Neurornuscular Pharmacology in Rat Neonates: Development of Responsiveness to Prototypic Blocking and Reversal Drugs Michiko Okamoto, PhD, Jose L. Walewski, Walter F. Riker, Jr., MD
BS,
Joseph F. Artusio, Jr., MD, and
Departments of Pharmacology and Anesthesiology, Cornell University Medical College, New York, New York
The neonatal pharmacology of neuromuscular drugs was studied in vivo in newborn rats and in vitro in neonatal phrenic nerve-hemidiaphragm preparations. Drugs used to probe neuromuscular development in rat neonates were physostigmine, edrophonium, neostigmine, 4-aminopyridine, d-tubocurarine (dTc), and succinylcholine.The prejunctional actions of these drugs were monitored in relation to neonatal age by the appearance of stimulus-evoked repetitive discharge initiated by motor nerve endings and the occurrence and magnitude of the resulting enhancement of twitch tension. The occurrence and incidence of drug-induced fasciculations also served to track the development of functional motor nerve endings. Each of these prejunctional actions was inoperative until the third neonatal week, indicative of incom-
T
he postnatal development of amphibian and mammalian neuromuscular junctions has been well defined (1-19). The structural and functional development of the rat neuromuscular junction achieves an adult level by the fifth postnatal week. The human neuromuscular junction exhibits an adult function and morphology by approximately the 12th postnatal week (3). This maturation process includes completion of myelination, disappearance of polyneuronal innervation, aggregation of postjunctional acetylcholine (ACh) receptors to form end plates, sensitivity to ACh, acetylcholinesterase (AChE) localization, and completion of functional and pharmacologic responsiveness (1,949). Pharmacologically, however, classic neuromuscular drug effects have not been explored fully or quantitatively in either animal or human neonates. The This work was supported by Research Grant AA-07722 from the NIAAA. Accepted for publication April 10, 1992. Address correspondence to Dr. Okamoto, Department of Pharmacology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021. 01992 by the International Anesthesia Research Society 0003-2999/921$5.00
plete motor nerve development. In contrast, 4-aminopyridine, a nonanticholinesterase, evoked these prejunctional actions in 1-wk-old rat neonates. Neostigmine and edrophonium antagonized dTc as early as the first week; presumably, postsynaptic maturation had reached a functional level. 4-Aminopyridine also antagonized dTc at week 1. Rat neonates showed resistance to dTc blockade when tested by neonatal phrenic nerve-hemidiaphragm preparations in vitro. Relationships between age and 85%-95% transmission block declined to the adult level by week 5. This result indicates that in rat neonates, pharmacodynamic rather than pharmacokinetic mechanisms predominate in the development of responsiveness to dTc. (Anesth Analg 1992;75:361-71)
present work reports and discusses the use of neonatal rat neuromuscular preparations in vivo and in vitro to study the pre- and postjunctionai actions of d-tubocurarine (dTc), succinylcholine (SCh), neostigmine, edrophonium, physostigmine, and 4aminopyridine (4-AP) on developing junctions. The principal aims were twofold: (a) to use well-defined pharmacologic probes in assessing developmental stages of mammalian neuromuscular junctions in vivo; and (b) to define the neonatal pharmacology of drugs having classical actions at neuromuscular junctions.
Methods Rat pups aged 1-5 wk, born to Sprague-Dawley dams (Hill Top Farm, N.J.) were studied. All of the procedures used were in accord with AALAC guidelines. Dams in their last week of pregnancy were housed individually. Pup body weights were recorded at birth. These progressed from 10 to 50 g during the first three neonatal weeks. Pups were raised by their dams during the first three postnatal weeks. To ensure uniform nutritive conditions of the pups, the Anesth Analg 1992;75:361-71
361
362
ANESTH ANALG
PEDIATRIC ANESTHESIA OKAMOTO ET AL. B L O C K " AND REVERSAL DRUGS ON RAT NEONATES
number of pups per dam was limited to 10 at birth, 7 at postnatal week 2, and 4 at postnatal week 3. The pups started eating on their own by postnatal day 17 or 18. The findings obtained in the rat neonates were compared with those from adult control SpragueDawley male and female rats, weighing between 200 and 250 g (i.e., >7 wk of age). Pups and adults were anesthetized with 25% urethane solution intraperitoneally, 6 pL/g body wt for a body weight 4 0 0 g and 5 pL/g body wt for a weight >lo0 g. The anesthetized animal was secured on a polystyrene board, and an unobstructed airway was ensured by tracheostomy. A carotid artery was catheterized for intraarterial (IA) drug injection. The carotid arteries were the only blood vessels accessible for cannulation in 1-wk-old rat neonates. Isometric muscle contraction was obtained as follows. A leg was immobilized by fixation of the knee and foot to the polystyrene board. A gastrocnemius muscle and its innervation were exposed in s i b . The corresponding sciatic nerve was cut centrally. The gastrocnemius tendon was detached from its insertion and connected to a force transducer (model FT-3, Grass Instruments Co., Quincy, Mass.). The output from the transducer was amplified and recorded (transducer amplifier model 13-4218 and recorder model 220, Gould, Cleveland, Ohio). The exposed tissue was protected with paraffin oil and the preparation temperature thermostatically held at 37°C by radiant heat. Indirect muscle contractions were evoked by supramaximal single stimuli applied to the sciatic nerve through a bipolar platinum electrode from a stimulator (model S-88, Grass Instruments Co.) via an isolation unit. For direct muscle stimulation, needle electrodes were inserted into the gastrocnemius muscle heads, and neural transmission was blocked by dTc. Both indirect and direct stimuli consisted of 0.1-ms square-wave pulses delivered every 4 s . Tetanic stimuli of 5 4 0 0 Hz were delivered for 30 s . To record muscle fasciculations, the output from the transducer was amplified 10 times that for recording normal twitch tension. Nerve stimulation was discontinued when recording muscle fasciculations. Fasciculations, when these occurred, were observed in exposed muscle and in the intact animal. Their incidence was recorded with regard to age at the physostigmine doses shown in Table 1. To record motor nerve ending" activity, thoracic and lumbar laminectomies exposed the S-1 and L-6 ventral roots; these contain gastrocnemius motor axons. In these experiments, the sciatic nerve was left *Motor nerve endings include the distal Ranvier node, the myelinated axon leading to the unmyelinated nerve terminals, and the unmyelinated terminals.
1992;75361-71
Table 1. Percent Incidence of Physostigmine-Induced Fasciculation in Neonatal and Adult Rats Dose" (mgikg SC) 0.5 1.0
2.0 5.0 10.0
Postnatal week Week2
Week3
Week4
0 0 0
0 0
50.0 72.7 75.0 100.0 100.0
0 0
12.5 27.2 40.0
Week5
Adult
100.0
100.0 100.0 100.0 -
100.0
100.0 -
-
SC, subcutaneous. '11 > 5 for each subcutaneous dose.
intact. The S-1 and L-6 roots were disconnected at their origin from the spinal cord and separated into small filaments. A reactive filament was placed on bipolar platinum recording electrodes leading to differential preamplifiers (model AM 502, Tektronix, Beaverton, Ore.) and displayed on a storage oscilloscope (model 4072, Gould). Thus, prejunctional actions of gastrocnemius motor nerve terminals were monitored antidromically, as detailed by other studies from this laboratory (20-23). Compound action potentials of gastrocnemius muscle, elicited by motor nerve stimulation, were recorded by bipolar platinum microelectrodes. The output was amplified through a differential preamplifier (model AM 502, Tektronix) and displayed simultaneously with the nerve action potentials on the storage oscilloscope described. The drugs administered IA were edrophonium chloride, neostigmine bromide, dTc chloride, and SCh chloride; physostigmine sulfate was administered subcutaneously (SC). Atropine sulfate (2 mgkg IP) was used to prevent muscarinic effects. All drugs were purchased from Sigma Chemical Co. (St. Louis, Mo.), and the solutions were prepared in 0.9% NaC1. Concentrations of each drug, with the exception of dTc and SCh, were prepared to limit injection volumes to 25 pL/lO g body wt for weights 40 g. The dTc was prepared in 200- or 4OO-pg/mL concentrations; SCh was prepared in 1- or 2-mg/mL concentrations. The dTc and SCh were infused through a carotid artery at 15-20 pL/min with a microinjection pump (model CMA/100, Bioanalytical Systems, Lafayette, Ind.), and dTc or SCh was infused until an 80%-95% neuromuscular block resulted. The dTc or SCh doses required for this were calculated from the total volumes injected. During the dTc or SCh experiments, respiration was maintained by a rodent ventilator (model 683, Harvard Apparatus and Instruments, South Natick, Mass.). At the end of all experiments, the animals were killed with sodium pentobarbital (>50 mg/kg IP).
A.
PHY8OBTIGNINE ¶
Figure 1. Physostigmine (A) and edrophonium (Bleffects in a 2-wk-old rat neonate and an adult iai. Twitch potentiation and muscle fasciculation are absent in the rat neonate but are welldeveloped effects in the adult rat. In the druginduced potentiations, the sciatic nerve was supramaximally stimulated at 0.25 Hz. Physostigmine and edrophonium injections are indicated by urrows. Muscle fasciculations were recorded in the absence of nerve stimulation and at a gain five times that used to record twitch tension. sc, subcutaneous; ia, intraarterial.
363
PEDIATRIC ANESTHESIA OKAMOTO ET AL. BLOCKING AND REVERSAL DRUGS O N RAT NEONATES
ANESTH ANALG 1992;75:361-71
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The neuromuscular blocking potency of dTc was studied in rat neonates of various ages, utilizing phrenic nerve-hemidiaphragm preparations in vitro. The muscle with its nerve was isolated and mounted in a Lucite chamber. The preparation was then bathed in Krebs’ solution containing NaCl, 118 mM; KC1, 5 mM; CaCI,, 2.5 mM; NaHCO,, 30 mM; KH,PO,, 1 mM; MgS04, 1 mM; glucose, 11 mM. The pH was maintained at 7.4 when aerated with a mixture of 95% O2 and 5% CO, (vol/vol%). The temperature of the bath was maintained at 36°C. The dTc concentrations studied were 1 x and 1 x M. The degree of neuromuscular block was calculated at the peak plateau effect of dTc; twitch
d.
tensions were measured before and after dTc and expressed as a percent block of twitch. Statistical calculation for estimating group mean, standard error, and correlation coefficient was done by analysis of variance.
Results Zndirect Enhancement of Twitch Tension Indirectly evoked twitch responses to the anticholinesterase drugs physostigmine and edrophonium are illustrated in Figure 1. The enhancement of
364
ANESTH ANALG 1992;75361-71
PEDIATRIC ANESTllliSIA OKAMOTO ET AL. BLOCKING AND R€VERSAL DRUGS ON RAT NEONATES
A. EDROPHONIUM 140t
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B. 350 z
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To determine whether the lack of potentiation in the 1-2-wk-old rat neonates is unique for these drugs, the nonanticholinesterase drug 4-AP was tested. Dose-twitch potentiation relationships, with respect to neonatal age, are plotted in Figure 2 for IA edrophonium, neostigmine, and 4AP. These dose-range studies disclosed an insignificant twitch potentiation by neostigmine and edrophonium in the 2-wk-old rat pups compared with 3-wk-old pups and adults. The results of neostigmine were similar to those for physostigmine and edrophonium (Figures 1 and 2). At 3 wk and beyond, neostigmine and physostigmine twitch potentiations significantly exceeded those for edrophonium. in contrast to the threshold responses to physostigmine, edrophonium, and neostigmine with respect to enhancement of twitch tension in the 1- and 2-wk-old rat neonates, the threshold response of IA 4-AP was positive. The dose-response curves for edrophonium, neostigmine, and 4-AP in Figure 2 are seen to be dose and age dependent.
100-
50L
Actions on Neonatal Motor Nerve Endings
0
5
10 -
100
NEOSTIGMINE DOSE (rng/kg)
C. 4-AMINOPYRIDINE
225
+
1
1
5 10 20 4-AMINOPYRIDINE DOSE (mg/kg)
2
I
Neostigmine action on neonatal rat motor nerve terminals was investigated in 3-wk-old rat neonates given a lOO-pg/kg dose IA. Figure 3 illustrates the repetitive action potentials recorded from ventral root fibers and a paired innervated muscle fiber after neostigmine dosing. As seen in Figure 3, repetitive motor nerve action potentials created a repetitive response in muscle. These events did not occur in 1and 2-wk-old rat neonates. In contrast to neostigmine, 4-AP in all 1- and 2-wk-old rat neonates tested caused both repetitive prejunctional firing (Figure 3) and significant twitch potentiation (Figure 2).
50
Figure 2. Dose- and age-related twitch potentiations plotted for intraarterial doses of edrophonium (A), neostigmine (B), and 4-aminopyridine (C). In all instances, sciatic nerve stimulation was supramaximal at 0.25 Hz. Maximal twitch potentiation (mean 2 SE) is calculated as percent potentiation above control twitch. Each point represents the average of n 2 7 experiments.
twitch tension (potentiation) caused by physostigmine, as seen in adult rats, was absent in 1- and 2-wk-oId rat pups. Subcutaneous physostigmine doses as large as 10 mgkg failed to produce potentiation in these rat neonates. By postnatal week 3, physostigmine potentiation appeared but was less than the response seen in the adult rat. The twitch potentiation evoked by edrophonium in the adult rats, like that of physostigmine, was absent in pups
BS,
Joseph F. Artusio, Jr., MD, and
Departments of Pharmacology and Anesthesiology, Cornell University Medical College, New York, New York
The neonatal pharmacology of neuromuscular drugs was studied in vivo in newborn rats and in vitro in neonatal phrenic nerve-hemidiaphragm preparations. Drugs used to probe neuromuscular development in rat neonates were physostigmine, edrophonium, neostigmine, 4-aminopyridine, d-tubocurarine (dTc), and succinylcholine.The prejunctional actions of these drugs were monitored in relation to neonatal age by the appearance of stimulus-evoked repetitive discharge initiated by motor nerve endings and the occurrence and magnitude of the resulting enhancement of twitch tension. The occurrence and incidence of drug-induced fasciculations also served to track the development of functional motor nerve endings. Each of these prejunctional actions was inoperative until the third neonatal week, indicative of incom-
T
he postnatal development of amphibian and mammalian neuromuscular junctions has been well defined (1-19). The structural and functional development of the rat neuromuscular junction achieves an adult level by the fifth postnatal week. The human neuromuscular junction exhibits an adult function and morphology by approximately the 12th postnatal week (3). This maturation process includes completion of myelination, disappearance of polyneuronal innervation, aggregation of postjunctional acetylcholine (ACh) receptors to form end plates, sensitivity to ACh, acetylcholinesterase (AChE) localization, and completion of functional and pharmacologic responsiveness (1,949). Pharmacologically, however, classic neuromuscular drug effects have not been explored fully or quantitatively in either animal or human neonates. The This work was supported by Research Grant AA-07722 from the NIAAA. Accepted for publication April 10, 1992. Address correspondence to Dr. Okamoto, Department of Pharmacology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021. 01992 by the International Anesthesia Research Society 0003-2999/921$5.00
plete motor nerve development. In contrast, 4-aminopyridine, a nonanticholinesterase, evoked these prejunctional actions in 1-wk-old rat neonates. Neostigmine and edrophonium antagonized dTc as early as the first week; presumably, postsynaptic maturation had reached a functional level. 4-Aminopyridine also antagonized dTc at week 1. Rat neonates showed resistance to dTc blockade when tested by neonatal phrenic nerve-hemidiaphragm preparations in vitro. Relationships between age and 85%-95% transmission block declined to the adult level by week 5. This result indicates that in rat neonates, pharmacodynamic rather than pharmacokinetic mechanisms predominate in the development of responsiveness to dTc. (Anesth Analg 1992;75:361-71)
present work reports and discusses the use of neonatal rat neuromuscular preparations in vivo and in vitro to study the pre- and postjunctionai actions of d-tubocurarine (dTc), succinylcholine (SCh), neostigmine, edrophonium, physostigmine, and 4aminopyridine (4-AP) on developing junctions. The principal aims were twofold: (a) to use well-defined pharmacologic probes in assessing developmental stages of mammalian neuromuscular junctions in vivo; and (b) to define the neonatal pharmacology of drugs having classical actions at neuromuscular junctions.
Methods Rat pups aged 1-5 wk, born to Sprague-Dawley dams (Hill Top Farm, N.J.) were studied. All of the procedures used were in accord with AALAC guidelines. Dams in their last week of pregnancy were housed individually. Pup body weights were recorded at birth. These progressed from 10 to 50 g during the first three neonatal weeks. Pups were raised by their dams during the first three postnatal weeks. To ensure uniform nutritive conditions of the pups, the Anesth Analg 1992;75:361-71
361
362
ANESTH ANALG
PEDIATRIC ANESTHESIA OKAMOTO ET AL. B L O C K " AND REVERSAL DRUGS ON RAT NEONATES
number of pups per dam was limited to 10 at birth, 7 at postnatal week 2, and 4 at postnatal week 3. The pups started eating on their own by postnatal day 17 or 18. The findings obtained in the rat neonates were compared with those from adult control SpragueDawley male and female rats, weighing between 200 and 250 g (i.e., >7 wk of age). Pups and adults were anesthetized with 25% urethane solution intraperitoneally, 6 pL/g body wt for a body weight 4 0 0 g and 5 pL/g body wt for a weight >lo0 g. The anesthetized animal was secured on a polystyrene board, and an unobstructed airway was ensured by tracheostomy. A carotid artery was catheterized for intraarterial (IA) drug injection. The carotid arteries were the only blood vessels accessible for cannulation in 1-wk-old rat neonates. Isometric muscle contraction was obtained as follows. A leg was immobilized by fixation of the knee and foot to the polystyrene board. A gastrocnemius muscle and its innervation were exposed in s i b . The corresponding sciatic nerve was cut centrally. The gastrocnemius tendon was detached from its insertion and connected to a force transducer (model FT-3, Grass Instruments Co., Quincy, Mass.). The output from the transducer was amplified and recorded (transducer amplifier model 13-4218 and recorder model 220, Gould, Cleveland, Ohio). The exposed tissue was protected with paraffin oil and the preparation temperature thermostatically held at 37°C by radiant heat. Indirect muscle contractions were evoked by supramaximal single stimuli applied to the sciatic nerve through a bipolar platinum electrode from a stimulator (model S-88, Grass Instruments Co.) via an isolation unit. For direct muscle stimulation, needle electrodes were inserted into the gastrocnemius muscle heads, and neural transmission was blocked by dTc. Both indirect and direct stimuli consisted of 0.1-ms square-wave pulses delivered every 4 s . Tetanic stimuli of 5 4 0 0 Hz were delivered for 30 s . To record muscle fasciculations, the output from the transducer was amplified 10 times that for recording normal twitch tension. Nerve stimulation was discontinued when recording muscle fasciculations. Fasciculations, when these occurred, were observed in exposed muscle and in the intact animal. Their incidence was recorded with regard to age at the physostigmine doses shown in Table 1. To record motor nerve ending" activity, thoracic and lumbar laminectomies exposed the S-1 and L-6 ventral roots; these contain gastrocnemius motor axons. In these experiments, the sciatic nerve was left *Motor nerve endings include the distal Ranvier node, the myelinated axon leading to the unmyelinated nerve terminals, and the unmyelinated terminals.
1992;75361-71
Table 1. Percent Incidence of Physostigmine-Induced Fasciculation in Neonatal and Adult Rats Dose" (mgikg SC) 0.5 1.0
2.0 5.0 10.0
Postnatal week Week2
Week3
Week4
0 0 0
0 0
50.0 72.7 75.0 100.0 100.0
0 0
12.5 27.2 40.0
Week5
Adult
100.0
100.0 100.0 100.0 -
100.0
100.0 -
-
SC, subcutaneous. '11 > 5 for each subcutaneous dose.
intact. The S-1 and L-6 roots were disconnected at their origin from the spinal cord and separated into small filaments. A reactive filament was placed on bipolar platinum recording electrodes leading to differential preamplifiers (model AM 502, Tektronix, Beaverton, Ore.) and displayed on a storage oscilloscope (model 4072, Gould). Thus, prejunctional actions of gastrocnemius motor nerve terminals were monitored antidromically, as detailed by other studies from this laboratory (20-23). Compound action potentials of gastrocnemius muscle, elicited by motor nerve stimulation, were recorded by bipolar platinum microelectrodes. The output was amplified through a differential preamplifier (model AM 502, Tektronix) and displayed simultaneously with the nerve action potentials on the storage oscilloscope described. The drugs administered IA were edrophonium chloride, neostigmine bromide, dTc chloride, and SCh chloride; physostigmine sulfate was administered subcutaneously (SC). Atropine sulfate (2 mgkg IP) was used to prevent muscarinic effects. All drugs were purchased from Sigma Chemical Co. (St. Louis, Mo.), and the solutions were prepared in 0.9% NaC1. Concentrations of each drug, with the exception of dTc and SCh, were prepared to limit injection volumes to 25 pL/lO g body wt for weights 40 g. The dTc was prepared in 200- or 4OO-pg/mL concentrations; SCh was prepared in 1- or 2-mg/mL concentrations. The dTc and SCh were infused through a carotid artery at 15-20 pL/min with a microinjection pump (model CMA/100, Bioanalytical Systems, Lafayette, Ind.), and dTc or SCh was infused until an 80%-95% neuromuscular block resulted. The dTc or SCh doses required for this were calculated from the total volumes injected. During the dTc or SCh experiments, respiration was maintained by a rodent ventilator (model 683, Harvard Apparatus and Instruments, South Natick, Mass.). At the end of all experiments, the animals were killed with sodium pentobarbital (>50 mg/kg IP).
A.
PHY8OBTIGNINE ¶
Figure 1. Physostigmine (A) and edrophonium (Bleffects in a 2-wk-old rat neonate and an adult iai. Twitch potentiation and muscle fasciculation are absent in the rat neonate but are welldeveloped effects in the adult rat. In the druginduced potentiations, the sciatic nerve was supramaximally stimulated at 0.25 Hz. Physostigmine and edrophonium injections are indicated by urrows. Muscle fasciculations were recorded in the absence of nerve stimulation and at a gain five times that used to record twitch tension. sc, subcutaneous; ia, intraarterial.
363
PEDIATRIC ANESTHESIA OKAMOTO ET AL. BLOCKING AND REVERSAL DRUGS O N RAT NEONATES
ANESTH ANALG 1992;75:361-71
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B. EDROPHONIUM
1o Adult
The neuromuscular blocking potency of dTc was studied in rat neonates of various ages, utilizing phrenic nerve-hemidiaphragm preparations in vitro. The muscle with its nerve was isolated and mounted in a Lucite chamber. The preparation was then bathed in Krebs’ solution containing NaCl, 118 mM; KC1, 5 mM; CaCI,, 2.5 mM; NaHCO,, 30 mM; KH,PO,, 1 mM; MgS04, 1 mM; glucose, 11 mM. The pH was maintained at 7.4 when aerated with a mixture of 95% O2 and 5% CO, (vol/vol%). The temperature of the bath was maintained at 36°C. The dTc concentrations studied were 1 x and 1 x M. The degree of neuromuscular block was calculated at the peak plateau effect of dTc; twitch
d.
tensions were measured before and after dTc and expressed as a percent block of twitch. Statistical calculation for estimating group mean, standard error, and correlation coefficient was done by analysis of variance.
Results Zndirect Enhancement of Twitch Tension Indirectly evoked twitch responses to the anticholinesterase drugs physostigmine and edrophonium are illustrated in Figure 1. The enhancement of
364
ANESTH ANALG 1992;75361-71
PEDIATRIC ANESTllliSIA OKAMOTO ET AL. BLOCKING AND R€VERSAL DRUGS ON RAT NEONATES
A. EDROPHONIUM 140t
O
t
10 EDROPHONIUM DOSE @g/kg)
B. 350 z
O-
FZ
NEOSTIGMINE
4 1
300-
I
250Z O w u
g
p)
n Z
I$ 0 tb9
z-
200.150-
To determine whether the lack of potentiation in the 1-2-wk-old rat neonates is unique for these drugs, the nonanticholinesterase drug 4-AP was tested. Dose-twitch potentiation relationships, with respect to neonatal age, are plotted in Figure 2 for IA edrophonium, neostigmine, and 4AP. These dose-range studies disclosed an insignificant twitch potentiation by neostigmine and edrophonium in the 2-wk-old rat pups compared with 3-wk-old pups and adults. The results of neostigmine were similar to those for physostigmine and edrophonium (Figures 1 and 2). At 3 wk and beyond, neostigmine and physostigmine twitch potentiations significantly exceeded those for edrophonium. in contrast to the threshold responses to physostigmine, edrophonium, and neostigmine with respect to enhancement of twitch tension in the 1- and 2-wk-old rat neonates, the threshold response of IA 4-AP was positive. The dose-response curves for edrophonium, neostigmine, and 4-AP in Figure 2 are seen to be dose and age dependent.
100-
50L
Actions on Neonatal Motor Nerve Endings
0
5
10 -
100
NEOSTIGMINE DOSE (rng/kg)
C. 4-AMINOPYRIDINE
225
+
1
1
5 10 20 4-AMINOPYRIDINE DOSE (mg/kg)
2
I
Neostigmine action on neonatal rat motor nerve terminals was investigated in 3-wk-old rat neonates given a lOO-pg/kg dose IA. Figure 3 illustrates the repetitive action potentials recorded from ventral root fibers and a paired innervated muscle fiber after neostigmine dosing. As seen in Figure 3, repetitive motor nerve action potentials created a repetitive response in muscle. These events did not occur in 1and 2-wk-old rat neonates. In contrast to neostigmine, 4-AP in all 1- and 2-wk-old rat neonates tested caused both repetitive prejunctional firing (Figure 3) and significant twitch potentiation (Figure 2).
50
Figure 2. Dose- and age-related twitch potentiations plotted for intraarterial doses of edrophonium (A), neostigmine (B), and 4-aminopyridine (C). In all instances, sciatic nerve stimulation was supramaximal at 0.25 Hz. Maximal twitch potentiation (mean 2 SE) is calculated as percent potentiation above control twitch. Each point represents the average of n 2 7 experiments.
twitch tension (potentiation) caused by physostigmine, as seen in adult rats, was absent in 1- and 2-wk-oId rat pups. Subcutaneous physostigmine doses as large as 10 mgkg failed to produce potentiation in these rat neonates. By postnatal week 3, physostigmine potentiation appeared but was less than the response seen in the adult rat. The twitch potentiation evoked by edrophonium in the adult rats, like that of physostigmine, was absent in pups
