 2). The effect of venom was not reversed even after wash-
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Fig. 2. Bar graphs showing the time required to produce 50% (T-50) depression of spinal reflexes at various concentrations of venom (0.1–1.0 g/ml). The mean ± SEM values are obtained from 5 to 10 experiments. The depression of MSR/PSR at different concentrations of venom is significantly different from one another (P < 0.05; oneway ANOVA). An asterisk (*) shows P < 0.05 as compared to MSR (Student’s t-test for unpaired observations).
ing the cord with normal physiological solution for 30 min (Fig. 1C). Superfusion of 1.0 g/ml of MBT-venom depressed MSR and PSR very rapidly. Within 5 min the depression of reflexes was greater than 65%. At 7 min the depression was about 90% and remained at that level up to 30 min (Fig. 1A and D). The time to produce 50% of depression for MSR was 3.6 ± 0.5 min and PSR was 2.87 ± 1.2 min (Fig. 2). After washing the cord with venom-free physiological solution for 30 min, the magnitude of MSR was 63.0 ± 10.3% of the initial and PSR was 34.0 ± 24.7% of the initial (Fig. 1D). Since, 0.3 g/ml of venom produced about 65% depression in a relatively less rapid time (10 min), we selected this concentration for experiments with APV. After exposure to APV (10.0 M), the MSR was depressed by 25% within 10 min and PSR was abolished. The depression was not increased any further even when the exposure time was increased to 30 min (26% of the initial) (Fig. 3). The effect of APV was similar to that reported earlier [3,13,19,21]. Superfusion of MBT-venom (0.3 g/ml) in the presence of APV, depressed the MSR in a timedependent manner (Fig. 3). The depression was 34% at 5 min, 60% at 10 min and remained at that level up to 30 min. The time–response relationship of venom-induced depression of MSR in the presence of APV was not different from venom only group (Fig. 3). In APV treated group, MSR returned to the initial level after washing with standard physiological solution for 30 min and was significantly different from venom only group (Fig. 3; P < 0.05; Student’s t-test). The findings of the present experiments reveal that MBTvenom depressed the spinal segmental reflexes in a time- and a concentration-dependent manner. The depression produced by venom is irreversible and does not involve NMDA receptors. In the spinal cord, the decreased synaptic activity can be produced by the activation of inhibitory pathway or the inhibition of excitatory pathway or by continuous depolarization of axonal terminals. It has been shown that scorpion venom increases (50–70 times) repolarization time and refractory period of amphibian axons [4]. Such prolonged repolarization/refractoriness may eventually block the conduction of impulses at afferent/efferent nerve terminals. Iberiotoxin (IbTx), a high conductance Ca2+ activated K+ channel blocker, was isolated from Indian red scorpion venom has been implicated in this action [10]. Thus, K+ channel blocker should mimic the response. In the earlier report, the blockade of K+ channel by 3,4-diaminopyridine (3,4-DAP) prolonged the repolarization time of MSR [7]. However in the present results, there was no prolongation of the duration of MSR/PSR at any concentration and
Fig. 3. APV failed to block the venom-induced depression of MSR. In the top, actual reflex recordings of an experiment with DL-2-amino-5-phosphonovaleric acid (APV) are shown at different time intervals. The cord was pretreated with APV (10 M) for 30 min (0), subsequently the cord was exposed to venom (0.3 g/ml) in the presence of APV. At the end, the preparation was washed (W) for 30 min with normal physiological solution. Horizontal calibration is 10 ms and vertical calibration is 1 mV. In the lower graph, mean ± SEM values from five different preparations after APV are shown and the venom only data is taken from Fig. 1. The response in the presence of APV was not different form the ‘venom only’ group.
at any time. Thus, the depression may not be due to action at K+ channel blocking activity. In the previous studies, it has been shown that Ptychodiscus brevis toxin (PbTx), 3-nitropropionic acid (3-NPA) and aglycemia/ischemia depressed the spinal reflexes [11,14,21]. Further, the NMDA receptors are involved in the depression produced by PbTx or aglycemia/ischemia [13,21]. Hence, the depression of reflexes by MBT-venom may also involve similar mechanisms. The present results exclude the involvement of NMDA receptors, as APV did not block the MBT-venom-induced depression (Fig. 3). However, the greater depression of PSR at 0.3 g/ml of venom indicates the partial involvement of NMDA receptors as PSR is mediated through NMDA receptors [19,21]. In addition, the total recovery of MSR in wash experiments after APV pre-treatment further supports it (Fig. 3). The recovery in APV pretreated cord can be explained on the basis of a competitive antagonism of APV with NMDA receptors. In conclusion, scorpion venom depresses MSR/PSR via a mechanism independent of NMDA receptors. However, the involvement of inhibitory system in depressing the MSR has to be explored. Acknowledgement ANM wishes to thank Indian Council of Medical Research, New Delhi for providing financial assistance. References [1] H.S. Bawaskar, Diagnostic cardiac premonitory signs and symptoms of red scorpion sting, Lancet 1 (1982) 552–554. [2] H.S. Bawaskar, P.H. Bawaskar, Management of the cardiovascular manifestations of poisoning by the Indian red scorpion, Br. Heart J. 68 (1992) 478–480. [3] S.B. Deshpande, Significance of monosynaptic and polysynaptic reflexes in neonatal rat spinal cord in vitro, Ind. J. Exp. Biol. 31 (1993) 850–854. [4] S.B. Deshpande, Indian red scorpion (Mesobuthus tamulus concanesis, Pocock) venom prolongs repolarization time and refractoriness of the compound action potential of frog sciatic nerve in vitro through calcium dependent mechanism, Ind. J. Exp. Biol. 36 (1998) 1108–1113.
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[5] S.B. Deshpande, J.E. Warnick, Temperature dependence of reflex transmission in the neonatal rat spinal cord, in vitro: influence strychnine and bicuculline sensitive inhibitions, Neuropharmacology 27 (1988) 1033–1037. [6] S.B. Deshpande, R. Pandey, A.K. Tiwari, Pathophysiological approach to the management of scorpion envenomation, Ind. J. Physiol. Pharmacol. 52 (2008) 311–314. [7] S.B. Deshpande, J.E. Warnick, Interaction of thyrotropin-releasing hormone and methysergide on the monosynaptic reflex in isolated mammalian spinal cord, Neurosci. Lett. 116 (1990) 141–148. [8] S.B. Deshpande, N.S. Pilotte, J.E. Warnick, Gender-specific action of thyrotropinreleasing hormone in the mammalian spinal cord, FASEB J. 1 (1987) 478–482. [9] B.B. Gaitonde, S.S. Jadhav, H.S. Bawaskar, Pulmonary oedema after scorpion sting, Lancet 1 (1978) 445–446. [10] A. Galvez, G. Gimenez-Gallego, J.P. Reuben, L. Roy-Contancin, P. Feigenbaum, G.L. Kaczorowski, M.L. Garcia, Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus, J. Biol. Chem. 265 (1990) 11083–11090. [11] R. Gupta, S.B. Deshpande, 3-Nitropropionic acid-induced depression of spinal reflexes involves mechanisms different from ischemia-induced depression, Br. Res. Bull. 77 (2008) 382–387. [12] C.E. Jahr, K. Yoshioka, Ia afferent excitation of motoneurone in the new born rat spinal cord is sensitively antagonized by kynurenate, J. Physiol. 370 (1986) 515–530. [13] A. Jha, S.B. Deshpande, Aglycemia and ischemia depress spinal synaptic transmission via inhibitory systems involving NMDA receptors, Eur. J. Pharmacol. 481 (2003) 189–196.
[14] A. Jha, S. Dasgupta, S.B. Deshpande, Effect of ischaemia and aglycaemia on the synaptic transmission in neonatal rat spinal cord in vitro, Ind. J. Med. Res. 118 (2003) 172–177. [15] K.R.K. Murthy, Z. Hossein, Increased osmotic fragility of red cells in dogs with acute myocarditis produced by scorpion (Buthus tamulus) venom, Ind. J. Physiol. Pharmacol. 30 (1986) 215–222. [16] K.R.K. Murthy, Z. Hossein, Increased osmotic fragility of red cells after incubation at 37 ◦ C for 24 h in dogs with acute myocarditis produced by scorpion (Buthus tamulus) venom, Ind. J. Exp. Biol. 24 (1986) 464–467. [17] K.R.K. Murthy, M.A. Zare, Effect of Indian red scorpion (Mesobuthus tamulus concanesis, Pocock) venom on thyroxine and triiodothyronine in experimental acute myocarditis and its reversal by species specific antivenom, Ind. J. Exp. Biol. 36 (1998) 16–21. [18] K.R.K. Murthy, R. Shenoy, P. Vaidyanathan, K. Kelkar, N. Sharma, N. Birevar, S. Rao, M.N. Mehta, Insulin reverses haemodynamic changes and pulmonary oedema in children stung by the Indian red scorpion Mesobuthus tamulus concanesis, Pocock, Ann. Trop. Med. Parasitol. 85 (1991) 651–657. [19] Y. Ohno, J.E. Warnick, Selective depression of the segmental polysynaptic reflex by phencyclidine and its analogs in the rat in vitro: interaction with N-methyld-aspartate receptors, J. Pharmacol. Exp. Ther. 252 (1990) 246–252. [20] R. Pandey, S.B. Deshpande, Aprotinin reverses ECG abnormalities induced by Mesobuthus tamulus concanesis, Pocock venom in adult rats, Ind. J. Exp. Biol. 45 (2007) 949–953. [21] J.N. Singh, S.B. Deshpande, Involvement of N-methyl-d-aspartate receptors for the Ptychodiscus brevis toxin-induced depression of monosynaptic and polysynaptic reflexes in neonatal rat spinal cord in vitro, Neuroscience 115 (2002) 1189–1197. [22] A.K. Tiwari, S.B. Deshpande, Toxicity of scorpion (Buthus tamulus) venom in mammals is influenced by the age and species, Toxicon 31 (1993) 1619–1622.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Indian red scorpion venom depresses spinal synaptic transmission without involving NMDA receptors Amar N. Maurya, Shripad B. Deshpande ∗ Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, UP 221005, India
a r t i c l e
i n f o
Article history: Received 8 January 2010 Received in revised form 17 March 2010 Accepted 20 March 2010 Keywords: Spinal cord Synaptic transmission MSR PSR NMDA receptor
a b s t r a c t Stings of Indian red scorpion (Mesobuthus tamulus, MBT) produce neurological abnormalities such as convulsions and paralysis. These parameters indicate the activity at ␣-motoneuron. The present study was therefore, undertaken to evaluate the effect of MBT-venom on spinal reflexes and the involvement of N-methyl-d-aspartate (NMDA) receptors. The experiments were performed on isolated hemisected spinal cords from 4 to 6 days old rats. Stimulation of a dorsal root with supramaximal strength at 0.1 Hz evoked monosynaptic (MSR) and polysynaptic reflex (PSR) potentials in the corresponding segmental ventral root. Superfusion of MBT-venom depressed the spinal reflexes in a time- and a concentrationdependent (0.1–1 g/ml) manner. MBT-venom at 0.1, 0.3 and 1.0 g/ml produced maximal depression of 55, 75 and 90% at 30, 10 and 7 min, respectively. The time required to produce 50% depression (T-50) of MSR was 19.0, 8.0, and 3.6 min and for PSR was 15.0, 5.6, and 2.9 min at 0.1, 0.3 and 1 g/ml of venom, respectively. Pre-treatment with DL-␣-2-amino-5-phosphonovaleric acid (APV) decreased MSR by 26% and abolished PSR. In the presence of APV, the MBT-venom-induced depression of MSR was not different from the venom only group. The results indicate that venom-induced depression of spinal reflexes did not involve NMDA receptors. © 2010 Elsevier Ireland Ltd. All rights reserved.
The Indian red scorpion (Mesobuthus tamulus, MBT) stings produce fatal manifestations with abnormalities in nervous, cardiopulmonary, endocrine and haematological systems [1,6,15,16,20]. The neural manifestations are characterized by local pain, paralysis, autonomic storm, alteration in neuronal excitability, convulsions, etc. [1,2,9,17,18,22]. The convulsions or paralysis represent the increased or decreased motoneuron activity, respectively. These motor alterations involve Ia-␣-motoneuron synapse in the spinal cord. The increased or decreased activity at this synapse can be produced either by the inhibition of excitatory system or by the excitation of inhibitory system [3,5,13,21]. In our previous studies, the Ptychodiscus brevis toxin (PbTx), aglycemia/ischemia and 3-nitropropionic acid (3-NPA) depressed the spinal reflexes. Further, NMDA receptors are shown to be involved in the depression induced by PbTx or aglycemia/ischemia [13,21]. It is also shown that the venom increases the excitability of peripheral axons [4]. However, the effect of venom on spinal reflexes is not known. Therefore, this study was undertaken to examine the effect of venom on spinal reflexes in isolated spinal cord. Since glutamate is a putative transmitter at Ia-␣ motoneuron synapse [12], the role of N-methyld-aspartate (NMDA) receptors in mediating these responses was also ascertained.
∗ Corresponding author. Tel.: +91 542 2309551/6703274; fax: +91 542 2367568. E-mail address: [email protected] (S.B. Deshpande). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.03.052
Methods for isolation and preparation of spinal cord have been described earlier [8,21]. Briefly, the neonatal rats (4–6 days) of Charles-Foster strain were anesthetized with diethyl ether and the vertebral column was quickly removed and placed in a sylgard plated petridish containing physiological solution bubbled with 100% O2 (pH 7.3). By dorsal laminectomy, the spinal cord was carefully removed from mid thoracic to mid sacral region along with the roots. Care was taken to keep the corresponding dorsal and ventral roots intact during dissection. The spinal cord was then placed ventral surface upwards, pinned and hemisected sagitally. The hemisected cord was then transferred to a small Plexiglas bath (total volume about 1 ml) which was continuously superfused with oxygenated physiological solution (3–5 ml/min) at 25.0 ± 0.5 ◦ C. All the experiments were performed according to the guidelines of the Institute of Medical Sciences, Banaras Hindu University, Varanasi for conducting the animal experiments. Suction electrodes were prepared by using borosilicate glass capillary tube (1.2 mm OD; 0.9 mm ID). By fire-polishing, the diameter of the capillary opening was reduced to the size slightly lesser than the dorsal or ventral root. The cut ends of the corresponding dorsal/ventral roots between L3–5 segments were sucked gently in the capillary tubes prefilled with physiological solution. The physiological solution had the following composition (mM) (NaCl, 124.0; KCl, 5.0; KH2 PO4 , 1.2; CaCl2 , 2.5; NaHCO3 , 4.5 and glucose-11.0; pH, 7.3). The temperature of the physiological solution was maintained at 25 ± 0.5 ◦ C. The tissue was
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Fig. 1. Time–response effect of various concentration of MBT-venom (0.1–1.0 g/ml) on spinal reflexes in vitro. In the left panel, actual reflex recordings at different time intervals after exposure to three concentrations of venom (0.1–1.0 g/ml) are shown. Horizontal calibration is 5 ms and vertical calibration is 1 mV. The preparation was washed (W) with normal physiological solution for 30 min. Right panel from B to D shows, mean ± SEM values after exposing to 0.1 g/ml (n = 9), 0.3 g/ml (n = 10) and 1.0 g/ml (n = 5) of MBT-venom. MSR, monosynaptic reflex, PSR, polysynaptic reflex. ‘*’ indicates P < 0.05 (Student–Newman–Keul test for multiple comparisons). Note the time- and concentration-dependent depression of MSR and PSR.
allowed to stabilize in the experimental chamber for 2 h for recovery. The stimulation of a dorsal root with rectangular pulses of 0.5 ms at 0.1 Hz evoked monosynaptic (MSR) and polysynaptic reflex (PSR) potentials in the segmental ventral root. The voltage was adjusted to get the maximal response (40–50 V). The potentials were amplified by Harvard AC–DC amplifier and digitized by DMS 708A AD converter (Dynalog Microsystems, Bombay) and stored in a personal computer for on-line or off-line analysis. The averaged potential of five consecutive reflexes was recorded and analyzed using UnkelScope software (MIT, MA, USA). The experiments were divided into two groups. In group-I, the concentration response of venom was determined. In this group, control reflex recordings were obtained after stabilization, then the cord was exposed to venom for 60 min (for 0.1 g/ml) or 30 min (for 0.3 or 1.0 g/ml) and the recordings were performed at every 5 min. In addition, the recordings at 3 and 7 min were also performed for 1.0 g/ml of venom. At the end, the cord was washed with normal physiological solution for 30 min and then the reflex potentials were recorded. In a given experiment the cord was exposed to only one concentration of venom. In time-matched control experiments, the recordings of reflexes were made up to 60 min with normal physiological solution. In group-II, effect of DL-␣-2-amino-5-phosphonovaleric acid (APV) on MBT-venom-induced alterations on the spinal reflexes was examined. After the control recordings, the cord was exposed to APV (10 M) for 30 min and then the reflex potentials were recorded. Subsequently, the preparation was exposed to MBTvenom (0.3 g/ml) in the presence of APV and the recordings were made at every 5 min till 30 min. The cord was washed with normal physiological solution for 30 min and the recordings were made. MBT-venom was obtained from Haffkine Institute, Bombay, India. APV was obtained from Sigma Chemicals, St. Louis, MO, USA.
Stock solutions were prepared in distilled water and stored in a freezer. Reflex activity was quantified by measuring the peak amplitude of the reflex potentials. The responses after stabilization were taken as the initial response for normalisation. The data were pooled to obtain mean ± SEM. Differences between the groups were compared by using one-way/two-way analysis of variance (ANOVA). Student’s t-test was used whenever required. A P < 0.05 was considered significant. Superfusion of physiological solution containing MBT-venom (0.1 g/ml) produced depression of spinal reflexes in a timedependent manner (Fig. 1A and B). The depression of MSR began immediately and reached maximum (about 55%) by 30 min and remained at that level up to 60 min. On the other hand, the depression of PSR began after 5 min, the maximum depression (about 54%) was seen at 30 min. Time to produce 50% depression of MSR and PSR was 19.0 ± 2.7 and 15.0 ± 2.4 min, respectively (Fig. 2). The effect of venom was not reversed by washing the cord with normal physiological solution for 30 min (Fig. 1A and B). In time-matched control experiments (n = 4), the MSR and PSR magnitude were not altered even up to 60 min. Superfusion of 0.3 g/ml of MBT-venom depressed both MSR and PSR in a time-dependent manner (Fig. 1A and C). The maximum depression for MSR (about 60%) and PSR (about 75%) was seen at 10 min and remained at that level up to 30 min. The depression of PSR was greater than MSR between 10 and 20 min (Fig. 1C). The duration of MSR before and after venom was 3.46 ± 0.44 and 4.51 ± 0.37 ms, respectively. In case of PSR, the values were 4.11 ± 0.4 and 3.11 ± 1.0 ms, respectively for before and after exposure to venom. The durations of reflexes after venom (0.3 g/ml) were not significantly different from before values (P > 0.1; Student’s t-test for paired observations). The time to produce 50% depression of MSR was 8.0 ± 0.8 min and PSR was 5.6 ± 0.4 min (Fig. 2). The effect of venom was not reversed even after wash-
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Fig. 2. Bar graphs showing the time required to produce 50% (T-50) depression of spinal reflexes at various concentrations of venom (0.1–1.0 g/ml). The mean ± SEM values are obtained from 5 to 10 experiments. The depression of MSR/PSR at different concentrations of venom is significantly different from one another (P < 0.05; oneway ANOVA). An asterisk (*) shows P < 0.05 as compared to MSR (Student’s t-test for unpaired observations).
ing the cord with normal physiological solution for 30 min (Fig. 1C). Superfusion of 1.0 g/ml of MBT-venom depressed MSR and PSR very rapidly. Within 5 min the depression of reflexes was greater than 65%. At 7 min the depression was about 90% and remained at that level up to 30 min (Fig. 1A and D). The time to produce 50% of depression for MSR was 3.6 ± 0.5 min and PSR was 2.87 ± 1.2 min (Fig. 2). After washing the cord with venom-free physiological solution for 30 min, the magnitude of MSR was 63.0 ± 10.3% of the initial and PSR was 34.0 ± 24.7% of the initial (Fig. 1D). Since, 0.3 g/ml of venom produced about 65% depression in a relatively less rapid time (10 min), we selected this concentration for experiments with APV. After exposure to APV (10.0 M), the MSR was depressed by 25% within 10 min and PSR was abolished. The depression was not increased any further even when the exposure time was increased to 30 min (26% of the initial) (Fig. 3). The effect of APV was similar to that reported earlier [3,13,19,21]. Superfusion of MBT-venom (0.3 g/ml) in the presence of APV, depressed the MSR in a timedependent manner (Fig. 3). The depression was 34% at 5 min, 60% at 10 min and remained at that level up to 30 min. The time–response relationship of venom-induced depression of MSR in the presence of APV was not different from venom only group (Fig. 3). In APV treated group, MSR returned to the initial level after washing with standard physiological solution for 30 min and was significantly different from venom only group (Fig. 3; P < 0.05; Student’s t-test). The findings of the present experiments reveal that MBTvenom depressed the spinal segmental reflexes in a time- and a concentration-dependent manner. The depression produced by venom is irreversible and does not involve NMDA receptors. In the spinal cord, the decreased synaptic activity can be produced by the activation of inhibitory pathway or the inhibition of excitatory pathway or by continuous depolarization of axonal terminals. It has been shown that scorpion venom increases (50–70 times) repolarization time and refractory period of amphibian axons [4]. Such prolonged repolarization/refractoriness may eventually block the conduction of impulses at afferent/efferent nerve terminals. Iberiotoxin (IbTx), a high conductance Ca2+ activated K+ channel blocker, was isolated from Indian red scorpion venom has been implicated in this action [10]. Thus, K+ channel blocker should mimic the response. In the earlier report, the blockade of K+ channel by 3,4-diaminopyridine (3,4-DAP) prolonged the repolarization time of MSR [7]. However in the present results, there was no prolongation of the duration of MSR/PSR at any concentration and
Fig. 3. APV failed to block the venom-induced depression of MSR. In the top, actual reflex recordings of an experiment with DL-2-amino-5-phosphonovaleric acid (APV) are shown at different time intervals. The cord was pretreated with APV (10 M) for 30 min (0), subsequently the cord was exposed to venom (0.3 g/ml) in the presence of APV. At the end, the preparation was washed (W) for 30 min with normal physiological solution. Horizontal calibration is 10 ms and vertical calibration is 1 mV. In the lower graph, mean ± SEM values from five different preparations after APV are shown and the venom only data is taken from Fig. 1. The response in the presence of APV was not different form the ‘venom only’ group.
at any time. Thus, the depression may not be due to action at K+ channel blocking activity. In the previous studies, it has been shown that Ptychodiscus brevis toxin (PbTx), 3-nitropropionic acid (3-NPA) and aglycemia/ischemia depressed the spinal reflexes [11,14,21]. Further, the NMDA receptors are involved in the depression produced by PbTx or aglycemia/ischemia [13,21]. Hence, the depression of reflexes by MBT-venom may also involve similar mechanisms. The present results exclude the involvement of NMDA receptors, as APV did not block the MBT-venom-induced depression (Fig. 3). However, the greater depression of PSR at 0.3 g/ml of venom indicates the partial involvement of NMDA receptors as PSR is mediated through NMDA receptors [19,21]. In addition, the total recovery of MSR in wash experiments after APV pre-treatment further supports it (Fig. 3). The recovery in APV pretreated cord can be explained on the basis of a competitive antagonism of APV with NMDA receptors. In conclusion, scorpion venom depresses MSR/PSR via a mechanism independent of NMDA receptors. However, the involvement of inhibitory system in depressing the MSR has to be explored. Acknowledgement ANM wishes to thank Indian Council of Medical Research, New Delhi for providing financial assistance. References [1] H.S. Bawaskar, Diagnostic cardiac premonitory signs and symptoms of red scorpion sting, Lancet 1 (1982) 552–554. [2] H.S. Bawaskar, P.H. Bawaskar, Management of the cardiovascular manifestations of poisoning by the Indian red scorpion, Br. Heart J. 68 (1992) 478–480. [3] S.B. Deshpande, Significance of monosynaptic and polysynaptic reflexes in neonatal rat spinal cord in vitro, Ind. J. Exp. Biol. 31 (1993) 850–854. [4] S.B. Deshpande, Indian red scorpion (Mesobuthus tamulus concanesis, Pocock) venom prolongs repolarization time and refractoriness of the compound action potential of frog sciatic nerve in vitro through calcium dependent mechanism, Ind. J. Exp. Biol. 36 (1998) 1108–1113.
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