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Diisopropylfluorophosphate-induced depression of segmental monosynaptic transmission in neonatal rat spinal cord is also mediated by increased axonal activity
Toxicology Letters ELSEVIER
Toxicology
Letters
90 (1997)
177-182
Diisopropylfluorophosphate-induced depression of segmental monosynaptic transmission in neonatal rat spinal cord is also mediated by increased axonal activity S.B. Deshpande”,*, S. DasGupta”b bDivision
“Department of Physiology, Institute of Medical Sciences, Banaras of Pharmacology and Toxicology. Defence Research and Development
Hindu University, Establishment,
Varanasi, India Jhansi Road, Gu’alior,
India
Received 20 May 1996; revised 31 October 1996; accepted 6 November 1996
Abstract Involvement of dorsal and ventral root activity for the depressant action of diisopropylfluorophosphate (DFP) on synaptic transmission was examined using in vitro spinal cord/root preparations. Superfusion of DFP produced a dose-dependent depression of monosynaptic reflex (MSR) and maximal depression of about 80% occurred at 1000 ,uM. The concentration to produce 50% of the maximal inhibition was about 100 PM of DFP. The DFP (100 PM)-induced depression of MSR was reversed by atropine (0.5 PM) but not by mecamylamine (0.5 ,uM). Contrary to the action on MSR, DFP potentiated the ventral root potential and 1st peak of dorsal root potential. The maximal potentiation was about 25% of control in both the root potentials at 100 ,uM of DFP. However, the second peak of dorsal root potential was slightly depressed (lo-20% of control) by DFP (l-1000 PM). Further, the cords treated with DFP (100 ,uM) showed significant decrease in the cholinesterase (ChE) activity (27% of control). Results suggest that the DFP-induced depression was mediated at least by two different mechanisms, one through the inhibition of ChE activity and the other through the activation axonal activity having inhibitory inputs to the segmental synaptic transmission. These inputs mediate their action through muscarinic receptors. 0 1997 Elsevier Science Ireland Ltd. Kq~ords: potential
Organophosphates;
Cholinesterase
inhibitors;
Monosynaptic
reflex;
Dorsal
root;
Ventral
root;
Root
* Corresponding author, Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India. Tel.: 91 542 317629; fax: 91 542 317074. ’ Present address: Executive Secretary, Indian Science Congress Association, 14-Biresh Guha Street, Calcutta-700017, India. 037%4274/97/$17.00 PII
8 1997 Elsevier
SO378-4274(96)03846-S
Science
Ireland
Ltd.
All rights
reserved
178
S. R. Deshpande.
S. DasGupta
/ Toxicology
Letters
90 (1997)
1. Introduction
2. Materials
Organophosphorous (OP) compounds and reversible cholinesterase (ChE) agents are shown to depress the spinal monosynaptic reflex (MSR) transmission in neonatal rats [l-4]. The inhibition of ChE activity by these compounds has been proposed to be responsible for the depressant action on MSR [2,4]. Atropine antagonized the depression caused by reversible as well as irreversible ChE agents [ 1,2]. Similarly, thyrotropin-releasing hormone also reversed the OPinduced depression of the synaptic transmission [ 11. In both situations, the acetylcholinesterase (AChE) activity remained inhibited despite the reversal of the reflex activity [1,2]. Thus, the mechanisms involved in the inhibition of MSR may be independent of AChE activity. Nevertheless, the accumulation of acetylcholine (ACh) resulting from the inhibition of AChE is one of the factors involved in the action of OP compounds [1,2,4]. The interneurons which receive cholinergic inputs from recurrent collaterals are first described by Renshaw by the antidromic activation of motor axon collaterals producing an inhibition to the motoneurons [5]. Subsequently, disynaptic pathway for recurrent inhibition of la-a motoneuron synapse involving motor axon collaterals and Renshaw interneuron was reported [6-81. The transmitter released at this synapse was shown to be acetylcholine (ACh) [6-S]. The inhibition of ChE thus results in the accumulation of ACh which will enhance the activity of Renshaw interneurons resulting in inhibition of motoneurons [9]. However, the direct effect of OP compounds on motor axons is not known. Further, the activation of afferent axons carrying cutaneous sensations (primary afferents) also produced inhibition of the segmental MSR and is antagonized by atropine [lo]. The action of OP compounds on these afferent axonal inputs is also not known. The present study, therefore, was undertaken to investigate the action of DFP on MSR with reference to the afferent (dorsal root) and efferent (ventral root) axonal activity.
2.1. Chemicals
177-
182
and methods
DFP was synthesized in the Synthetic Chemistry Division of Defence Research and Development Establishment, Gwalior, India. Atropine sulfate and mecamylamine were from Sigma, USA. 2.2. Recording monosynaptic reflex
The method for isolation of spinal cord and recording of monosynaptic reflex (MSR) has been described elsewhere [1,4]. Briefly, the spinal cord from anaesthetized rats (6- lo-day-old) was dissected free from the column, hemisected sagitally and transferred to a small Plexiglas bath (volume < 1 ml) which was superfused with the oxygenated physiological solution (3- 5 ml/min) maintained at 25 f 0.5”C. Suction electrodes were applied to corresponding dorsal and ventral roots (L& using a common bath electrode (Ag-AgCl). Stimulation of a dorsal root with supramaximal strength ( < 0.2 ms duration; maximal volt; at 0.1 Hz) elicited a potential (MSR) in the corresponding ventral root. The root potentials were recorded by applying suction electrodes to the cut ends of dorsal or ventral root. The electrode at one end of the root was used for stimulation (0.2 ms; maximal volt; 0.1 Hz) and the other end for recording the potential. The generated potentials were amplified, monitored and saved in a computer. The latency, rise time, total duration, peak amplitude and refractory period were determined. 2.3. Experimental protocol for DFP effects
After application of suction electrodes, the cord/root preparation was kept for at least 1 h for stabilization and the cumulative dose response of DFP (0.01-1000 PM) was performed as mentioned below. The preparation was superfused with physiological solution containing a particular concentration of DFP for 30 min and the average of 5-6 successive potentials was saved in a computer. The spinal cord was then
S.R.
Dpshptmde,
S. DnsGupta
/ Toxicology
exposed to the next higher concentration of DFP for a similar period and the cord was exposed to not more than 3 concentrations of DFP. 2.4. Estimation
of acetylcholinesterase
(AChE)
AChE activity was determined by spectrophotometric method described by Ellman et al. [l 11. Briefly, the spinal cords from 7-g-day-old rats were weighed, homogenized in an ice-cold phosphate buffer (pH 8.0) and the homogenate was centrifuged at 1000 x g for 15 min at 0-3°C. The supernatant was taken and AChE was measured at 412 nm in a spectrophotometer. Some cords were pretreated with 100 PM DFP for 15 min and the AChE activity was determined as above. The AChE activity was expressed as nmol of ACh hydrolyzed per mg wet weight of the spinal cord tissue per min. 2.5. Analysis of data
The data is presented as mean + S.E.M. values. Significant difference in the dose-response curves was tested by using ANOVA and Student’s t-test for comparing between various treatments and P < 0.05 was considered significant.
Letters
90 (1997)
179
177-182
3.2. Effect of DFP on root poten.tials 3.2.1. Ventral root potential
(VRP)
Stimulation of one end of the ventral root evoked a monophasic potential with a latency of 0.14 f 0.025 ms corresponding to a conduction velocity of 71 m/s. DFP potentiated the VRP and the maximal potentiation was about 25% of control at 100 ,uM of DFP (Figs. 1 and 2). The potentiation did not increase even when the concentration was increased to 1000 ,uM. 3.2.2. Dorsal root potential
(DRP)
Stimulation of dorsal root produced compound potential with two peaks. The first peak which appeared after a latency was 0.45 + 0.096 ms and approximate conduction velocity was around 22 m/s (DRP-Pkl). The latency of the second peak was 2.13 + 0.13 ms with the conduction velocity of these fibers around 5 m/s (DRP-Pk2). The threshold for the second peak was greater than the first peak (data not given). Superfusion of DFP potentiated DRP-Pkl to the same magnitude that was seen with VRP. However, the sec-
MSR
DRP
I'RP
3. Results 3.1. Dose-dependent depression of MSR by DFP
DFP (O.Ol- 1000 ,LM) produced a dose-dependent depression of the MSR (Figs. 1 and 2). The depression was about 5% with 1 ,uM of DFP while the maximal depression (about 80%) was seen at 1000 ,uM. The concentration to produce 50% of the maximal inhibition was about 100 PM of DFP (Fig. 2). The action of DFP on MSR or on root potentials could not be reversed by washing with physiological solution for up to 60 min (Table 1). Superfusion of atropine (0.5 ,uM) reversed the DFP (100 pM)-induced depression of MSR (Table 2). However, mecamylamine (nicotine receptor antagonist; 0.5 ,uM) failed to reverse the depressant action, rather the depression was enhanced further (Table 2).
Fig. I. Effect of diisopropylphosphorofluoridate (DFP) on monosynaptic reflex (MSR) and root potentials. Actual reflex/ root potentials before (0) and after superfusion of DFP (IO and 1000 PM) on MSR, dorsal root (DRP) and ventral root (VRP) potential. Area of the reflex/potential before superfusion of DFP is taken as control (100%). Note the depression of MSR while the 1st peak of DRP (DRP-Pkl) as well as VRP were potentiated, the 2nd peak of DRP (DRP-Pk2) was depressed. Vertical calibration = 1 mV (for all); horizontal calibration = 5 ms for MSR; 2 ms for DRPiVRP.
180
S.B. Deshpande,
1401
I
I
S. DasGupta
i Toxicology
TI
I
80 -
40 - A MSR
1
1000 (pl)
177-182
Table 2 Effect of DFP on monosynaptic reflex (MSR) in the presence of muscarinic or nicotinic receptor antagonists Condition
n
MSR area (% of control)
DFP alone DFP + atropine DFP + mecamylamine
10 5 5
48 + 5.6 104&7.1* 12 i 4.2*
root potentials were not altered in the presence of DFP (data not shown). 3.3. DFP on spinal cord ChE activity
Fig. 2. Dose-response of DFP on MSR and root potentials. DFP produced a dose-dependent depression of the MSR (ANOVA; P < 0.05). There was potentiation of the 1st peak of DRP (DRP-Pkl) and VRP. The second peak of DRP was depressed slightly but significantly. Mean k S.E.M. values were obtained from 4-6 different preparations for each point.
ond peak of DRP was significantly depressed (Figs. 1 and 2). Rise time, threshold, conduction velocity and depolarization time and refractory period of the Table 1 The DFP-induced changes on root potential and monosynaptic reflex (MSR) could not be reversed after washing MSR area (% of control)
MSR Ventral root potential Dorsal root potential Peak 1 (Pkl) Peak 2 (Pk2)
90 (1997)
The spinal cord was first exposed to 100 pM of DFP for 30 min then physiological solution containing DFP in the presence or absence of cholinergic antagonists (0.5 ,uM) for 30 min. The mean k S.E.M. values were from n number of observations. *Significant difference (P~0.05) as compared to DFP alone.
60 -
IA
Letters
In presence of DFP
After wash
52.4 k 5.7 121.1 * 4.8
54.8 + 6.2 125.4 + 5.2
119.2 f 8.1 88.1 i 4.4
122.1 + 7.0 90.0 * 5.1
The cord/root was exposed to DFP (100 PM) for 30 min then the cords were washed with normal physiological solution for 60 min before the recordings were made. The mean k S.E.M. is obtained from 4 different observations. The values after wash were not statistically different from those recorded after exposing to DFP (P>O.l; paired t-test).
Control ChE activity of the spinal cord was 16.3 a 1.96 nmol/min/g tissue and it was 4.4 f 0.33 nmol/min/g tissue in DFP (100 PM)-treated groups. These values were significantly different (P < 0.05; unpaired t-test).
4. Discussion
The observations of this study while confirming the depressant action of DFP on MSR [1,3] further revealed that the DFP potentiated the magnitude of afferent or efferent axonal potentials. The DFP-induced depression of MSR was associated with the inhibition of AChE activity and could be reversed by muscarinic receptor antagonist. Irreversible inhibition of AChE by OP compounds is known to occur through phosphorylation or phophonylation of the serine hydroxyl at the active site of the enzyme. The resultant accumulation of ACh at both peripheral and central sites produces a variety of cholinergically related manifestations [1,2,4,12]. Thus, OP compounds possess their activity primarily to the effects of ChE inhibition, although DFP is shown to block the ion channels of the nicotinic ACh receptor directly [12]. However, it was observed that the
S.B.
Deshpande,
S. DasGupta
i Toxicology
muscarinic antagonists (atropine and benactyzine) effectively reversed the DFP/sarin-induced depression of the MSR in neonatal rat spinal cord while mecamylamine (nicotinic receptor antagonists) failed to reverse the depression [2,4]. Thus, the present results are also consistent with muscarinic receptor involvement for the neurotoxic action of OP compound in the spinal cord. The magnitude of inhibition of AChE activity by DFP is similar to the magnitude of inhibition seen elsewhere [I]. Such an inhibition will increase the ACh levels at cholinergic synapses. Elsewhere, the cholinergic agonists depressed MSR [ 10,131. The cholinergic projections in the spinal cord are distributed at the recurrent collateral projecting on the Renshaw interneuron [5-71. The cholinergic transmission at primary afferent terminals in the spinal cord is not known [14] but, the primary afferent projection on tachykininergic/dopaminergic interneurons modulating the cholinergic transmission has been reported [lo]. The recurrent collateral from the motor axons excite the Renshaw interneurons and inhibit the motoneuron activity [5-71. The transmitter at the collateral and Renshaw interneuron is ACh [6-81. The presence of choline acetyl transferase (ChAT) and AChE activity further confirms this point [15]. Inhibition of ChE results in the accumulation of ACh as mentioned before. The nicotinic and muscarinic receptors are identified in the spinal cord [8,9,13]. Further, the results of this study or elsewhere indicate that, the muscarinic receptor antagonists reversed the depression of MSR by ChE inhibitors [ 1,2,4]. Similarly, the muscarinic agonists exhibited similar inhibition of MSR which could be blocked by atropine [ 131. The muscarinic agonists are shown to elicit two types of responses on motoneurons such as depolarization of the motoneurons via Ml receptors and the depression of MSR mediated by M2 receptors [13]. The Ml receptors are presynaptically located and activation of these receptors modulate the release of transmitters by depolarizing these terminals [13]. Since the observed increase in dorsal root potential by DFP, action at the presynaptic terminals by the agonist (ACh) cannot be ruled out. The increased activity of efferent terminals as observed in this study along with the inhibition of
Letters
90 (1997)
177-182
181
ChE activity (ACh accumulation) produce greater excitation of Renshaw-interneurons. This interneuronal activity will produce the inhibition of MSR. The above results indicate that the depression was because of accumulation of ACh acting on muscarinic receptors. Reversal of OP-induced depression of the MSR by thyrotropin-releasing hormone without altering AChE inhibition will also favor the non-involvement of enzyme directly in mediating this action [l]. Thus, the toxic action of OP compounds may be due to the accumulation of ACh at the site. The present results further indicate that the activity of fast conducting afferent fibers was potentiated while that of the slow conducting afferent fibers was depressed. Inhibition of MSR brought about by the activation of primary afferents involve inhibitory cholinergic interneurons in the pathway. Stimulation of group C fibers excite the tachykininergic neurons resulting in inhibition through the cholinergic system [lo]. However, the threshold and latency of either 1st peak or 2nd peak do not correspond to that for group C fibers reported elsewhere [lo]. Conduction velocity of 2nd peak also does not correspond with group C fiber velocity. Further, the depression of the 2nd peak of DRP in the present study may indicate the inhibition of excitatory neurons in producing the depression of MSR. In summary, the inhibition of AChE by DFP results in accumulation of ACh. The increased activity of recurrent collaterals further enhance this inhibition. However, the exact nature of action of OP compounds on afferent nerve terminals that are directly inhibitory to the MSR requires further investigation.
Acknowledgements
The authors thank Dr. R.V. Swamy, Director, Defence Research and Development Establishment (DRDE), Gwalior for constant encouragement and discussion during this work. They also thank Dr. S.N. Dube for useful discussions. This work was supported by a grant from DRDE, Gwalior.
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S. DasGupta
/ Toxicology
References [l]
[2]
[3]
[4]
[5]
[6]
[7]
DasGupta, S., Deshpande, S.B. and Warnick, J.E. (1988) Segmental synaptic depression caused by diisopropylphosphorofluoridate and sarin is reversed by thyrotropinreleasing hormone in the neonatal rat spinal cord. Toxicol. Appl. Pharmacol. 99, 499-506. DasGupta. S., Bass, K. and Wamick, J.E. (1989) Interaction of reversible and irreversible cholinesterase inhibitors on the monosynaptic reflex in neonatal rats. Toxicol. Appl. Pharmacol. 99, 28-36. DasGupta, S. and Warnick, J.E. (1993) Effect of organophosphates and carbamates on the monosynaptic reflex recorded from neonatal rat spinal cord. In: S.K. Manchanda, W. Selvamurthy and V. Mohan Kumar (Eds.), Advances in Physiological sciences, Macmillan India, New Delhi, pp. 6244630. Warnick, J.E., Deshpande, S.B.. Yang, Q.Z. and DasGupta, S. (1993) Biphasic action of sarin on monosynaptic reflex in the neonatal rat spinal cord in vitro. Arch. Toxicol. 67, 3022306. Renshaw, B.C. (1946) Central effects of centripetal impulses in axons of spinal ventral roots. J. Neurophysiol. 9, 191-194. Eccles, J.C., Fatt, P. and Koketsu, K. (1954) Cholinergic and inhibitory synapses in pathway from axons from motor axon collateral to motoneuron. J. Physiol. 126. 5244562. Eccles, J.C., Eccles, R.M. and Fatt. P. (1956) Pharmacological involvement on a central synapse by acetylcholine. J. Physiol. 131, 1544169.
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[8] Curtis, D.R. and Ryall, R.W. (1966) The acetylcholine receptors of Renshaw cell. Exp. Br. Res. 2, 6680. [9] Ryall, R.W. (1984) Cholinergic transmission in the spinal cord. In: R.A. Davidoff (Ed.), Handbook of Spinal cord. vol. 2-3, Marcel Dekker, New York, pp. 2033237. [lo] Yoshioka, K., Sakuma, M. and Otsuka, M. (1990) Cutaneous nerve evoked cholinegic inhibition of monosynaptic reflex in neonatal rat spinal cord: involvement of M2 receptors and tachykininergic primary afferents. Neuroscience 38, 195-203. [ll] Ehdn, G.L., Courtney, K.D.. Andres, V. Jr. and Featherstone, R.M. (1961) A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95. [12] Kuba, K., Albuquerque, E.X., Daly, J. and Barnard, E.A. (1974) A study of the irreversible cholinesterase inhibitor, diisopropylfluorophosphate, on time course of end-plate currents in frog sartorius muscle. J. Pharmacol. Exp. Ther. 189, 499-512. [13] Newberry, N.R. and Connolly, G.P. (1989) Muscarinic pharmacology of the spinal cord of neonatal rat in vitro. Nemopharmacology 28, 149- 152. [14] Kato, T. and Marushima, Y.L. (1985) Choline acetyl transferase activities in single motor neuron from the vertebrate spinal cord. J. Neurochem. 44, 675-679. [15] Potter, P.E. and Neef, N.H. (1984) Spinal cord acetylcholine content: the consequence of transection or treatment with the neurotoxin 6-aminonicotinamide. Br. Res. 303, 87-90.
Toxicology
Letters
90 (1997)
177-182
Diisopropylfluorophosphate-induced depression of segmental monosynaptic transmission in neonatal rat spinal cord is also mediated by increased axonal activity S.B. Deshpande”,*, S. DasGupta”b bDivision
“Department of Physiology, Institute of Medical Sciences, Banaras of Pharmacology and Toxicology. Defence Research and Development
Hindu University, Establishment,
Varanasi, India Jhansi Road, Gu’alior,
India
Received 20 May 1996; revised 31 October 1996; accepted 6 November 1996
Abstract Involvement of dorsal and ventral root activity for the depressant action of diisopropylfluorophosphate (DFP) on synaptic transmission was examined using in vitro spinal cord/root preparations. Superfusion of DFP produced a dose-dependent depression of monosynaptic reflex (MSR) and maximal depression of about 80% occurred at 1000 ,uM. The concentration to produce 50% of the maximal inhibition was about 100 PM of DFP. The DFP (100 PM)-induced depression of MSR was reversed by atropine (0.5 PM) but not by mecamylamine (0.5 ,uM). Contrary to the action on MSR, DFP potentiated the ventral root potential and 1st peak of dorsal root potential. The maximal potentiation was about 25% of control in both the root potentials at 100 ,uM of DFP. However, the second peak of dorsal root potential was slightly depressed (lo-20% of control) by DFP (l-1000 PM). Further, the cords treated with DFP (100 ,uM) showed significant decrease in the cholinesterase (ChE) activity (27% of control). Results suggest that the DFP-induced depression was mediated at least by two different mechanisms, one through the inhibition of ChE activity and the other through the activation axonal activity having inhibitory inputs to the segmental synaptic transmission. These inputs mediate their action through muscarinic receptors. 0 1997 Elsevier Science Ireland Ltd. Kq~ords: potential
Organophosphates;
Cholinesterase
inhibitors;
Monosynaptic
reflex;
Dorsal
root;
Ventral
root;
Root
* Corresponding author, Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India. Tel.: 91 542 317629; fax: 91 542 317074. ’ Present address: Executive Secretary, Indian Science Congress Association, 14-Biresh Guha Street, Calcutta-700017, India. 037%4274/97/$17.00 PII
8 1997 Elsevier
SO378-4274(96)03846-S
Science
Ireland
Ltd.
All rights
reserved
178
S. R. Deshpande.
S. DasGupta
/ Toxicology
Letters
90 (1997)
1. Introduction
2. Materials
Organophosphorous (OP) compounds and reversible cholinesterase (ChE) agents are shown to depress the spinal monosynaptic reflex (MSR) transmission in neonatal rats [l-4]. The inhibition of ChE activity by these compounds has been proposed to be responsible for the depressant action on MSR [2,4]. Atropine antagonized the depression caused by reversible as well as irreversible ChE agents [ 1,2]. Similarly, thyrotropin-releasing hormone also reversed the OPinduced depression of the synaptic transmission [ 11. In both situations, the acetylcholinesterase (AChE) activity remained inhibited despite the reversal of the reflex activity [1,2]. Thus, the mechanisms involved in the inhibition of MSR may be independent of AChE activity. Nevertheless, the accumulation of acetylcholine (ACh) resulting from the inhibition of AChE is one of the factors involved in the action of OP compounds [1,2,4]. The interneurons which receive cholinergic inputs from recurrent collaterals are first described by Renshaw by the antidromic activation of motor axon collaterals producing an inhibition to the motoneurons [5]. Subsequently, disynaptic pathway for recurrent inhibition of la-a motoneuron synapse involving motor axon collaterals and Renshaw interneuron was reported [6-81. The transmitter released at this synapse was shown to be acetylcholine (ACh) [6-S]. The inhibition of ChE thus results in the accumulation of ACh which will enhance the activity of Renshaw interneurons resulting in inhibition of motoneurons [9]. However, the direct effect of OP compounds on motor axons is not known. Further, the activation of afferent axons carrying cutaneous sensations (primary afferents) also produced inhibition of the segmental MSR and is antagonized by atropine [lo]. The action of OP compounds on these afferent axonal inputs is also not known. The present study, therefore, was undertaken to investigate the action of DFP on MSR with reference to the afferent (dorsal root) and efferent (ventral root) axonal activity.
2.1. Chemicals
177-
182
and methods
DFP was synthesized in the Synthetic Chemistry Division of Defence Research and Development Establishment, Gwalior, India. Atropine sulfate and mecamylamine were from Sigma, USA. 2.2. Recording monosynaptic reflex
The method for isolation of spinal cord and recording of monosynaptic reflex (MSR) has been described elsewhere [1,4]. Briefly, the spinal cord from anaesthetized rats (6- lo-day-old) was dissected free from the column, hemisected sagitally and transferred to a small Plexiglas bath (volume < 1 ml) which was superfused with the oxygenated physiological solution (3- 5 ml/min) maintained at 25 f 0.5”C. Suction electrodes were applied to corresponding dorsal and ventral roots (L& using a common bath electrode (Ag-AgCl). Stimulation of a dorsal root with supramaximal strength ( < 0.2 ms duration; maximal volt; at 0.1 Hz) elicited a potential (MSR) in the corresponding ventral root. The root potentials were recorded by applying suction electrodes to the cut ends of dorsal or ventral root. The electrode at one end of the root was used for stimulation (0.2 ms; maximal volt; 0.1 Hz) and the other end for recording the potential. The generated potentials were amplified, monitored and saved in a computer. The latency, rise time, total duration, peak amplitude and refractory period were determined. 2.3. Experimental protocol for DFP effects
After application of suction electrodes, the cord/root preparation was kept for at least 1 h for stabilization and the cumulative dose response of DFP (0.01-1000 PM) was performed as mentioned below. The preparation was superfused with physiological solution containing a particular concentration of DFP for 30 min and the average of 5-6 successive potentials was saved in a computer. The spinal cord was then
S.R.
Dpshptmde,
S. DnsGupta
/ Toxicology
exposed to the next higher concentration of DFP for a similar period and the cord was exposed to not more than 3 concentrations of DFP. 2.4. Estimation
of acetylcholinesterase
(AChE)
AChE activity was determined by spectrophotometric method described by Ellman et al. [l 11. Briefly, the spinal cords from 7-g-day-old rats were weighed, homogenized in an ice-cold phosphate buffer (pH 8.0) and the homogenate was centrifuged at 1000 x g for 15 min at 0-3°C. The supernatant was taken and AChE was measured at 412 nm in a spectrophotometer. Some cords were pretreated with 100 PM DFP for 15 min and the AChE activity was determined as above. The AChE activity was expressed as nmol of ACh hydrolyzed per mg wet weight of the spinal cord tissue per min. 2.5. Analysis of data
The data is presented as mean + S.E.M. values. Significant difference in the dose-response curves was tested by using ANOVA and Student’s t-test for comparing between various treatments and P < 0.05 was considered significant.
Letters
90 (1997)
179
177-182
3.2. Effect of DFP on root poten.tials 3.2.1. Ventral root potential
(VRP)
Stimulation of one end of the ventral root evoked a monophasic potential with a latency of 0.14 f 0.025 ms corresponding to a conduction velocity of 71 m/s. DFP potentiated the VRP and the maximal potentiation was about 25% of control at 100 ,uM of DFP (Figs. 1 and 2). The potentiation did not increase even when the concentration was increased to 1000 ,uM. 3.2.2. Dorsal root potential
(DRP)
Stimulation of dorsal root produced compound potential with two peaks. The first peak which appeared after a latency was 0.45 + 0.096 ms and approximate conduction velocity was around 22 m/s (DRP-Pkl). The latency of the second peak was 2.13 + 0.13 ms with the conduction velocity of these fibers around 5 m/s (DRP-Pk2). The threshold for the second peak was greater than the first peak (data not given). Superfusion of DFP potentiated DRP-Pkl to the same magnitude that was seen with VRP. However, the sec-
MSR
DRP
I'RP
3. Results 3.1. Dose-dependent depression of MSR by DFP
DFP (O.Ol- 1000 ,LM) produced a dose-dependent depression of the MSR (Figs. 1 and 2). The depression was about 5% with 1 ,uM of DFP while the maximal depression (about 80%) was seen at 1000 ,uM. The concentration to produce 50% of the maximal inhibition was about 100 PM of DFP (Fig. 2). The action of DFP on MSR or on root potentials could not be reversed by washing with physiological solution for up to 60 min (Table 1). Superfusion of atropine (0.5 ,uM) reversed the DFP (100 pM)-induced depression of MSR (Table 2). However, mecamylamine (nicotine receptor antagonist; 0.5 ,uM) failed to reverse the depressant action, rather the depression was enhanced further (Table 2).
Fig. I. Effect of diisopropylphosphorofluoridate (DFP) on monosynaptic reflex (MSR) and root potentials. Actual reflex/ root potentials before (0) and after superfusion of DFP (IO and 1000 PM) on MSR, dorsal root (DRP) and ventral root (VRP) potential. Area of the reflex/potential before superfusion of DFP is taken as control (100%). Note the depression of MSR while the 1st peak of DRP (DRP-Pkl) as well as VRP were potentiated, the 2nd peak of DRP (DRP-Pk2) was depressed. Vertical calibration = 1 mV (for all); horizontal calibration = 5 ms for MSR; 2 ms for DRPiVRP.
180
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I
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i Toxicology
TI
I
80 -
40 - A MSR
1
1000 (pl)
177-182
Table 2 Effect of DFP on monosynaptic reflex (MSR) in the presence of muscarinic or nicotinic receptor antagonists Condition
n
MSR area (% of control)
DFP alone DFP + atropine DFP + mecamylamine
10 5 5
48 + 5.6 104&7.1* 12 i 4.2*
root potentials were not altered in the presence of DFP (data not shown). 3.3. DFP on spinal cord ChE activity
Fig. 2. Dose-response of DFP on MSR and root potentials. DFP produced a dose-dependent depression of the MSR (ANOVA; P < 0.05). There was potentiation of the 1st peak of DRP (DRP-Pkl) and VRP. The second peak of DRP was depressed slightly but significantly. Mean k S.E.M. values were obtained from 4-6 different preparations for each point.
ond peak of DRP was significantly depressed (Figs. 1 and 2). Rise time, threshold, conduction velocity and depolarization time and refractory period of the Table 1 The DFP-induced changes on root potential and monosynaptic reflex (MSR) could not be reversed after washing MSR area (% of control)
MSR Ventral root potential Dorsal root potential Peak 1 (Pkl) Peak 2 (Pk2)
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The spinal cord was first exposed to 100 pM of DFP for 30 min then physiological solution containing DFP in the presence or absence of cholinergic antagonists (0.5 ,uM) for 30 min. The mean k S.E.M. values were from n number of observations. *Significant difference (P~0.05) as compared to DFP alone.
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After wash
52.4 k 5.7 121.1 * 4.8
54.8 + 6.2 125.4 + 5.2
119.2 f 8.1 88.1 i 4.4
122.1 + 7.0 90.0 * 5.1
The cord/root was exposed to DFP (100 PM) for 30 min then the cords were washed with normal physiological solution for 60 min before the recordings were made. The mean k S.E.M. is obtained from 4 different observations. The values after wash were not statistically different from those recorded after exposing to DFP (P>O.l; paired t-test).
Control ChE activity of the spinal cord was 16.3 a 1.96 nmol/min/g tissue and it was 4.4 f 0.33 nmol/min/g tissue in DFP (100 PM)-treated groups. These values were significantly different (P < 0.05; unpaired t-test).
4. Discussion
The observations of this study while confirming the depressant action of DFP on MSR [1,3] further revealed that the DFP potentiated the magnitude of afferent or efferent axonal potentials. The DFP-induced depression of MSR was associated with the inhibition of AChE activity and could be reversed by muscarinic receptor antagonist. Irreversible inhibition of AChE by OP compounds is known to occur through phosphorylation or phophonylation of the serine hydroxyl at the active site of the enzyme. The resultant accumulation of ACh at both peripheral and central sites produces a variety of cholinergically related manifestations [1,2,4,12]. Thus, OP compounds possess their activity primarily to the effects of ChE inhibition, although DFP is shown to block the ion channels of the nicotinic ACh receptor directly [12]. However, it was observed that the
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muscarinic antagonists (atropine and benactyzine) effectively reversed the DFP/sarin-induced depression of the MSR in neonatal rat spinal cord while mecamylamine (nicotinic receptor antagonists) failed to reverse the depression [2,4]. Thus, the present results are also consistent with muscarinic receptor involvement for the neurotoxic action of OP compound in the spinal cord. The magnitude of inhibition of AChE activity by DFP is similar to the magnitude of inhibition seen elsewhere [I]. Such an inhibition will increase the ACh levels at cholinergic synapses. Elsewhere, the cholinergic agonists depressed MSR [ 10,131. The cholinergic projections in the spinal cord are distributed at the recurrent collateral projecting on the Renshaw interneuron [5-71. The cholinergic transmission at primary afferent terminals in the spinal cord is not known [14] but, the primary afferent projection on tachykininergic/dopaminergic interneurons modulating the cholinergic transmission has been reported [lo]. The recurrent collateral from the motor axons excite the Renshaw interneurons and inhibit the motoneuron activity [5-71. The transmitter at the collateral and Renshaw interneuron is ACh [6-81. The presence of choline acetyl transferase (ChAT) and AChE activity further confirms this point [15]. Inhibition of ChE results in the accumulation of ACh as mentioned before. The nicotinic and muscarinic receptors are identified in the spinal cord [8,9,13]. Further, the results of this study or elsewhere indicate that, the muscarinic receptor antagonists reversed the depression of MSR by ChE inhibitors [ 1,2,4]. Similarly, the muscarinic agonists exhibited similar inhibition of MSR which could be blocked by atropine [ 131. The muscarinic agonists are shown to elicit two types of responses on motoneurons such as depolarization of the motoneurons via Ml receptors and the depression of MSR mediated by M2 receptors [13]. The Ml receptors are presynaptically located and activation of these receptors modulate the release of transmitters by depolarizing these terminals [13]. Since the observed increase in dorsal root potential by DFP, action at the presynaptic terminals by the agonist (ACh) cannot be ruled out. The increased activity of efferent terminals as observed in this study along with the inhibition of
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ChE activity (ACh accumulation) produce greater excitation of Renshaw-interneurons. This interneuronal activity will produce the inhibition of MSR. The above results indicate that the depression was because of accumulation of ACh acting on muscarinic receptors. Reversal of OP-induced depression of the MSR by thyrotropin-releasing hormone without altering AChE inhibition will also favor the non-involvement of enzyme directly in mediating this action [l]. Thus, the toxic action of OP compounds may be due to the accumulation of ACh at the site. The present results further indicate that the activity of fast conducting afferent fibers was potentiated while that of the slow conducting afferent fibers was depressed. Inhibition of MSR brought about by the activation of primary afferents involve inhibitory cholinergic interneurons in the pathway. Stimulation of group C fibers excite the tachykininergic neurons resulting in inhibition through the cholinergic system [lo]. However, the threshold and latency of either 1st peak or 2nd peak do not correspond to that for group C fibers reported elsewhere [lo]. Conduction velocity of 2nd peak also does not correspond with group C fiber velocity. Further, the depression of the 2nd peak of DRP in the present study may indicate the inhibition of excitatory neurons in producing the depression of MSR. In summary, the inhibition of AChE by DFP results in accumulation of ACh. The increased activity of recurrent collaterals further enhance this inhibition. However, the exact nature of action of OP compounds on afferent nerve terminals that are directly inhibitory to the MSR requires further investigation.
Acknowledgements
The authors thank Dr. R.V. Swamy, Director, Defence Research and Development Establishment (DRDE), Gwalior for constant encouragement and discussion during this work. They also thank Dr. S.N. Dube for useful discussions. This work was supported by a grant from DRDE, Gwalior.
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