8
Brain Research, 579 (1992) 8-16 (~) 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92l$05.00
BRES 17658
Activation of 5-HTlc/2 receptors depresses polysynaptic reflexes and excitatory amino acid-induced motoneuron responses in frog spinal cord A.M. Holohean, J.C. Hackman, S.B. Shope and R.A. Davidoff Neurophysiology and Spinal Cord Pharmacology Laboratories, Veterans Affairs Medical Center, and the Department of Neurology, University of Miami School of Medicine, Miami, FL 33101 (USA) (Accepted 10 December 1991) Key words: Serotonin; Spinal cord; Frog; Motoneuron; Excitatory amino acid
Sucrose gap recordings from the ventral roots of isolated, hemisected frog spinal cords were used to evaluate the effects of high concentrations of serotonin (5-HT) and a-methyl-5-HT (a-Me-5-HT) on the changes in motoneuron potential produced by dorsal root stimulation and by excitatory amino acids and agonists. Bath application of 5-HT in concentrations of 10 pM or greater produced a concentration-dependent motoneuron depolarization. Polysynaptic ventral root potentials evoked by dorsal root stimuli were reduced in both amplitude and area by 5-HT or a-Me-5-HT (both 100/~M). This may result from a reduction of the postsynaptic sensitivity of motoneurons to excitatory amino acid transmitters because 5-HT significantly depressed motoneuron depolarizations produced by addition of L-glutamate and L-aspartate to the superfusate. Similarly, 5-HT reduced depolarizations produced by the excitatory amino acid agonists Nomethyl-o-aspartate (NMDA), quisqualate, a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA), and kainate, a-Me-5-HT reduced NMDA depolarizations. Tetrodotoxin (TTX) did not affect the ability of 5-HT to attenuate NMDA or kainate depolarizations, but did eliminate the 5-HTinduced attenuation of quisqualate and AMPA depolarizations. The glycine receptor site associated with the NMDA receptor did not appear to be affected by 5-HT because saturation of the site by excess glycine did not alter the 5-HT-induced depression of NMDA responses. The 5-HTIc~2 antagonist ketanserin and the 5-HTxAj2 antagonist spiperone significantly attenuated the 5-HT-induced depression of NMDA-depolarizations. The relatively selective 5-HT1A antagonist spiroxatrine and the selective 5-HT3 blocker MDL 72222 were without effect against the depressive effects of 5-HT. These observations support the notion that 5-HT putatively released from descending bulbospinal fibers can alter frog motoneuronal output. The data indicate that 5-HTlc/2 receptors affect the afferent input to motoneurons and do so by attenuation of NMDA receptor-mediated synaptic processes. INTRODUCTION In both m a m m a l s and amphibians large numbers of fibers containing serotonin (5-hydroxytryptamine, 5-HT) originate in the raphe nuclei of the brainstem and descend to the spinal cord to terminate throughout the spinal gray m a t t e r 1'3'4~. Fibers and terminals containing s e rotonin are present in the ventral horn 3°'39 where distinctive fascicles of 5-HT fibers a p p e a r to terminate near interneurons and m o t o n e u r o n s 2s. The precise action of the descending bulbospinal 5-HT fiber system upon spinal m o t o n e u r o n s and segmental reflexes is elusive. F o r example, direct application of 5-HT has been r e p o r t e d to have both depolarizing and hyperpolarizing effects on m a m m a l i a n and amphibian spinal m o t o n e u r o n s 22'35'4°'44. In addition, both depressive and facilitatory interactions between serotonin and L-glutamate, the excitatory amino acid neurotransmitter putatively mediating segmental spinal reflexes, have been de-
scribed, but the pharmacological identity of the 5-HT receptors responsible for the interactions is disputed (cf. refs. 27, 31, 36, 43, 45). F u r t h e r m o r e , 5-HT can activate multiple r e c e p t o r subtypes located on the same neurons 5 and radioligand binding studies have d e m o n s t r a t e d multiple subtypes of 5-HT binding sites in the spinal cord 7' 15,26,29,33.Presumably, many of the r e p o r t e d inconsistencies concerning the physiological actions of 5-HT on spinal m o t o n e u r o n s , reflexes, and on the postsynaptic actions of excitatory amino acids result from the activation of distinct subtypes of 5-HT receptors by different concentrations of 5-HT applied under diverse experimental conditions. In this regard, we have recently shown that 5-HT caused two distinctive changes in frog mot o n e u r o n m e m b r a n e potential: hyperpolarizations caused by a direct action of low concentrations of the amine which activated 5-HT1A receptors and depolarizations by high 5-HT concentrations which activated 5-HT 2 and/or 5-HT~c receptors on m o t o n e u r o n s and interneurons 22.
Correspondence: R.A. Davidoff, MD, Department of Neurology (D4-5), P.O. Box 016960, University of Miami School of Medicine, Miami, FL 33101, USA.
9 In the present study, we have investigated some of the interactions between specific 5-HT and excitatory amino acid receptors on spinal motoneurons in the isolated frog spinal cord. Some of the results have appeared in preliminary forln 9'16.
NMDA and quisqualate from Cambridge Research Biochemicals, AMPA from Tocris Neuramin, TTX from Calbiochem, and mianserin, spiperone, and spiroxatrine from Research Biochemicals Inc. Ketanserintartrate was generously donated by Janssen, a-methyl5-hydroxytryptamine (a-Me-5-HT) by Sandoz, and MDL 72222 by Merrell Dow.
MATERIALS AND METHODS
RESULTS
Experiments were performed on adult frogs (Rana pipiens) weighing 30-55 g. Frogs were deeply anesthetized by placing them on crushed ice until they ceased to respond to noxious stimuli. After decapitation and laminectomy, the spinal cord was removed with attached dorsal (DR) and ventral (VR) roots and hemisected saglttally. Half of a lumbar cord with separated DRs and VRs was placed in a sucrose gap chamber and continuously superfused at a rate of 10 ml/min with amphibian Ringer solution of the following composition (mM): NaCI 114, KC1 2.0, CaC12 1.9, NaHCO 3 10, and glucose 5.5. The Ringer solution was kept at pH 7.4 by gassing with 95% 02/5% CO2. The temperature of the Ringer solution was adjusted to 18°C by a thermoelectric cooling unit. Mg2+ was omitted from the superfusate to permit investigation of responses involving N-methyl-D-aspartate (NMDA) receptors. In some experiments, indirect responses were blocked by the inclusion of tetrodotoxin (TI'X) at a concentration (0.625/~M) sufficient to prevent interneuron discharges and action potential conduction in afferent fibers. The IXth ventral root was placed across a 3 mm sucrose gap to record the membrane potential of motoneurons electrotonically conducted along the population of axons contained in the root. Differential DC recordings were obtained between the spinal cord bath and the distal end of the ventral root with calomel electrodes connected via agar-Ringer bridges to each recording site. The preparation was left ungrounded. After amplification, the signals were displayed on an oscilloscope and recorded using a rectilinear pen writer. The fibers contained in the IXth dorsal root were stimulated with rectangular pulses delivered via biopolar silver/silver chloride electrodes. The ventral root potential changes evoked by DR stimulation were stored in an IBM AT computer. The integral (the area under the change in ventral root potential in mV.s) was determined by reprogrammed Asyst software and the potentials were plotted on a laser printer. Statistical analyses were carried out using Student's t-test. Results are expressed as mean + S.E.M. Excitatory amino acids and excitatory amino acid agonists were applied for 10 s at 15-20 min intervals in the following concentrations: L-glutamate (1.0 mM), L-aspartate (1.0 mM), NMDA (100 #M), quisqualate (10-30/~M), a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA, 10/~M), and kainate (30/~M). The 10 s applications of these concentrations of agonists at these intervals were shown by preliminary experimentation to produce submaximal responses without reduction of later responses. In a given cord all responses for both control and experimental conditions were replicated at least twice. The number (n) reflects the number of different cords used for each experiment, Most drugs were dissolved in Ringer solution shortly before use to minimize chemical degradation. However, spiperone, spiroxatrine, ketanserin, and MDL 72222 (laH,3a,5aH-tropan-3-yl-3dichlorobenzoate) were insoluble in distilled water or Ringer solution. Spiperone was dissolved in glacial acetic acid, spiroxatrine in ethanol and 1 N HCI, ketanserin in 1 N HC1, and MDL 72222 in ethanol. These solutions could then be diluted with normal Ringer solution without causing precipitation. The pH was adjusted when necessary. Control solutions containing solvent alone were tested and produced no change in motoneuron membrane potential. Solution changes were accomplished by means of an electronicallytimed solenoid valve assembly which rapidly admitted different solutions to the chamber containing the cord. In all experiments drugs were administered at known concentrations to the bath. 5-HT creatinine sulfate and kainate were purchased from Sigma,
In the frog spinal cord polysynaptic potentials (ven-
tral root potentials, VRPs) produced in m o t o n e u r o n s and electronically conducted along the V R are generated by submaximal stimulation of the D R which excites only
Aa and Aft fibers. Larger polysynaptic VRPs with short latency monosynaptic components in some spinal cords are evoked by supramaximal stimuli which excite both
A and C fibers (Fig. 1). As seen in Fig. 1A, application of low concentrations of 5-HT (0.1 to 1.0 #M) facilitated the V R P 9. The facilitatory effects of 5-HT are the subject of a separate communication. In contrast, application of higher concentrations ( > 1 0 / t M ) significantly at-
tenuated both the amplitude and the duration of the slow polysynaptic c o m p o n e n t s of VRPs generated by su-
pramaximal stimulation of the DR and reduced the same V R P components elicited by submaximal stimuli in all spinal cords (Table I) (cf. refs. 24, 38, 42). The depressive effects of 5-HT were d e p e n d e n t u p o n the concen-
tration of the amine the medium and maximum effects were seen after application of 100/~M 5-HT (Fig. 1). For
this reason, a 100/~M concentration of 5-HT was used in most of the experiments described below. Effects were noticeable within a few min after exposure of the
spinal cord to the amine. The reduction in reflex responses was unaccompanied by any change in the latency of the VRP. The effects of 5-HT were reversible when the cord was returned to control Ringer solution for more than 30 min. In agreement with our previous results 22, bath application of 5-HT in concentrations of 10 /zM or greater caused a dose-dependent depolarization of the memb r a n e potential of m o t o n e u r o n s electronically conducted down the ventral root (Fig. 2A). In the present investi-
gations, the mean amplitude of the depolarizations produced by 100/~M 5-HT was 1.72 + 0.22 mV (n = 17, 60 s applications). The 5-HT depolarizations were pro-
longed in duration (up to 5 min) and reversible, a-Me5-HT (100/~M) elicited similar depolarizations (Fig. 2A). In contrast, low concentrations of 5-HT ( < 1 0 / ~ M ) hyperpolarized m o t o n e u r o n s (not illustrated) (see ref. 22). In m a n y spinal cords, the 5-HT-induced depolarization caused by high concentrations of the amine was preceded by a small hyperpolarization (Fig. 2A) (cf. ref. 22).
10
A'control
0.1uM, 5-HT
1.0,uM
.j
_
10uM
30~M
B. Control
100~M
+ a -methyl-5-HT
2s
0.3 s
Fig. 1. Low concentrations of 5-HT facilitate and high concentrations of 5-HT depress ventral root potentials (VRPs). Traces are sucrose gap recordings from the IXth VR of VRPs evoked by single supramaximal shocks applied to the afferent fibers in dorsal root (DR). A: control VRP recorded in normal Ringer solution. Other traces are VRPs recorded during exposure to increasing concentrations of 5-HT (0.1 to 100 /aM). The cord was exposed to the various concentrations of 5-HT for at least 10 min. B: control VRP recorded in normal Ringer solution and VRP recorded during exposure to a-Me-5-HT (100/aM, 10 min). In these, and in all subsequent records, negativity is indicated by an upward pen deflection and signifies a depolarization of motoneurons whose axons exit from the cord in the IXth VR. A and B are from experiments performed in separate spinal cords.
Motoneuron depolarizations produced by activation o f excitatory amino acid receptors It is possible that high concentrations of 5-HT inhibit spinal reflexes by reducing the sensitivity of postsynaptic neuronal m e m b r a n e s to endogenously released excitatory amino acid transmitters. A s seen in Fig. 3, 100/~M 5-HT significantly depressed m o t o n e u r o n depolarizations caused by applications of both L-glutamate (1.0 m M , 10 s) and L-aspartate (1.0 m M , 10 s) in normal Ringer so-
TABLE I
lution (Table II). F u r t h e r m o r e , the depolarizations produced by the excitatory amino acid agonists N M D A (100 /~M), A M P A (10 # M ) , quisqualate (30/~M), and kainate (30 ktM) applied in normal Ringer solution (10 s) were significantly reduced by 5-HT (100 /~M) (Fig. 4, Table II). A l l effects of 5-HT on excitatory amino acidand excitatory amino acid agonist depolarizations were reversed after washing with control Ringer solution for m o r e than 30 min. The 5-HT-induced depolarization of m o t o n e u r o n s was not responsible for the amine-induced depression of eXcitatory amino acid agonist effects because depolariza-
Effects of 5-HT and a-Me-5-HT on VRPs evoked by sciatic nerve volleys
tion (2.5 mV) of m o t o n e u r o n s by elevation of [K+]o (2.4 mM) did not mimic the depressive effects of high con-
Control values are integrals of VRPs (mean + S.E.M.) expressed in mV.s. Values in 5-HT and a-Me-5-HT are expressed as % of control VRPs (mean + S.E.M.). Numbers of preparations tested are in parentheses. 5-HT and a-Me-5-HT applied for 10-20 rain.
centrations of 5-HT ( n - - 2 ) . F u r t h e r m o r e , addition of a saturating concentration of glycine (5/~M) to the Ringer solution a u g m e n t e d N M D A - i n d u c e d m o t o n e u r o n depo-
Fibers stimulated Aa + Aft
A + C
larizations, but it did not affect the ability of 100 p M 5-HT to depress N M D A responses (n = 2; not illustrated).
Control VRPs 7.6 _+ 0.8 mV.s (17) 10.6 + 0.9 mV.s (19) 5-HT 100/aM 85 + 10% (10) 73 + 9% (11)* a-Me-5-HT 100/aM 64 + 5% (4)** 78 + 3 (4)**
Direct and indirect effects o f 5 - H T The effects of 5-HT on excitatory amino acid agonistinduced responses could be caused by a direct postsynaptic action of the amine on m o t o n e u r o n m e m b r a n e s
Significantly different from control responses: *P < 0.5, **P < 0.01.
and/or by an indirect action involving interneurons. The presence of T T X in the Ringer solution did not affect
A,
11
B. 5-HT
+TTX
i
a -METHYL-5-HT
+TTX
,=,
2min
Fig. 2. Alterations in motoneuron membrane potential produced by 5-HT and a-Me-5-HT in normal and TTX-containing medium within the same cord. A: sucrose gap recordings from the VR of depolarizing changes in motoneuron potential caused by 5-HT (100/~M, 60 s application) and by a-Me-5-HT (100 ~M, 60 s) in normal Ringer solution. Note small hyperpolarization preceding 5-HT-induced depolarization. B: repeat applications of 5-HT and a-Me-5-HT in Ringer solution containing TFX (0.625/tM). The upward 'spike-like' deflections of the baseline represent spontaneous VRPs. Applications of 5-HT and a-Me-5-HT are indicated by bars below the baseline.
the ability of 100/tM 5-HT to attenuate motoneuron depolarizations produced by addition of NMDA (100/~M, 10 s) or kainate (30/~M, 10 s) to the medium. In contrast, the 5-HT-induced attenuation of depolarizations
A GLUTAMATE
t .
~
~,
caused by AMPA (10/~M, 10 s) or quisqualate (30/~M, 10 s) (Fig. 5, Table II) was eliminated in medium containing TTX (cf. ref. 22). Furthermore, motoneuron depolarizations evoked by 5-HT were substantially diminished by TTX (Fig. 2B).
B. +5-HT
5-HT and after-hyperpolarizations 5-HT increased the amplitude of the after-hyperpolarizations (AHPs) that followed the depolarizations evoked
l
TABLE II
.._.~
|
• ~
•
ASPARTATE
+5-HT
&
Effects of 100 I~M 5-HT on motoneuron depolarizations produced by excitatory amino acids and excitatory amino acid agonists
.
~
All applications of excitatory amino acids or excitatory amino acid agonists were 10 s in duration. Values in 5-HT are expressed as percentages of control motoneuron depolarizations (mean + S.E.M.) produced by excitatory amino acids or excitatory amino acid agonists before exposure to 5-HT (100 /~M, ~
2.5V
2min Fig. 3.5-HT depresses motoneuron depolarizations induced by excitatory amino acids. A: changes in motoneuron potential produced by L-glutamate (1.0 mM, 10 s) and L-aspartate (1.0 mM, 10 s) in normal Ringer solution. B: reduction of L-glutamate- and L-aspartate-induced depolarizations by 5-HT (100 gtM, 20 min). The applications of the excitatory amino acids are indicated by arrows below the~baseline. The L-glutamate and L-aspartate traces are from experiments performed in separate spinal cords.
tested. 25-45 min). Numbers in parentheses Motoneuron Normal are Ringer numbers depolarizations ofTTX-Ringer preparations
L-Glutamate (1 mM) L-Aspartate (1 mM) NMDA (100/~M) AMPA (10/~M) Quisqualate (30/tM) Kainate (10/zM)
80 82 81 76 80 80
_+ 4 + 5 + 3 _+ 3 + 4 _+ 3
(5)* (5)* (3)* (6)** (4)** (3)*
79 105 101 88
+ 7 (4)* _+ 11 (3) + 8 (4) + 3 (5)*
Significantly different from control responses: *P < 0.05, **P
Brain Research, 579 (1992) 8-16 (~) 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92l$05.00
BRES 17658
Activation of 5-HTlc/2 receptors depresses polysynaptic reflexes and excitatory amino acid-induced motoneuron responses in frog spinal cord A.M. Holohean, J.C. Hackman, S.B. Shope and R.A. Davidoff Neurophysiology and Spinal Cord Pharmacology Laboratories, Veterans Affairs Medical Center, and the Department of Neurology, University of Miami School of Medicine, Miami, FL 33101 (USA) (Accepted 10 December 1991) Key words: Serotonin; Spinal cord; Frog; Motoneuron; Excitatory amino acid
Sucrose gap recordings from the ventral roots of isolated, hemisected frog spinal cords were used to evaluate the effects of high concentrations of serotonin (5-HT) and a-methyl-5-HT (a-Me-5-HT) on the changes in motoneuron potential produced by dorsal root stimulation and by excitatory amino acids and agonists. Bath application of 5-HT in concentrations of 10 pM or greater produced a concentration-dependent motoneuron depolarization. Polysynaptic ventral root potentials evoked by dorsal root stimuli were reduced in both amplitude and area by 5-HT or a-Me-5-HT (both 100/~M). This may result from a reduction of the postsynaptic sensitivity of motoneurons to excitatory amino acid transmitters because 5-HT significantly depressed motoneuron depolarizations produced by addition of L-glutamate and L-aspartate to the superfusate. Similarly, 5-HT reduced depolarizations produced by the excitatory amino acid agonists Nomethyl-o-aspartate (NMDA), quisqualate, a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA), and kainate, a-Me-5-HT reduced NMDA depolarizations. Tetrodotoxin (TTX) did not affect the ability of 5-HT to attenuate NMDA or kainate depolarizations, but did eliminate the 5-HTinduced attenuation of quisqualate and AMPA depolarizations. The glycine receptor site associated with the NMDA receptor did not appear to be affected by 5-HT because saturation of the site by excess glycine did not alter the 5-HT-induced depression of NMDA responses. The 5-HTIc~2 antagonist ketanserin and the 5-HTxAj2 antagonist spiperone significantly attenuated the 5-HT-induced depression of NMDA-depolarizations. The relatively selective 5-HT1A antagonist spiroxatrine and the selective 5-HT3 blocker MDL 72222 were without effect against the depressive effects of 5-HT. These observations support the notion that 5-HT putatively released from descending bulbospinal fibers can alter frog motoneuronal output. The data indicate that 5-HTlc/2 receptors affect the afferent input to motoneurons and do so by attenuation of NMDA receptor-mediated synaptic processes. INTRODUCTION In both m a m m a l s and amphibians large numbers of fibers containing serotonin (5-hydroxytryptamine, 5-HT) originate in the raphe nuclei of the brainstem and descend to the spinal cord to terminate throughout the spinal gray m a t t e r 1'3'4~. Fibers and terminals containing s e rotonin are present in the ventral horn 3°'39 where distinctive fascicles of 5-HT fibers a p p e a r to terminate near interneurons and m o t o n e u r o n s 2s. The precise action of the descending bulbospinal 5-HT fiber system upon spinal m o t o n e u r o n s and segmental reflexes is elusive. F o r example, direct application of 5-HT has been r e p o r t e d to have both depolarizing and hyperpolarizing effects on m a m m a l i a n and amphibian spinal m o t o n e u r o n s 22'35'4°'44. In addition, both depressive and facilitatory interactions between serotonin and L-glutamate, the excitatory amino acid neurotransmitter putatively mediating segmental spinal reflexes, have been de-
scribed, but the pharmacological identity of the 5-HT receptors responsible for the interactions is disputed (cf. refs. 27, 31, 36, 43, 45). F u r t h e r m o r e , 5-HT can activate multiple r e c e p t o r subtypes located on the same neurons 5 and radioligand binding studies have d e m o n s t r a t e d multiple subtypes of 5-HT binding sites in the spinal cord 7' 15,26,29,33.Presumably, many of the r e p o r t e d inconsistencies concerning the physiological actions of 5-HT on spinal m o t o n e u r o n s , reflexes, and on the postsynaptic actions of excitatory amino acids result from the activation of distinct subtypes of 5-HT receptors by different concentrations of 5-HT applied under diverse experimental conditions. In this regard, we have recently shown that 5-HT caused two distinctive changes in frog mot o n e u r o n m e m b r a n e potential: hyperpolarizations caused by a direct action of low concentrations of the amine which activated 5-HT1A receptors and depolarizations by high 5-HT concentrations which activated 5-HT 2 and/or 5-HT~c receptors on m o t o n e u r o n s and interneurons 22.
Correspondence: R.A. Davidoff, MD, Department of Neurology (D4-5), P.O. Box 016960, University of Miami School of Medicine, Miami, FL 33101, USA.
9 In the present study, we have investigated some of the interactions between specific 5-HT and excitatory amino acid receptors on spinal motoneurons in the isolated frog spinal cord. Some of the results have appeared in preliminary forln 9'16.
NMDA and quisqualate from Cambridge Research Biochemicals, AMPA from Tocris Neuramin, TTX from Calbiochem, and mianserin, spiperone, and spiroxatrine from Research Biochemicals Inc. Ketanserintartrate was generously donated by Janssen, a-methyl5-hydroxytryptamine (a-Me-5-HT) by Sandoz, and MDL 72222 by Merrell Dow.
MATERIALS AND METHODS
RESULTS
Experiments were performed on adult frogs (Rana pipiens) weighing 30-55 g. Frogs were deeply anesthetized by placing them on crushed ice until they ceased to respond to noxious stimuli. After decapitation and laminectomy, the spinal cord was removed with attached dorsal (DR) and ventral (VR) roots and hemisected saglttally. Half of a lumbar cord with separated DRs and VRs was placed in a sucrose gap chamber and continuously superfused at a rate of 10 ml/min with amphibian Ringer solution of the following composition (mM): NaCI 114, KC1 2.0, CaC12 1.9, NaHCO 3 10, and glucose 5.5. The Ringer solution was kept at pH 7.4 by gassing with 95% 02/5% CO2. The temperature of the Ringer solution was adjusted to 18°C by a thermoelectric cooling unit. Mg2+ was omitted from the superfusate to permit investigation of responses involving N-methyl-D-aspartate (NMDA) receptors. In some experiments, indirect responses were blocked by the inclusion of tetrodotoxin (TI'X) at a concentration (0.625/~M) sufficient to prevent interneuron discharges and action potential conduction in afferent fibers. The IXth ventral root was placed across a 3 mm sucrose gap to record the membrane potential of motoneurons electrotonically conducted along the population of axons contained in the root. Differential DC recordings were obtained between the spinal cord bath and the distal end of the ventral root with calomel electrodes connected via agar-Ringer bridges to each recording site. The preparation was left ungrounded. After amplification, the signals were displayed on an oscilloscope and recorded using a rectilinear pen writer. The fibers contained in the IXth dorsal root were stimulated with rectangular pulses delivered via biopolar silver/silver chloride electrodes. The ventral root potential changes evoked by DR stimulation were stored in an IBM AT computer. The integral (the area under the change in ventral root potential in mV.s) was determined by reprogrammed Asyst software and the potentials were plotted on a laser printer. Statistical analyses were carried out using Student's t-test. Results are expressed as mean + S.E.M. Excitatory amino acids and excitatory amino acid agonists were applied for 10 s at 15-20 min intervals in the following concentrations: L-glutamate (1.0 mM), L-aspartate (1.0 mM), NMDA (100 #M), quisqualate (10-30/~M), a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA, 10/~M), and kainate (30/~M). The 10 s applications of these concentrations of agonists at these intervals were shown by preliminary experimentation to produce submaximal responses without reduction of later responses. In a given cord all responses for both control and experimental conditions were replicated at least twice. The number (n) reflects the number of different cords used for each experiment, Most drugs were dissolved in Ringer solution shortly before use to minimize chemical degradation. However, spiperone, spiroxatrine, ketanserin, and MDL 72222 (laH,3a,5aH-tropan-3-yl-3dichlorobenzoate) were insoluble in distilled water or Ringer solution. Spiperone was dissolved in glacial acetic acid, spiroxatrine in ethanol and 1 N HCI, ketanserin in 1 N HC1, and MDL 72222 in ethanol. These solutions could then be diluted with normal Ringer solution without causing precipitation. The pH was adjusted when necessary. Control solutions containing solvent alone were tested and produced no change in motoneuron membrane potential. Solution changes were accomplished by means of an electronicallytimed solenoid valve assembly which rapidly admitted different solutions to the chamber containing the cord. In all experiments drugs were administered at known concentrations to the bath. 5-HT creatinine sulfate and kainate were purchased from Sigma,
In the frog spinal cord polysynaptic potentials (ven-
tral root potentials, VRPs) produced in m o t o n e u r o n s and electronically conducted along the V R are generated by submaximal stimulation of the D R which excites only
Aa and Aft fibers. Larger polysynaptic VRPs with short latency monosynaptic components in some spinal cords are evoked by supramaximal stimuli which excite both
A and C fibers (Fig. 1). As seen in Fig. 1A, application of low concentrations of 5-HT (0.1 to 1.0 #M) facilitated the V R P 9. The facilitatory effects of 5-HT are the subject of a separate communication. In contrast, application of higher concentrations ( > 1 0 / t M ) significantly at-
tenuated both the amplitude and the duration of the slow polysynaptic c o m p o n e n t s of VRPs generated by su-
pramaximal stimulation of the DR and reduced the same V R P components elicited by submaximal stimuli in all spinal cords (Table I) (cf. refs. 24, 38, 42). The depressive effects of 5-HT were d e p e n d e n t u p o n the concen-
tration of the amine the medium and maximum effects were seen after application of 100/~M 5-HT (Fig. 1). For
this reason, a 100/~M concentration of 5-HT was used in most of the experiments described below. Effects were noticeable within a few min after exposure of the
spinal cord to the amine. The reduction in reflex responses was unaccompanied by any change in the latency of the VRP. The effects of 5-HT were reversible when the cord was returned to control Ringer solution for more than 30 min. In agreement with our previous results 22, bath application of 5-HT in concentrations of 10 /zM or greater caused a dose-dependent depolarization of the memb r a n e potential of m o t o n e u r o n s electronically conducted down the ventral root (Fig. 2A). In the present investi-
gations, the mean amplitude of the depolarizations produced by 100/~M 5-HT was 1.72 + 0.22 mV (n = 17, 60 s applications). The 5-HT depolarizations were pro-
longed in duration (up to 5 min) and reversible, a-Me5-HT (100/~M) elicited similar depolarizations (Fig. 2A). In contrast, low concentrations of 5-HT ( < 1 0 / ~ M ) hyperpolarized m o t o n e u r o n s (not illustrated) (see ref. 22). In m a n y spinal cords, the 5-HT-induced depolarization caused by high concentrations of the amine was preceded by a small hyperpolarization (Fig. 2A) (cf. ref. 22).
10
A'control
0.1uM, 5-HT
1.0,uM
.j
_
10uM
30~M
B. Control
100~M
+ a -methyl-5-HT
2s
0.3 s
Fig. 1. Low concentrations of 5-HT facilitate and high concentrations of 5-HT depress ventral root potentials (VRPs). Traces are sucrose gap recordings from the IXth VR of VRPs evoked by single supramaximal shocks applied to the afferent fibers in dorsal root (DR). A: control VRP recorded in normal Ringer solution. Other traces are VRPs recorded during exposure to increasing concentrations of 5-HT (0.1 to 100 /aM). The cord was exposed to the various concentrations of 5-HT for at least 10 min. B: control VRP recorded in normal Ringer solution and VRP recorded during exposure to a-Me-5-HT (100/aM, 10 min). In these, and in all subsequent records, negativity is indicated by an upward pen deflection and signifies a depolarization of motoneurons whose axons exit from the cord in the IXth VR. A and B are from experiments performed in separate spinal cords.
Motoneuron depolarizations produced by activation o f excitatory amino acid receptors It is possible that high concentrations of 5-HT inhibit spinal reflexes by reducing the sensitivity of postsynaptic neuronal m e m b r a n e s to endogenously released excitatory amino acid transmitters. A s seen in Fig. 3, 100/~M 5-HT significantly depressed m o t o n e u r o n depolarizations caused by applications of both L-glutamate (1.0 m M , 10 s) and L-aspartate (1.0 m M , 10 s) in normal Ringer so-
TABLE I
lution (Table II). F u r t h e r m o r e , the depolarizations produced by the excitatory amino acid agonists N M D A (100 /~M), A M P A (10 # M ) , quisqualate (30/~M), and kainate (30 ktM) applied in normal Ringer solution (10 s) were significantly reduced by 5-HT (100 /~M) (Fig. 4, Table II). A l l effects of 5-HT on excitatory amino acidand excitatory amino acid agonist depolarizations were reversed after washing with control Ringer solution for m o r e than 30 min. The 5-HT-induced depolarization of m o t o n e u r o n s was not responsible for the amine-induced depression of eXcitatory amino acid agonist effects because depolariza-
Effects of 5-HT and a-Me-5-HT on VRPs evoked by sciatic nerve volleys
tion (2.5 mV) of m o t o n e u r o n s by elevation of [K+]o (2.4 mM) did not mimic the depressive effects of high con-
Control values are integrals of VRPs (mean + S.E.M.) expressed in mV.s. Values in 5-HT and a-Me-5-HT are expressed as % of control VRPs (mean + S.E.M.). Numbers of preparations tested are in parentheses. 5-HT and a-Me-5-HT applied for 10-20 rain.
centrations of 5-HT ( n - - 2 ) . F u r t h e r m o r e , addition of a saturating concentration of glycine (5/~M) to the Ringer solution a u g m e n t e d N M D A - i n d u c e d m o t o n e u r o n depo-
Fibers stimulated Aa + Aft
A + C
larizations, but it did not affect the ability of 100 p M 5-HT to depress N M D A responses (n = 2; not illustrated).
Control VRPs 7.6 _+ 0.8 mV.s (17) 10.6 + 0.9 mV.s (19) 5-HT 100/aM 85 + 10% (10) 73 + 9% (11)* a-Me-5-HT 100/aM 64 + 5% (4)** 78 + 3 (4)**
Direct and indirect effects o f 5 - H T The effects of 5-HT on excitatory amino acid agonistinduced responses could be caused by a direct postsynaptic action of the amine on m o t o n e u r o n m e m b r a n e s
Significantly different from control responses: *P < 0.5, **P < 0.01.
and/or by an indirect action involving interneurons. The presence of T T X in the Ringer solution did not affect
A,
11
B. 5-HT
+TTX
i
a -METHYL-5-HT
+TTX
,=,
2min
Fig. 2. Alterations in motoneuron membrane potential produced by 5-HT and a-Me-5-HT in normal and TTX-containing medium within the same cord. A: sucrose gap recordings from the VR of depolarizing changes in motoneuron potential caused by 5-HT (100/~M, 60 s application) and by a-Me-5-HT (100 ~M, 60 s) in normal Ringer solution. Note small hyperpolarization preceding 5-HT-induced depolarization. B: repeat applications of 5-HT and a-Me-5-HT in Ringer solution containing TFX (0.625/tM). The upward 'spike-like' deflections of the baseline represent spontaneous VRPs. Applications of 5-HT and a-Me-5-HT are indicated by bars below the baseline.
the ability of 100/tM 5-HT to attenuate motoneuron depolarizations produced by addition of NMDA (100/~M, 10 s) or kainate (30/~M, 10 s) to the medium. In contrast, the 5-HT-induced attenuation of depolarizations
A GLUTAMATE
t .
~
~,
caused by AMPA (10/~M, 10 s) or quisqualate (30/~M, 10 s) (Fig. 5, Table II) was eliminated in medium containing TTX (cf. ref. 22). Furthermore, motoneuron depolarizations evoked by 5-HT were substantially diminished by TTX (Fig. 2B).
B. +5-HT
5-HT and after-hyperpolarizations 5-HT increased the amplitude of the after-hyperpolarizations (AHPs) that followed the depolarizations evoked
l
TABLE II
.._.~
|
• ~
•
ASPARTATE
+5-HT
&
Effects of 100 I~M 5-HT on motoneuron depolarizations produced by excitatory amino acids and excitatory amino acid agonists
.
~
All applications of excitatory amino acids or excitatory amino acid agonists were 10 s in duration. Values in 5-HT are expressed as percentages of control motoneuron depolarizations (mean + S.E.M.) produced by excitatory amino acids or excitatory amino acid agonists before exposure to 5-HT (100 /~M, ~
2.5V
2min Fig. 3.5-HT depresses motoneuron depolarizations induced by excitatory amino acids. A: changes in motoneuron potential produced by L-glutamate (1.0 mM, 10 s) and L-aspartate (1.0 mM, 10 s) in normal Ringer solution. B: reduction of L-glutamate- and L-aspartate-induced depolarizations by 5-HT (100 gtM, 20 min). The applications of the excitatory amino acids are indicated by arrows below the~baseline. The L-glutamate and L-aspartate traces are from experiments performed in separate spinal cords.
tested. 25-45 min). Numbers in parentheses Motoneuron Normal are Ringer numbers depolarizations ofTTX-Ringer preparations
L-Glutamate (1 mM) L-Aspartate (1 mM) NMDA (100/~M) AMPA (10/~M) Quisqualate (30/tM) Kainate (10/zM)
80 82 81 76 80 80
_+ 4 + 5 + 3 _+ 3 + 4 _+ 3
(5)* (5)* (3)* (6)** (4)** (3)*
79 105 101 88
+ 7 (4)* _+ 11 (3) + 8 (4) + 3 (5)*
Significantly different from control responses: *P < 0.05, **P
