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Effects of substance P analogues on spinal dorsal horn neurons
Peptides, Vol. 9, pp. 651-660. ©PergamonPress plc, 1988. Printedin the U.S.A.
0196-9781/88$3.00 + .00
Effects of Substance P Analogues on Spinal Dorsal Horn Neurons M. R A N D I C , * S. J E F T I N I J A , ? L. U R B A N , *x C. R A S P A N T I N I * A N D K. F O L K E R S ~
Department of *Veterinary Physiology and Pharmacology and tDepartment of Veterinary Anatomy Iowa State University, Ames, IA 50011 and ~Institute of Biomedical Research the University of Texas, Austin, TX 78712 R e c e i v e d 11 A u g u s t 1987 RANDIC, M., S. JEFTINIJA, L. URBAN, C. RASPANTINI AND K. FOLKERS. Effects of substance P analogues on spinal dorsal horn neurons. PEPTIDES 9(3) 651-660, 1988.--The effects of iontophoretically applied (D-Proz, D-Phe7, D-Trpg)-SP and (D-Pro2, D-TrpT.9)-SPon the spontaneous and evoked activity of functionally identified cat spinal dorsal horn neurons have been investigated in vivo by means of extracellular single unit recording technique. In addition, the rat spinal cord slice preparation has been used to study the actions of (D-Proz, D-Trp7'9)-SPand (D-Argl, D-Pro2, D-Trp7,9, Leu~)-SP on the resting membrane potential of dorsal horn neurons and also on their responses to dorsal root stimulation and exogenous SP application. We have observed that both (D-Pro2, D-Phe7, D-Trpa)-SP and (D-Pro2, D-TrpT'9)-SP produced an excitation of about 15% of all neurons tested and had a weak antagonistic effect against SP in the cat spinal cord. (D-Pro2, D-TrpT.9)-SPsuppressed the SP-induced excitation in 63% of examined cells. In addition, depression of the glutamate-induced excitation and spontaneous activity was evident in 10% and 19% of the cat dorsal horn neurons tested, respectively. In the spinal cord slice preparation (D-Arg~, D-Pro2, D-Trp7,9, Leu~a)-SP proved to be a more potent antagonist of the SP-induced depolarization and the dorsal root-elicited slow depolarization, if compared with (D-Pro2, D-TrpT.a)-SP. Putative substance P antagonists
Spinal dorsal horn neurons
SEVERAL structural analogues of substance P (SP) containing D-amino acids substitutions in position 2 (Pro), 7 (Phe or Trp) and 9 (Trp), and having antagonistic properties against synthetic SP, have been developed [2, 6, 32]. Two most studied analogues, i.e., (D-Pro 2, D-TrpT.9)-SP and (DArg 1, D-Pro 2, D-Trp ~,9, Leul~)-SP have been claimed to be competitive antagonists of SP in different peripheral organs [4, 15, 16, 19, 31] and also in mammalian central nervous system [1, 5, 8, 25, 30, 33, 39]. However, the major disadvantage of the three SP analogues is that besides exerting weak antagonistic properties they also possess agonistic activity [19] a~id some nonspecific local anesthetic effects [12, 23, 24, 30]. Our previous work showed that SP has an excitatory action on cat dorsal horn neurons in vivo and depolarizes the membrane of rat dorsal horn neurons in vitro [20, 21, 26]. In addition, we have reported that high intensity repetitive stimulation of a lumbar dorsal root elicits a slow depolarizing potential in more than half of rat spinal dorsal horn neurons. The experimental evidence obtained led us to suggest that SP, or a SP-related peptide, may in some manner be involved in the generation of this response [27, 28, 35, 36]. The present study was undertaken in order to examine the effects of SP analogues on the SP-induced excitation of the cat dorsal
Cat spinal cord
Rat spinal cord slice
horn neurons in vivo and the rat dorsal horn neurons in vitro, and on the slow excitatory transmission in the rat spinal dorsal horn. Preliminary results have been communicated [29]. METHOD
Cat Dorsal Horn Neurons Studied In Vivo Experiments were performed on adult cats of either sex weighing between 2 and 4 kg. Animals were initially anesthetized by halothane. The brain was anemically destroyed by bilateral occlusion of the common carotid and vertebral arteries. The spinal cord was transected at the first cervical level. Thereafter, the animal was artificially respired and immobilized by constant infusion of succinylcholine chloride. Body temperature, blood pressure and end-tidal CO2 concentration were monitored and maintained at optimal levels throughout the experiment. The spinal cord was exposed by laminectomy at the lumbosacral and caudal spinal levels. High intensity mechanical stimuli were delivered to the skin of the tail or left hindlimb (e.g., pressure from a sharply pointed object, grasping a fold of skin with a calibrated forceps). Low intensity mechanical stimuli (touching by resting a finger lightly on the skin, brushing the skin with a camel-hair brush) were also used. The spontaneous and
1Permanent address: Department of Anatomy, University Medical School, Debrecen H-4012, Hungary.
651
R A N D I C ET AL.
652 evoked activity (the latter either by noxious mechanical stimulation or by an input in sensitive mechanoreceptors to the spinal neurons in laminae I-VII), was extracellularly recorded through the central barrel of a " p a r a l l e l " multibarrelled glass micropipette filled with a solution of Fast Green dye in 3 M sodium chloride. The site of the recording was marked by iontophoresis of the dye. The tip of the recording electrode protruded by about 15-20 ~ m beyond the multibarrelled drug-containing assembly. Using electrodes of this type both the size and the stability of recorded units were greatly improved. Extracellular unitary activity was amplified and displayed on an oscilloscope. Action potentials were separated from the background noise by means of a spike amplitude discriminator, and the frequency of the discharge integrated and recorded on a paper record. Conventional microiontophoretic technique was used to study the effects of synthetic SP (3,7 mM in 20 mM acetic acid, pH 5.5, Beckman), SP analogues (D-Pro 2, D-TrpT,9)-SP and D-Pro 2, D-Phe 7, D-Trpg)-SP (3,7 mM in 20 mM acetic acid, pH 5.5) synthesized in Dr. Folkers laboratory, and L-glutamate (1 M in 165 mM NaC1, pH 7.2, Calbiochem) on the spontaneous firing of the functionally identified spinal dorsal horn neurons. SP as well as SP analogues were ejected as cations, while L-glutamate was ejected as an anion. In all experiments, an adjacent barrel of the micropipette drug assembly routinely contained a solution of 20 mM sodium acetate at a similar pH as for the peptides used in order to eliminate any possible current or pH artifacts, and to provide a current return path while ejecting a chemical. The iontophoretic unit used (Medical Instruments) contained a current balancing circuit. A retaining current of 5-10 nA was applied to drug barrels between the periods of ejection of chemicals. This current was sufficient to prevent drug diffusion, since raising the retaining current did not modify cell activity. The excitatory effect of SP on the cat dorsal horn neurons was quantified in three ways: (1) by determining the proportions of responsive and unresponsive units; (2) by measuring the maximum firing frequency occurring during and just after SP administration; and (3) by measuring the duration of the excitatory response, starting from the end of application of SP in neurons showing the maximal increase of their firing rate of at least 50%. In each examined cell the criterion for a depressant effect of the SP analogues was a decrease of at least 50% in the maximal discharge and/or duration of the SP-elicited excitatory response.
Rat Dorsal Horn Neurons Studied In Vitro Experiments were performed on 10- to 20-day-old Sprague-Dawley rats. The animals were anesthetized with ether, and a laminectomy performed to expose the lowerthoracic and lumbosacral spinal cord together with dorsal roots. Following laminectomy, about 1-1.5 cm long segment of lumbosacral spinal cord with attached dorsal rootlets was quickly excised and immersed into aerated (95% 02 and 5% CO2) Krebs solution kept at 34°C. The composition of the solution was (mM): NaC1 124; KC1 5; KH2PO4 1.2; CaCI2 2.4; MgSO4 1.3; NaHCO3 26; glucose 10; pH 7.4. After the removal of the pia mater on the lateral aspects of the spinal cord, the spinal segment was cut manually into 5 mm blocks, and one of the blocks of tissue affixed with cyanoacrylic glue (Borden, Inc.) to the bottom of a Plexiglas cutting chamber of an Oxford Vibratome. The bath of the Vibratome was filled with the Krebs solution, maintained at 30°C and bubbled with a mixture of oxygen and carbon dioxide. The blade
of the Vibratome was positioned 300/xm below the dorsal surface of the spinal cord, and the spinal segment sectioned to yield one 300/xm-thick horizontal spinal cord slice. We have also used transverse slices to study neurons located in the deeper laminae of the spinal dorsal horn. The duration of the entire procedure from the removal of the spinal cord until the slice was made rarely exceeded 5 min. The slices were incubated in Krebs solution at 37°C for about an hour. After incubation, a slice was transferred into the recording chamber where it was continuously perfused with oxygenated modified Krebs solution (NaC1 127 mM; KC1 1.9 mM; CaC12 2.4 mM; MgSO4 1.3 mM; N a H C Q 26 mM; glucose 10 mM, at 33-1°C at a flow rate of about 2.8-3 ml/min. The recording chamber had a capacity of 0.5 ml. Intracellular recordings were performed with micropipettes filled with 1-3 M potassium acetate having DC resistances of 80-120 MI~. Good intracellular recordings from slices could be obtained for a period of up to 12 hr after isolation of the spinal cord. A high-input impedance bridge amplifier (WP Instruments, M707) was used to inject current through the recording microelectrode. The amplitude of the recorded voltage produced by rectangular hyperpolarizing current pulses (0.1-0.3 nA, 50-100 msec duration applied at 0.2 Hz) was used as a measure of the neuronal membrane input resistance. Data were recorded on a Gould-Brush pen recorder (Model 2200S) or stored on the diskettes of a Nicolet 4094 digital oscilloscope. For synaptic activation of dorsal horn neurons a coaxial stainless steel stimulating electrode (inner and outer electrodes being 25 and 200/xm, respectively) was positioned on the dorsal and/or on the ventral root. Substance P (Cambridge Research Biochemicals, CRB; Sigma), cholecystokinin octapeptide (CRB; Peninsula Labs.) and vasoactive intestinal polypeptide (Beckman; Peninsula Labs.) were applied by bath perfusion in known concentrations. Substance P analogues (D-Pro 2, D-TrpT.9)-SP and (DArg 1, D-Pro 2, D-Trp 7'9, LeulI)-SP were purchased from the CRB and Peninsula Labs or were obtained from Dr. Folkers. Peptides were dissolved in 20 mM acetic acid at the concentration of 5 x 10-3 M and distributed to 8 ~zl aliquots. The stock solution was kept frozen at -20°C until used in experiments. To test effects of the SP analogues on the SP-induced depolarization and on the slow depolarization elicited by dorsal root stimulation, the SP analogues were added to the perfusing solution 1 to 6 min prior to the SP application or electrical stimulation of the dorsal root. RESULTS
Effects of lontophoretic Application of(D-Pro 2, D-Trpr'9)-SP and (D-Pro 2, D-Phe 7, D-Trpg)-SP on the Spontaneous and SP-Evoked Activity of the Cat Spinal Dorsal Horn Neurons In Vivo Effects of iontophoretic application of (D-Pro 2, DTrpT'9)-SP and (D-Pro 2, D-PhC, D-Trpg)-SP, applied with currents of 25-100 nA for periods up to 6 min, on the spontaneous and the SP-elicited activity of cat spinal dorsal horn neurons were examined in 52 cells. Data from these experiments are summarized in Table 1. Both SP-analogues produced excitation of about 15% of all neurons tested. Excitation was manifested as appearance of firing in a previously silent cell or as an increase in the rate of spontaneous firing. (D-Pro 2, D-TrpT.9)-SP appeared to be more effective in blocking the SP-induced excitation than (D-Pro 2, D-PhC, D-Trpg)-SP. In 7 out of 11 neurons tested
SP A N A L O G U E S A N D D O R S A L H O R N N E U R O N S
653 TABLE 1
THE EFFECTS OF (D-Pro 2, D-TrpT'9)-SP AND (D-Pro 2, D-Phe r, D-Trp~)-SP ON THE SPONTANEOUS AND SP- OR L-GLUTAMATE-EVOKED ACTIVITY OF THE CAT DORSAL HORN NEURONS
SP Analogue
Spontaneous Activity
SP-Excitation
g
Dlpro 2, DITrpT.e)-sP D-Pro 2, D-Phe r, D-Trpg)-SP
W touch pinch
I
o
0J
o
o
~
•_
"3
o
`5 o
~
Z
Z
~
~
Z
z
~
~.
Z
4
5
4
0
17 23
11 14
7 3
0 2
4 9
17 11
2 0
0 1
15 10
Z
~
26 27
I SP 25 nA
:,lhl[lll[,i[l l,[-L---[ I lllllillllil, [ l
L-Glutamate-Excitation
I ]
l lll ~llill[lllll llll l~llllllllllll~ll~l[l
l
(D-Pro 2, D-Trp',')-SP 50nA
II
'®"
Ill III l[1]..... LtJl
I SP 25 nA
i SP 25nA
}
so
0
I 1 min.
FIG. 1. Iontophoretic application of (D-Pro2, D-Trpr'9)-SP depresses the SP-induced excitation in a reversible manner in a cat spinal dorsal horn neuron activated by both sensitive mechanoreceptors and nociceptors. Substances were applied during the periods indicated by horizontal bars. Rate-meter records of extracellularly recorded unitary activity.
the SP-induced excitation was depressed or abolished by c o n c u r r e n t application of (D-Pro z, D-TrpT'a)-SP. In 3 out of 7 cells exhibiting the SP antagonism, the SP analogue also elicited a reduction in s p o n t a n e o u s activity. In one of the cells (Fig. 2) glutamate-induced excitation was reduced to a small degree. (D-Pro 2, D - P h E , D-Trpa)-sP, however, produced depression or total blockade of the SP-induced excitation only in about 21% of e x a m i n e d n e u r o n s . (D-Pro z, D-Trpr'a)-SP p r o d u c e d depression of glutamate-induced excitation in about 10% of n e u r o n s tested (n=17).
Typical examples of the depressant effect of (D-Pro 2, D-TrpT'a)-sP on s p o n t a n e o u s and the SP-evoked activity of cat spinal dorsal horn n e u r o n s are illustrated in Figs. 1 and 2. Iontophoretic application of the SP-analogue with a current of 50 n A for more than 2 min markedly depressed the SPinduced excitation in a reversible m a n n e r in this n e u r o n activated by sensitive m e c h a n o r e c e p t o r s and nociceptors. The depressant effect was manifested by an absence of the SP excitatory response if the peptide was given for an equivalent period as in the control record (upper trace). Doubling
654
RANDIC E T A L .
t IIIIII I Ut..J
I
i
hair
kUlkiUm,,
,J',
,i
1
1
GLU 13nA
[,
I
!
liilllll
~ I
--b'=,a, ,in J I
Na+50nA
I
tJ
SP 25nA
li
SP 25nA
i
l
I [
iililll
il
l
,,
I
_1
J
I
I
J
(D-Pro 2, D-Trp',0)-SP50nA
GLU 13nA
SP 25
SP 25
,i
u
hair
so 1 t
0
"K
l
SP 25
I
I
I min
FIG. 2. lontophoretic application of (D-Pro2, D-TrpT'9)-SPcompletely blocked the SP-induced excitation of a cat dorsal horn neuron activated by sensitive mechanoreceptors only. This effect was relatively specific for SP since the excitation elicited by L-glutamate or natural stimulation was not reduced to the same degree.
t.....J
i
touch
i
Na+100nA ~LU
i
-
-
|
J
(D-Pro 2, D-TrpT,g)-SP 100nA m
mm
25 nA
,ram
m
mm
a
Na+100nA =m
mm mm
mm
mm
mm
, I rain
FIG. 3. lontophoretic application of (D-Pro2, D-TrpT'~)-SPcompletely blocked L-glutamate-induced excitation of a dorsal horn neuron activated by sensitive mechanoreceptors in a rapidly reversible manner. Positive current of a similar magnitude was ineffective.
the period of the administration of SP still produced a smaller excitatory response if compared to the control response (middle trace). The SP-evoked excitation recovered to the control level within 10 min following the termination of the (D-Pro 2, D-TrpT.a)-SP application. In this cell, but not in all tested cells, there was a depression of the background firing. Figure 2 illustrates a relative selectivity of the action of the SP analogue against the SP-induced excitation. Here a dorsal horn neuron, activated by sensitive mechanoreceptors only, was excited by SP (25 nA) and L-glutamate (13 nA). Iontophoretic application of the SP analogue with a current of 50 nA for almost 3 min reduced the spontaneous activity and blocked the SP-induced excitation during and several minutes after the application. However, the responsiveness of the same neuron to L-glutamate or natural stimulation was reduced only to a small degree. Within l0 min following the termination of the current ejecting the SP
analogue the SP excitatory response only partly recovered. To test for a possibility that observed depressant effect was due to a passage of an outward current, it was important to distinguish between the effects of the SP analogue itself, and the associated current flow. Although both activities, spontaneous and SP-induced, were depressed during outward current of 50 nA applied throught the control barrel (sodium ions) to a similar degree as after the SP analogue, the important difference is that the effect did not outlast the Na-ion application as it did in the case of the SP analogue application. Figure 3 illustrates a record obtained from a neuron activated by sensitive mechanoreceptors in which (D-Pro 2, D-Trpr.9)-SP produced a complete blockade of L-glutamate-induced excitation. Iontophoretic application of the SP analogue with a positive current of 100 nA depressed the spontaneous activity and abolished the excitation in-
SP A N A L O G U E S A N D D O R S A L H O R N N E U R O N S
655 TABLE 2
EFFECTS OF (D-Pro z, D-TrpT'9)-SPAND (D-Arc 1, D-Pro 2, D-Trpr'9)-SP ON RESTING MEMBRANE POTENTIAL OF RAT DORSAL HORN NEURONS AND THEIR DEPOLARIZING RESPONSES ELICITED BY DORSAL ROOT STIMULATION OR EXOGENOUS APPLICATION OF SP, VIP, AND CCK-8
Dorsal Root-Evoked Slow Depolarization
SP-Evoked Depolarization
No
No
No
Depolarization
Depression
Effect
Depression
Effect
(D-Pro 2, D-Trp7.9)-SP (2-4× 10 .5M)
5 (n=21)
7
4
10
4
(D-Arg ~, D-Pro z, D-Trp7% LeuH)-SP (1-4× 10'~M)
6 (n= 14)
10
1
8
0
SP-Analogue
VIP- or CCK-8-Evoked Depolarization* Depression
Effect
0
9
*VIP (n=5).
CCK (n=4).
duced by L-glutamate applied with a negative current of 25 nA. Positive current of 100 nA applied through a barrel containing Na-acetate (20 mM) did not modify significantly either the spontaneous activity or the L-glutamate-induced excitation. A relatively short latency for the onset of the depressant effect and a fast recovery should be noted.
Effects of(D-Pro z, D-Trpr'9)-SP and (D-Arc 1, D-Pro 2, D-Trp 7'9, Leu~)-SP on the SP-Induced Depolarization and on the Dorsal Root-Elicited Slow Depolarization in Rat Dorsal Horn Neurons In Vitro Forty-four neurons in laminae I I - V of the spinal dorsal horn were examined intracellularly. The mean value of the resting membrane potential was -64.5-+4 mV (mean-+S.D.). Data from these experiments are summarized in Table 2. Bath application of (D-Pro 2, D-Trpr'g)-SP in concentrations of 3-4 × 10-s M for periods of 1-6 min produced weak depolarization (1-3 mV) and an increase in synaptic activity in 5 out of 21 neurons tested. Similarly, as for SP, the analogue-evoked depolarization was accompanied by an increase in membrane resistance (n=2). In one cell a small hyperpolarization was observed. The agonistic (excitatory) effect of the SP analogue subsided within 3 to 5 min following its removal from the bath. The depolarizing effect of the SP analogue remained somewhat reduced when synaptic transmission was blocked by tetrodotoxin. However, the SP analogue, when used in a lower concentration range (12 × 10 '~ M), exhibited a reproducible antagonism against SPinduced depolarization in 64% of tested neurons ( n = l l , Table 2). The SP response was reduced to less than half of the peak response observed prior to the SP analogue admini stration (45 -+12%, mean -+S.D.). After the removal of the S P analogue, 10-15 min were needed for the SP response to fully recover. An antagonism against the slow depolarization elicited by high intensity repetitive stimulation of a dorsal root was also shown following application of (D-Pro 2, D-Trpr'9)-SP. In 71% of neurons tested (n = 14) we found a definite reduction in the peak amplitude (48-+ 17%) of the slow depolarization; the response almost fully recovered within 10-15 minutes after the removal of the SP analogue (Table 2). Typical depressant effect of (D-Pro z, D-TrpT'9)-SP on the SP-induced depolarization of a dorsal horn neuron is illus-
trated in Fig. 4. As shown in Fig. 4A, bath application of the SP analogue (2 × 10-5 M) for about 2 min reduced the SPinduced depolarization and synaptic noise in this dorsal horn neuron. The effect was relatively specific since the depolarizing response produced by L-glutamate was not affected 1 min following the SP analogue administration (Fig. 4B). Bath application of (D-Arc 1, D-Pro z, D-Trp 7,a, LeuH)-SP at concentrations of 1-4 × 10-5 M for 2-4 min evoked 1-3 mV dopolarization and increased synaptic activity in 43% of neurons tested (n = 14). The depolarization was slow in onset and of prolonged duration. (D-Arg 1, D-Pro z, D-Trp r,9, Leul~)-SP, if used at lower dosages of 1-2 x 10-5 M, suppressed the SP-induced depolarization in 10 out of 11 neurons tested (Table 2). The mean value for the reduction in the peak amplitude of the response was 75-+6.5% (n = 10). In two neurons the SP-induced depolarization was completely abolished. It is of interest that fast excitatory postsynaptic potentials were usually not depressed (Fig. 5). Since vasoactive intestinal polypeptide (VIP) and cholecystokinin octapeptide (CCK-8) are also present in the primary sensory neurons and the dorsal horn, and they are also known to elicit a slow membrane depolarization of a certain proportion of dorsal horn interneurons [9, 10, 21], we examined the effect of (D-Arg ~, D-Pro 2, D-Trp 7,a, Leu~)-SP on the responses induced by VIP (n =5) and CCK-8 (n =4). It is of interest that the SP analogue did not affect either of the depolarizing responses, as shown in Table 2 and Fig. 6C. This result shows the specificity of (D-Arg 1, D-Pro 2, D-Trp r,9, Leu11)-SP antagonistic effect against the SP-induced depolarization. We examined effect of (D-Arg ~, D-Pro 2, D-Trp 7"9, Leu 1~)-SP on the slow excitatory transmission in the dorsal horn as well. The slow depolarization induced by repetitive stimulation of the lumbar dorsal roots (5-20 V pulses of 0.2-0.5 msec duration applied at a rate of 10-20 Hz, for 3-5 sec) was suppressed in all 8 neurons examined (Table 2). The calculated percent of reduction of the peak depolarization of 83-+5%, is similar to the reduction described above for the SP-induced depolarization. Furthermore, as shown in Fig. 7, the time course of the antagonistic effect of (D-Arc 1, D-Pro 2, D-Trp 7,9, Leu~a)-SP against the depolarizing responses evoked by dorsal root stimulation and exogenous SP application was similar. Figure 6 demonstrates the typical effects
R A N D I C ET A L
656
A
B
SP IO-'M
I
t:
,
.'
L-GLUTAMATE IO-'M
i
lmln FIG. 4. Effects of bath-applied (D-Pro 2, D-TrpT,9)-SP (2 × 10-5 M for 2 min) on the responses of a dorsal horn neuron to SP (A) and L-glutamate (B) are illustrated. Upper traces represent control records, middle traces show responses obtained 1 min after stopping the perfusion with the SP analogue, and lower traces show the responses recorded 9 rain after the removal of the SP antagonist. Vm =52 mV.
A (D-Arg ~, D-Pro 2, D-Trp', Q, Leu')--SP
single
25 mV 15 ms B .
.
°
,
1 min 15 Hz 5s FIG. 5. Effects of (D-Arg 1, D-Pro2, D-Trp 7.a, L e u ' ) - S P (2 × 10-5 M for 2 min) on fast and slow excitatory synaptic potentials in a dorsal horn neuron. The SP antagonist reversibly depressed (B) the response of this cell to dorsal root stimulation (10 V, 0.5 msec, 15 Hz, 5 sec), while the response of the same cell to a single, high threshold stimulus (15 V, 0.5 msec) was not modified (A).
SP ANALOGUES
AND DORSAL HORN NEURONS (D-Arg', D-Pro ~, D'Trp T'', Leu')-SP
CONTROL A
657 RECOVERY
2o.z 5s
B
C
SP 5d0 aM
VIP 10-6M . . . . . . . . .
I10mV
1 min
FIG. 6. Effects of (D-Arg 1, D-Pro z, D-Trp 7"9, L e u ' ) - S P (2 x l0 5 M for 2 rain) on the responses of a dorsal horn neuron to dorsal root stimulation (A), SP (B), and vasoactive intestinal polypeptide (C). In each record, the left trace represents control response, the middle trace shows response obtained 1 min after stopping the perfusion with (D-Arg 1, D.Pro s, D.Trpr,9, Leu~l).Sp, and the right trace represents response obtained 9 rain after the removal of the SP analogue. Fourteen-day-old rat, Vm = - 6 4 mV. amplitude
%
100
50
o
s
1o
l"S
2o
FIG. 7. Depressant effect of (D-Arg 1, D-Pro s, D-Trp 7"9, Leull)-SP (2-4x10 -5 M) on the SP-induced depolarization ((3) and on the dorsal root-elicited slow postsynaptic depolarization (A) in 5 dorsal horn neurons. Black horizontal bar indicates application period of 2 min for the analogue (n =5). Standard deviations of the mean are presented with vertical bars. Almost complete recovery of both responses occurred within 10-15 min following the onset of administration of the SP analogue.
min
658
RANDIC E T A L . DORSAL ROOT
VENTRAL ROOT 15 Hz 5s
10 Hz 5s
(D-Arg', D-Pro 2, D-Trpr, g , L e u ' ) - - S P
L
.....
w-
~'l
....
z--AT"-
_L.__
5mV 1 rain
FIG. 8. Slow excitatory synaptic responses recorded from a dorsal horn neuron in response to electrical stimulation of a lumbar dorsal root (15 V; 0.2 msec; 10 Hz; 5 sec) or ventral root (15 V, 0.2 msec; 15 Hz; 5 sec). In each column the upper trace represents the control response, the middle trace, the response obtained 1 rain after stopping the perfnsion with (D-Arg1, D-Pro2, D-Trpr'9, Leu11)-SP (2 :x:10 s M for 2 rain), and the bottom trace represents response obtained 9 min after the removal of the SP antagonist. V,, =-62 mV.
of (D-Arg 1, D-Pro 2, D-Trp 7"0 Leull)-SP on the SP evoked depolarization (Fig. 6B) and the dorsal root-elicited slow depolarization of a dorsal horn neuron (Fig. 6A). The SP analogue significantly depressed both depolarizations, while the VIP-evoked depolarization (Fig. 6C) and the peak amplitude of the fast excitatory synaptic potential (not illustrated) were not affected. There is evidence that some small diameter primary afferent fibers reach the spinal cord via the ventral roots [3]. We have shown that repetitive stimulation of the ventral roots infrequently (about 10--20% of all tested cells) elicits slow postsynaptic depolarization in dorsal horn neurons [28]. As illustrated in Fig. 8 (D-Arg 1, D-Pro 2, D-Trp r'a, Leull)-SP at a concentration level of 2x10 -5 M suppressed both dorsal root- and ventral root-elicited slow depolarizations in a reversible manner. After the removal of the SP analogue 10-15 minutes were required for the responses to recover. DISCUSSION
The present work showed that the SP-induced excitatory responses of cat dorsal horn neurons examined in vivo and rat dorsal horn neurons examined in vitro, are reversibly depressed by (D-Pro 2, D-Phe 7, D-Trpg)-SP, (D-Pro z, D-TrpT,9)-SP and (D-Arg 1, D-Pro 2, D-Trp 7,9, L e u ' ) - S P . In addition, (D-Pro 2, D-Trpr'9)-SP and (D-Arg ~, D-Pro 2, D-Trp r'9, Leull)-SP significantly reduced the dorsal rootelicited slow depolarizing potential of rat dorsal horn neurons. However, potency and the degree of specificity of their antagonistic actions varied considerably between indi-
vidual SP analogues and in different cells. In addition, all three SP analogues tested retained a partial agonism. Weak antagonistic effect of (D-Pro z, D-Phe 7, D-Trpg)-sP observed in our study is in agreement with previously reported data in the mammalian ganglia [7]. Both, in cat and rat dorsal horn neurons (D-Pro 2, D-Trpr'a)-sP depressed SPinduced excitation in about two-thirds of all cells tested. The result obtained in the cat is in a good agreement with the findings observed in the cat locus coeruleus neurons where (D-Pro z, D-TrpT'o)-SP abolished the excitatory action of iontophoretically applied SP [5]. However, Salt et al. [33] showed that (D-Pro z, D-TrpT,a)-SP apparently had no specific antagonistic effect in the rat caudal trigeminal nucleus or in the newborn rat hemisected isolated spinal cord preparation. In the spinal cord slice preparation we found that (D-Arg 1, D-Pro z, D-Trp 7,9, Leull)-SP proved to be the most potent antagonist of SP-induced depolarization and dorsal rootevoked slow depolarization. Similar antagonism for this SP analogue has been observed in the isolated rat spinal cord [38,39] and in guinea pig sympathetic ganglia [14]. The specificity of the antagonistic action of the SP analogues towards SP has been a controversial issue. We have observed in several cat dorsal horn neurons that the excitatory response to L-glutamate was suppressed by (DPro z, D-Trpr'a)-SP. Similar finding was reported by Salt et al. [33] on a greater sample of trigeminal neurons. However, in Fig. 3 of our work and also in the experiment illustrated in Fig. 1 of Salt et al. [33], it does not appear that the depression of L-glutamate-induced excitation is a result of a postsynaptic receptor antagonism. The onset and recovery
SP A N A L O G U E S AND DORSAL HORN N E U R O N S of glutamate response is too rapid and appears to coincide with onset and turning off the current used to eject the SP analogue. Furthermore, a high degree of specificity toward SP for (D-Pro 2, D-TrpT.9)-SP has been observed in locus coeruleus neurons [5]. This result may also be interpreted that the depressant action of (D-Pro 2, D-TrpT"a)-sP in cat dorsal horn neurons is due to a presynaptic action involving the release of inhibitory substances. Surprenant et al. [34] have recently provided evidence that (D-Arg ~, D-Pro z, D-Trp 7,9, LeuH)-SP releases noradrenaline from sympathetic nerves in the submucosal plexus, with a consequent membrane hyperpolarization and a possible presynaptic inhibition of transmitter release. However, the failure of (D-Arg 1, D-Pro 2, D-Trp 7,9, Leun)-SP in our experiments to modify the depolarizing responses of dorsal horn neurons to CCK-8 and VIP demonstrates that in a certain range of dosages this SP analogue exhibits a relatively high degree of specificity towards SP receptors. The specificity of the SP analogues with D-amino acid substitutions has been also questioned since in high doses they can inhibit responses not only to tachykinins but also to bombesin [37] and exert a nonspecific neurosuppressive effect [23] at least in some tissues. In addition, they have also been shown to elicit motor impairment which can be persistent [23,30]. It is also apparent that peptides with increasing purity show the same effects, thereby excluding the possibility that this effect is mediated by some contaminant. Furthermore, it was demonstrated that the ventral parts of the spinal cord are more susceptible and that thyrotropin releasing hormone has a protective effect against the behavioral and histological effects of the SP analogue, spantide
659 [24]. The motor blockade observed in animals was possibly due to the observed local anesthetic effect of the SP analogues [12]. At histopathological examination it was found that rats showing paresis had motoneuronal necrosis [24]. Our observation that (D-Pro 2, D-Trp~'a)-sP and (D-Arg 1, D-Pro 2, D-Trp 7,a, LeuH)-SP produced a depression of the dorsal root-evoked slow depolarization is in agreement with earlier observations [38,39]. Furthermore, our finding that SP analogues depress the slow depolarization to about similar degree as the SPinduced depolarization support the notion that the slow depolarizing potential is possibly mediated by a SP, or a SP-like peptide. Recently, Matsuto et al. [18] showed that (D-Arg ~, D-Pro 2, D-Trp r,9, Leu~I)-SP depressed the responses of the ventral root to neurokinin A and neurokinin B, the two SPrelated peptides shown to occur in the mammalian spinal dorsal horn [11, 13, 17, 22]. Therefore, some of the endogenous tachykinins other than SP may have a physiological role similar to that of SP. To examine whether other tachykinins are also involved in the generation of the dorsal root-evoked slow depolarization further improvement of specificity of SP and other tachykinin antagonists is highly desirable.
ACKNOWLEDGEMENTS This work was supported in part by grants from the National Institute for Neurological and Communicative Disorders and Stroke (NS 17297); National Science Foundation (BNS 8418042); and the United States Department of Agriculture.
REFERENCES 1. Akerman, B., S. Rosell and K. Folkers. Intrathecal (D-Pro2, D-Trpr'9)-SP elicits hypoalgesia and motor blockade in the rat and antagonizes noxious responses induced by substance P. Acta Physiol Scand 114: 631-633, 1982. 2. Caranikas, S., J. Mizrahi, P. D'Orleans-Juste and D. Regoli. Antagonists of substance P. Eur J Pharmaco177" 205-220, 1982. 3. Coggeshall, R. E. and H. Ito. Sensory fibres in ventral roots L7 and S1 in the cat. J Physiol (Lond) 267: 215-235, 1977. 4. Delbro, D., L. Fandriks, S. Rosell and K. Folkers. Inhibition of antidromically induced stimulation of gastric motility by substance P receptor blockade. Acta Physiol Scand 118: 30%316, 1983. 5. Engberg, G., T. H. Svensson, S. Rosell and K. Folkers. A synthetic peptide as an antagonist of substance P. Nature 293: 222-223, 1981. 6. Folkers, K., J. Horig, S. Rosell and U. Bjorkroth. Chemical design of antagonists of substance P. Acta Physiol Scand 111: 505-506, 1981. 7. Hawcock, A. B., A. G. Hayes and M. B. Tyers. Agonist effects of (D-Pro2, D-Phe7, D-Trp9) substance P--Evidence for different receptors. Eur J Pharmacol 80: 135-138, 1982. 8. Henry, J. L. Substance P and pain: an updating. Trends Neurosci 3: 95-97, 1980. 9. Jeftinija, S., V. Miletic and M. Randic. Cholecystokinin octapeptide excites dorsal horn neurons both in vivo and in vitro. Brain Res 213: 231-236, 1981. 10. Jeftinija, S., K. Murase, V. Nedeljkov and M. Randic. Vasoactive intestinal polypeptide excites mammalian dorsal horn neurons both in vivo and in vitro. Brain Res 243: 158-164, 1982.
11. Kangawa, K., N. Minamino, A. Fukuda and H. Matsuo. Neuromedin K and B: Novel neuropeptides with smooth muscle stimulant activity identified in pig spinal cord. In: Peptide Chemistry, proceedings of the 21st symposium on peptide chemistry, edited by E. Munekata. Osaka: Protein Research Foundation, 1983, pp. 309--314. 12. Karlsson, J. A., M. J. B. Finney, C. G. A. Persson and C. Post. Substance P antagonists and the role of tachykinins in non-cholinergic bronchoconstriction. Life Sci 35: 2681-2691, 1984. 13. Kimura, S., M. Okada, Y. Sugita, I. Kanazawa and E. Munekata. Novel neuropeptides, neurokinin a and/3, isolated from porcine spinal cord. Proc Jpn Acad Sci (B) 59: 101-104, 1985. 14. Konishi, S. and M. Otsuka. Blockade of slow excitatory postsynaptic potential by substance P antagonists in guinea pig sympathetic ganglia. J Physiol (Lond) 361:115-130, 1985. 15. Leander, S., R. Hakanson, S. Rosell, K. Folkers, F. Sundler and K. Tornqvist. A specific substance P antagonist blocks smooth muscle contractions induced by non-cholinergic, nonadrenergic nerve stimulation. Nature 294: 467-469, 1981. 16. Lundberg, J. M., A. Saria, S. Rosell and K. Folkers. A substance P antagonist inhibits heat-induced oedema in the rat skin. Acta Physiol Scand 120" 145-146, 1984. 17. Maggio, J. E., B. E. B. Sandberg, C. V. Bradley, L. L. Iversen, S. Santikarn, D. H. Williams, J. C. Hunter and M. R. Henley. A novel tachykinin in mammalian spinal cord. Int J Med Sci 152: Suppl 1, 20-21, 1983. 18. Matsuto, T., M. Yanagisawa, Otsuka, M., I. Kanazawa and E. Munekata. The excitatory action of the newly discovered mammalian tachykinins, neurokinin a and neurokinin /3, on neurons of the isolated spinal cord of the newborn rat. Neurosci Res 2: 105-110, 1984.
660 19. Mizrahi, J., P. D'Orleans-Juste, G. Drapeau, E. Escher and D. Regoli. Partial agonists and antagonists for substance P. Ear J Pharmacol 98: 457-458, 1984. 20. Murase, K. and M. Randic. Actions of substance P on rat spinal dorsal horn neurones. J Physiol (Lond) 346: 203-217, 1984. 21. Murase, K., V. Nedeljkov and M. Randic. The actions of neuropeptides on dorsal horn neurons in the rat spinal cord slice preparation: an intracellular study. Brain Res 234: 170-176, 1982. 22. Nawa, H., T. Hirose, H. Takashima, S. Inayama and S. Nakanishi. Nucleotide sequences of cloned cDNAs for two types of bovine brain substance P precursor. Nature 306: 32-36, 1983. 23. Post, C., J. F. Butterworth, G. R. Strichartz, J-A. Karlsson and C. G. A. Persson. Tachykinin antagonists have potent local anaesthetic actions. Eur J Pharmacol 117: 347-354, 1985. 24. Post, C., J. Freedman, T. Hokfelt, J. Jonsson, I. Paulsson, E. Arwestrom, S. Leander, J. A. Fischer and A. Verhofstad. Neuronal damage induced by a substance P-antagonist is counteracted by thyrotropin releasing hormone. In: Tachykinin Antagonists, edited by R. Hakanson and F. Sundler. Amsterdam: Elsevier, 1985, pp. 383-391. 25. Post, C. and K. Folkers. Behavioral and antinociceptive effects of intrathecally injected substance P analogues in mice. Eur J Pharmacol 113: 335-343, 1985. 26. Randic, M. and V. Miletic. Effect of substance P in cat dorsal horn neurons activated by noxious stimuli. Brain Res 128: 164169, 1977. 27. Randic, M., P. D. Ryu and L. Urban. Effects of polyclonal and monoclonal antibodies to substance P on slow excitatory transmission in rat spinal dorsal horn. Brain Res 383: 15-27, 1986. 28. Randic, M. and L. Urban. Slow excitatory transmission in rat spinal dorsal horn and the effects of capsaicin. Acta Physio! Hung 69: 375-392, 1987. 29. Raspantini, C. D., L. Urban, S. Jeftinija, K. Folkers and M. Randic. Effects of substance P analogues on substance P excitation and slow excitatory transmission in the mammalian spinal dorsal horn. Soc Neurosci Abstr 9: 256, 1983.
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30. Rodriguez, R. E., T. E. Salt, P. M. B. Cahusac and R. G. Hill. The behavioral effects of intrathecaUy administered (D-Pro 2, D-Trpr'a)-SP, an analogue with presumed antagonist actions in the rat. Neuropharmacology 22: 173-176, 1983. 31. Rosell, S., U. Bjorkroth, D. Chang, I. Yamaguchi, Y.-P. Wan, G. Rackur, G. Fischer and K. Folkers. Effects of substance P analogues on isolated guinea-pig ileum. In: Substance P, edited by U. S. yon Euler and B. Pernow. New York: Raven Press, 1977, pp. 83-88. 32. Rosell, S. and K. Folkers. Substance P-antagonists: a new type of pharmacological tool. Trends Pharmacol Sci 3: 211-212, 1983. 33. Salt, T. E., G. J. De Vries, R. E. Rodriguez, P. M. B. Cahusac, R. Morris and R. G. Hill. Evaluation of (D-Pro 2, DTrpT'9)-substance P as an antagonist of substance P responses in the rat central nervous system. Neurosci Lett 30: 291-295, 1982. 34. Surprenant, A., R. A. North and Y. Katayama. Observations on the actions of substance P and [D-Arg 1, D-Pro 2, D-Trp r,9, Leu 11] substance P on single neurons of the guinea pig submucous plexus. Neuroscience 20: 189-199, 1987. 35. Urban, L. and M. Randic. Slow excitatory transmission in rat dorsal horn: possible mediation by peptides. Brain Res 290: 336-341, 1984. 36. Urban, L., J. Willets, M. Randic and R. E. Papka. The acute and chronic effects of capsaicin on slow excitatory transmission in rat dorsal horn. Brain Res 330: 390-396, 1985. 37. Yachnis, A. T., J. N. Crawley, R. T. Jensen, M. M. McGrane and T. W. Moody. The antagonism of bombesin in the CNS by SP analogues. Life Sci 35: 1963-1969, 1984. 38. Yanagisawa, M. and M. Otsuka. The effect of a substance P antagonist on chemically induced nociceptive reflex in the isolated spinal cord-tail preparation of newborn rat. Proc Jpn Acad (B) 60: 427-430, 1984. 39. Yanagisawa, M., M. Otsuka, S. Konishi, H. Akagi, K. Folkers and S. Rosell. A substance P antagonist inhibits a slow reflex response in the spinal cord of the newborn rat. Acta Physiol Scand 116: 109-112, 1982.
0196-9781/88$3.00 + .00
Effects of Substance P Analogues on Spinal Dorsal Horn Neurons M. R A N D I C , * S. J E F T I N I J A , ? L. U R B A N , *x C. R A S P A N T I N I * A N D K. F O L K E R S ~
Department of *Veterinary Physiology and Pharmacology and tDepartment of Veterinary Anatomy Iowa State University, Ames, IA 50011 and ~Institute of Biomedical Research the University of Texas, Austin, TX 78712 R e c e i v e d 11 A u g u s t 1987 RANDIC, M., S. JEFTINIJA, L. URBAN, C. RASPANTINI AND K. FOLKERS. Effects of substance P analogues on spinal dorsal horn neurons. PEPTIDES 9(3) 651-660, 1988.--The effects of iontophoretically applied (D-Proz, D-Phe7, D-Trpg)-SP and (D-Pro2, D-TrpT.9)-SPon the spontaneous and evoked activity of functionally identified cat spinal dorsal horn neurons have been investigated in vivo by means of extracellular single unit recording technique. In addition, the rat spinal cord slice preparation has been used to study the actions of (D-Proz, D-Trp7'9)-SPand (D-Argl, D-Pro2, D-Trp7,9, Leu~)-SP on the resting membrane potential of dorsal horn neurons and also on their responses to dorsal root stimulation and exogenous SP application. We have observed that both (D-Pro2, D-Phe7, D-Trpa)-SP and (D-Pro2, D-TrpT'9)-SP produced an excitation of about 15% of all neurons tested and had a weak antagonistic effect against SP in the cat spinal cord. (D-Pro2, D-TrpT.9)-SPsuppressed the SP-induced excitation in 63% of examined cells. In addition, depression of the glutamate-induced excitation and spontaneous activity was evident in 10% and 19% of the cat dorsal horn neurons tested, respectively. In the spinal cord slice preparation (D-Arg~, D-Pro2, D-Trp7,9, Leu~a)-SP proved to be a more potent antagonist of the SP-induced depolarization and the dorsal root-elicited slow depolarization, if compared with (D-Pro2, D-TrpT.a)-SP. Putative substance P antagonists
Spinal dorsal horn neurons
SEVERAL structural analogues of substance P (SP) containing D-amino acids substitutions in position 2 (Pro), 7 (Phe or Trp) and 9 (Trp), and having antagonistic properties against synthetic SP, have been developed [2, 6, 32]. Two most studied analogues, i.e., (D-Pro 2, D-TrpT.9)-SP and (DArg 1, D-Pro 2, D-Trp ~,9, Leul~)-SP have been claimed to be competitive antagonists of SP in different peripheral organs [4, 15, 16, 19, 31] and also in mammalian central nervous system [1, 5, 8, 25, 30, 33, 39]. However, the major disadvantage of the three SP analogues is that besides exerting weak antagonistic properties they also possess agonistic activity [19] a~id some nonspecific local anesthetic effects [12, 23, 24, 30]. Our previous work showed that SP has an excitatory action on cat dorsal horn neurons in vivo and depolarizes the membrane of rat dorsal horn neurons in vitro [20, 21, 26]. In addition, we have reported that high intensity repetitive stimulation of a lumbar dorsal root elicits a slow depolarizing potential in more than half of rat spinal dorsal horn neurons. The experimental evidence obtained led us to suggest that SP, or a SP-related peptide, may in some manner be involved in the generation of this response [27, 28, 35, 36]. The present study was undertaken in order to examine the effects of SP analogues on the SP-induced excitation of the cat dorsal
Cat spinal cord
Rat spinal cord slice
horn neurons in vivo and the rat dorsal horn neurons in vitro, and on the slow excitatory transmission in the rat spinal dorsal horn. Preliminary results have been communicated [29]. METHOD
Cat Dorsal Horn Neurons Studied In Vivo Experiments were performed on adult cats of either sex weighing between 2 and 4 kg. Animals were initially anesthetized by halothane. The brain was anemically destroyed by bilateral occlusion of the common carotid and vertebral arteries. The spinal cord was transected at the first cervical level. Thereafter, the animal was artificially respired and immobilized by constant infusion of succinylcholine chloride. Body temperature, blood pressure and end-tidal CO2 concentration were monitored and maintained at optimal levels throughout the experiment. The spinal cord was exposed by laminectomy at the lumbosacral and caudal spinal levels. High intensity mechanical stimuli were delivered to the skin of the tail or left hindlimb (e.g., pressure from a sharply pointed object, grasping a fold of skin with a calibrated forceps). Low intensity mechanical stimuli (touching by resting a finger lightly on the skin, brushing the skin with a camel-hair brush) were also used. The spontaneous and
1Permanent address: Department of Anatomy, University Medical School, Debrecen H-4012, Hungary.
651
R A N D I C ET AL.
652 evoked activity (the latter either by noxious mechanical stimulation or by an input in sensitive mechanoreceptors to the spinal neurons in laminae I-VII), was extracellularly recorded through the central barrel of a " p a r a l l e l " multibarrelled glass micropipette filled with a solution of Fast Green dye in 3 M sodium chloride. The site of the recording was marked by iontophoresis of the dye. The tip of the recording electrode protruded by about 15-20 ~ m beyond the multibarrelled drug-containing assembly. Using electrodes of this type both the size and the stability of recorded units were greatly improved. Extracellular unitary activity was amplified and displayed on an oscilloscope. Action potentials were separated from the background noise by means of a spike amplitude discriminator, and the frequency of the discharge integrated and recorded on a paper record. Conventional microiontophoretic technique was used to study the effects of synthetic SP (3,7 mM in 20 mM acetic acid, pH 5.5, Beckman), SP analogues (D-Pro 2, D-TrpT,9)-SP and D-Pro 2, D-Phe 7, D-Trpg)-SP (3,7 mM in 20 mM acetic acid, pH 5.5) synthesized in Dr. Folkers laboratory, and L-glutamate (1 M in 165 mM NaC1, pH 7.2, Calbiochem) on the spontaneous firing of the functionally identified spinal dorsal horn neurons. SP as well as SP analogues were ejected as cations, while L-glutamate was ejected as an anion. In all experiments, an adjacent barrel of the micropipette drug assembly routinely contained a solution of 20 mM sodium acetate at a similar pH as for the peptides used in order to eliminate any possible current or pH artifacts, and to provide a current return path while ejecting a chemical. The iontophoretic unit used (Medical Instruments) contained a current balancing circuit. A retaining current of 5-10 nA was applied to drug barrels between the periods of ejection of chemicals. This current was sufficient to prevent drug diffusion, since raising the retaining current did not modify cell activity. The excitatory effect of SP on the cat dorsal horn neurons was quantified in three ways: (1) by determining the proportions of responsive and unresponsive units; (2) by measuring the maximum firing frequency occurring during and just after SP administration; and (3) by measuring the duration of the excitatory response, starting from the end of application of SP in neurons showing the maximal increase of their firing rate of at least 50%. In each examined cell the criterion for a depressant effect of the SP analogues was a decrease of at least 50% in the maximal discharge and/or duration of the SP-elicited excitatory response.
Rat Dorsal Horn Neurons Studied In Vitro Experiments were performed on 10- to 20-day-old Sprague-Dawley rats. The animals were anesthetized with ether, and a laminectomy performed to expose the lowerthoracic and lumbosacral spinal cord together with dorsal roots. Following laminectomy, about 1-1.5 cm long segment of lumbosacral spinal cord with attached dorsal rootlets was quickly excised and immersed into aerated (95% 02 and 5% CO2) Krebs solution kept at 34°C. The composition of the solution was (mM): NaC1 124; KC1 5; KH2PO4 1.2; CaCI2 2.4; MgSO4 1.3; NaHCO3 26; glucose 10; pH 7.4. After the removal of the pia mater on the lateral aspects of the spinal cord, the spinal segment was cut manually into 5 mm blocks, and one of the blocks of tissue affixed with cyanoacrylic glue (Borden, Inc.) to the bottom of a Plexiglas cutting chamber of an Oxford Vibratome. The bath of the Vibratome was filled with the Krebs solution, maintained at 30°C and bubbled with a mixture of oxygen and carbon dioxide. The blade
of the Vibratome was positioned 300/xm below the dorsal surface of the spinal cord, and the spinal segment sectioned to yield one 300/xm-thick horizontal spinal cord slice. We have also used transverse slices to study neurons located in the deeper laminae of the spinal dorsal horn. The duration of the entire procedure from the removal of the spinal cord until the slice was made rarely exceeded 5 min. The slices were incubated in Krebs solution at 37°C for about an hour. After incubation, a slice was transferred into the recording chamber where it was continuously perfused with oxygenated modified Krebs solution (NaC1 127 mM; KC1 1.9 mM; CaC12 2.4 mM; MgSO4 1.3 mM; N a H C Q 26 mM; glucose 10 mM, at 33-1°C at a flow rate of about 2.8-3 ml/min. The recording chamber had a capacity of 0.5 ml. Intracellular recordings were performed with micropipettes filled with 1-3 M potassium acetate having DC resistances of 80-120 MI~. Good intracellular recordings from slices could be obtained for a period of up to 12 hr after isolation of the spinal cord. A high-input impedance bridge amplifier (WP Instruments, M707) was used to inject current through the recording microelectrode. The amplitude of the recorded voltage produced by rectangular hyperpolarizing current pulses (0.1-0.3 nA, 50-100 msec duration applied at 0.2 Hz) was used as a measure of the neuronal membrane input resistance. Data were recorded on a Gould-Brush pen recorder (Model 2200S) or stored on the diskettes of a Nicolet 4094 digital oscilloscope. For synaptic activation of dorsal horn neurons a coaxial stainless steel stimulating electrode (inner and outer electrodes being 25 and 200/xm, respectively) was positioned on the dorsal and/or on the ventral root. Substance P (Cambridge Research Biochemicals, CRB; Sigma), cholecystokinin octapeptide (CRB; Peninsula Labs.) and vasoactive intestinal polypeptide (Beckman; Peninsula Labs.) were applied by bath perfusion in known concentrations. Substance P analogues (D-Pro 2, D-TrpT.9)-SP and (DArg 1, D-Pro 2, D-Trp 7'9, LeulI)-SP were purchased from the CRB and Peninsula Labs or were obtained from Dr. Folkers. Peptides were dissolved in 20 mM acetic acid at the concentration of 5 x 10-3 M and distributed to 8 ~zl aliquots. The stock solution was kept frozen at -20°C until used in experiments. To test effects of the SP analogues on the SP-induced depolarization and on the slow depolarization elicited by dorsal root stimulation, the SP analogues were added to the perfusing solution 1 to 6 min prior to the SP application or electrical stimulation of the dorsal root. RESULTS
Effects of lontophoretic Application of(D-Pro 2, D-Trpr'9)-SP and (D-Pro 2, D-Phe 7, D-Trpg)-SP on the Spontaneous and SP-Evoked Activity of the Cat Spinal Dorsal Horn Neurons In Vivo Effects of iontophoretic application of (D-Pro 2, DTrpT'9)-SP and (D-Pro 2, D-PhC, D-Trpg)-SP, applied with currents of 25-100 nA for periods up to 6 min, on the spontaneous and the SP-elicited activity of cat spinal dorsal horn neurons were examined in 52 cells. Data from these experiments are summarized in Table 1. Both SP-analogues produced excitation of about 15% of all neurons tested. Excitation was manifested as appearance of firing in a previously silent cell or as an increase in the rate of spontaneous firing. (D-Pro 2, D-TrpT.9)-SP appeared to be more effective in blocking the SP-induced excitation than (D-Pro 2, D-PhC, D-Trpg)-SP. In 7 out of 11 neurons tested
SP A N A L O G U E S A N D D O R S A L H O R N N E U R O N S
653 TABLE 1
THE EFFECTS OF (D-Pro 2, D-TrpT'9)-SP AND (D-Pro 2, D-Phe r, D-Trp~)-SP ON THE SPONTANEOUS AND SP- OR L-GLUTAMATE-EVOKED ACTIVITY OF THE CAT DORSAL HORN NEURONS
SP Analogue
Spontaneous Activity
SP-Excitation
g
Dlpro 2, DITrpT.e)-sP D-Pro 2, D-Phe r, D-Trpg)-SP
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11 14
7 3
0 2
4 9
17 11
2 0
0 1
15 10
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26 27
I SP 25 nA
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FIG. 1. Iontophoretic application of (D-Pro2, D-Trpr'9)-SP depresses the SP-induced excitation in a reversible manner in a cat spinal dorsal horn neuron activated by both sensitive mechanoreceptors and nociceptors. Substances were applied during the periods indicated by horizontal bars. Rate-meter records of extracellularly recorded unitary activity.
the SP-induced excitation was depressed or abolished by c o n c u r r e n t application of (D-Pro z, D-TrpT'a)-SP. In 3 out of 7 cells exhibiting the SP antagonism, the SP analogue also elicited a reduction in s p o n t a n e o u s activity. In one of the cells (Fig. 2) glutamate-induced excitation was reduced to a small degree. (D-Pro 2, D - P h E , D-Trpa)-sP, however, produced depression or total blockade of the SP-induced excitation only in about 21% of e x a m i n e d n e u r o n s . (D-Pro z, D-Trpr'a)-SP p r o d u c e d depression of glutamate-induced excitation in about 10% of n e u r o n s tested (n=17).
Typical examples of the depressant effect of (D-Pro 2, D-TrpT'a)-sP on s p o n t a n e o u s and the SP-evoked activity of cat spinal dorsal horn n e u r o n s are illustrated in Figs. 1 and 2. Iontophoretic application of the SP-analogue with a current of 50 n A for more than 2 min markedly depressed the SPinduced excitation in a reversible m a n n e r in this n e u r o n activated by sensitive m e c h a n o r e c e p t o r s and nociceptors. The depressant effect was manifested by an absence of the SP excitatory response if the peptide was given for an equivalent period as in the control record (upper trace). Doubling
654
RANDIC E T A L .
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FIG. 2. lontophoretic application of (D-Pro2, D-TrpT'9)-SPcompletely blocked the SP-induced excitation of a cat dorsal horn neuron activated by sensitive mechanoreceptors only. This effect was relatively specific for SP since the excitation elicited by L-glutamate or natural stimulation was not reduced to the same degree.
t.....J
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mm mm
mm
mm
mm
, I rain
FIG. 3. lontophoretic application of (D-Pro2, D-TrpT'~)-SPcompletely blocked L-glutamate-induced excitation of a dorsal horn neuron activated by sensitive mechanoreceptors in a rapidly reversible manner. Positive current of a similar magnitude was ineffective.
the period of the administration of SP still produced a smaller excitatory response if compared to the control response (middle trace). The SP-evoked excitation recovered to the control level within 10 min following the termination of the (D-Pro 2, D-TrpT.a)-SP application. In this cell, but not in all tested cells, there was a depression of the background firing. Figure 2 illustrates a relative selectivity of the action of the SP analogue against the SP-induced excitation. Here a dorsal horn neuron, activated by sensitive mechanoreceptors only, was excited by SP (25 nA) and L-glutamate (13 nA). Iontophoretic application of the SP analogue with a current of 50 nA for almost 3 min reduced the spontaneous activity and blocked the SP-induced excitation during and several minutes after the application. However, the responsiveness of the same neuron to L-glutamate or natural stimulation was reduced only to a small degree. Within l0 min following the termination of the current ejecting the SP
analogue the SP excitatory response only partly recovered. To test for a possibility that observed depressant effect was due to a passage of an outward current, it was important to distinguish between the effects of the SP analogue itself, and the associated current flow. Although both activities, spontaneous and SP-induced, were depressed during outward current of 50 nA applied throught the control barrel (sodium ions) to a similar degree as after the SP analogue, the important difference is that the effect did not outlast the Na-ion application as it did in the case of the SP analogue application. Figure 3 illustrates a record obtained from a neuron activated by sensitive mechanoreceptors in which (D-Pro 2, D-Trpr.9)-SP produced a complete blockade of L-glutamate-induced excitation. Iontophoretic application of the SP analogue with a positive current of 100 nA depressed the spontaneous activity and abolished the excitation in-
SP A N A L O G U E S A N D D O R S A L H O R N N E U R O N S
655 TABLE 2
EFFECTS OF (D-Pro z, D-TrpT'9)-SPAND (D-Arc 1, D-Pro 2, D-Trpr'9)-SP ON RESTING MEMBRANE POTENTIAL OF RAT DORSAL HORN NEURONS AND THEIR DEPOLARIZING RESPONSES ELICITED BY DORSAL ROOT STIMULATION OR EXOGENOUS APPLICATION OF SP, VIP, AND CCK-8
Dorsal Root-Evoked Slow Depolarization
SP-Evoked Depolarization
No
No
No
Depolarization
Depression
Effect
Depression
Effect
(D-Pro 2, D-Trp7.9)-SP (2-4× 10 .5M)
5 (n=21)
7
4
10
4
(D-Arg ~, D-Pro z, D-Trp7% LeuH)-SP (1-4× 10'~M)
6 (n= 14)
10
1
8
0
SP-Analogue
VIP- or CCK-8-Evoked Depolarization* Depression
Effect
0
9
*VIP (n=5).
CCK (n=4).
duced by L-glutamate applied with a negative current of 25 nA. Positive current of 100 nA applied through a barrel containing Na-acetate (20 mM) did not modify significantly either the spontaneous activity or the L-glutamate-induced excitation. A relatively short latency for the onset of the depressant effect and a fast recovery should be noted.
Effects of(D-Pro z, D-Trpr'9)-SP and (D-Arc 1, D-Pro 2, D-Trp 7'9, Leu~)-SP on the SP-Induced Depolarization and on the Dorsal Root-Elicited Slow Depolarization in Rat Dorsal Horn Neurons In Vitro Forty-four neurons in laminae I I - V of the spinal dorsal horn were examined intracellularly. The mean value of the resting membrane potential was -64.5-+4 mV (mean-+S.D.). Data from these experiments are summarized in Table 2. Bath application of (D-Pro 2, D-Trpr'g)-SP in concentrations of 3-4 × 10-s M for periods of 1-6 min produced weak depolarization (1-3 mV) and an increase in synaptic activity in 5 out of 21 neurons tested. Similarly, as for SP, the analogue-evoked depolarization was accompanied by an increase in membrane resistance (n=2). In one cell a small hyperpolarization was observed. The agonistic (excitatory) effect of the SP analogue subsided within 3 to 5 min following its removal from the bath. The depolarizing effect of the SP analogue remained somewhat reduced when synaptic transmission was blocked by tetrodotoxin. However, the SP analogue, when used in a lower concentration range (12 × 10 '~ M), exhibited a reproducible antagonism against SPinduced depolarization in 64% of tested neurons ( n = l l , Table 2). The SP response was reduced to less than half of the peak response observed prior to the SP analogue admini stration (45 -+12%, mean -+S.D.). After the removal of the S P analogue, 10-15 min were needed for the SP response to fully recover. An antagonism against the slow depolarization elicited by high intensity repetitive stimulation of a dorsal root was also shown following application of (D-Pro 2, D-Trpr'9)-SP. In 71% of neurons tested (n = 14) we found a definite reduction in the peak amplitude (48-+ 17%) of the slow depolarization; the response almost fully recovered within 10-15 minutes after the removal of the SP analogue (Table 2). Typical depressant effect of (D-Pro z, D-TrpT'9)-SP on the SP-induced depolarization of a dorsal horn neuron is illus-
trated in Fig. 4. As shown in Fig. 4A, bath application of the SP analogue (2 × 10-5 M) for about 2 min reduced the SPinduced depolarization and synaptic noise in this dorsal horn neuron. The effect was relatively specific since the depolarizing response produced by L-glutamate was not affected 1 min following the SP analogue administration (Fig. 4B). Bath application of (D-Arc 1, D-Pro z, D-Trp 7,a, LeuH)-SP at concentrations of 1-4 × 10-5 M for 2-4 min evoked 1-3 mV dopolarization and increased synaptic activity in 43% of neurons tested (n = 14). The depolarization was slow in onset and of prolonged duration. (D-Arg 1, D-Pro z, D-Trp r,9, Leul~)-SP, if used at lower dosages of 1-2 x 10-5 M, suppressed the SP-induced depolarization in 10 out of 11 neurons tested (Table 2). The mean value for the reduction in the peak amplitude of the response was 75-+6.5% (n = 10). In two neurons the SP-induced depolarization was completely abolished. It is of interest that fast excitatory postsynaptic potentials were usually not depressed (Fig. 5). Since vasoactive intestinal polypeptide (VIP) and cholecystokinin octapeptide (CCK-8) are also present in the primary sensory neurons and the dorsal horn, and they are also known to elicit a slow membrane depolarization of a certain proportion of dorsal horn interneurons [9, 10, 21], we examined the effect of (D-Arg ~, D-Pro 2, D-Trp 7,a, Leu~)-SP on the responses induced by VIP (n =5) and CCK-8 (n =4). It is of interest that the SP analogue did not affect either of the depolarizing responses, as shown in Table 2 and Fig. 6C. This result shows the specificity of (D-Arg 1, D-Pro 2, D-Trp r,9, Leu11)-SP antagonistic effect against the SP-induced depolarization. We examined effect of (D-Arg ~, D-Pro 2, D-Trp 7"9, Leu 1~)-SP on the slow excitatory transmission in the dorsal horn as well. The slow depolarization induced by repetitive stimulation of the lumbar dorsal roots (5-20 V pulses of 0.2-0.5 msec duration applied at a rate of 10-20 Hz, for 3-5 sec) was suppressed in all 8 neurons examined (Table 2). The calculated percent of reduction of the peak depolarization of 83-+5%, is similar to the reduction described above for the SP-induced depolarization. Furthermore, as shown in Fig. 7, the time course of the antagonistic effect of (D-Arc 1, D-Pro 2, D-Trp 7,9, Leu~a)-SP against the depolarizing responses evoked by dorsal root stimulation and exogenous SP application was similar. Figure 6 demonstrates the typical effects
R A N D I C ET A L
656
A
B
SP IO-'M
I
t:
,
.'
L-GLUTAMATE IO-'M
i
lmln FIG. 4. Effects of bath-applied (D-Pro 2, D-TrpT,9)-SP (2 × 10-5 M for 2 min) on the responses of a dorsal horn neuron to SP (A) and L-glutamate (B) are illustrated. Upper traces represent control records, middle traces show responses obtained 1 min after stopping the perfusion with the SP analogue, and lower traces show the responses recorded 9 rain after the removal of the SP antagonist. Vm =52 mV.
A (D-Arg ~, D-Pro 2, D-Trp', Q, Leu')--SP
single
25 mV 15 ms B .
.
°
,
1 min 15 Hz 5s FIG. 5. Effects of (D-Arg 1, D-Pro2, D-Trp 7.a, L e u ' ) - S P (2 × 10-5 M for 2 min) on fast and slow excitatory synaptic potentials in a dorsal horn neuron. The SP antagonist reversibly depressed (B) the response of this cell to dorsal root stimulation (10 V, 0.5 msec, 15 Hz, 5 sec), while the response of the same cell to a single, high threshold stimulus (15 V, 0.5 msec) was not modified (A).
SP ANALOGUES
AND DORSAL HORN NEURONS (D-Arg', D-Pro ~, D'Trp T'', Leu')-SP
CONTROL A
657 RECOVERY
2o.z 5s
B
C
SP 5d0 aM
VIP 10-6M . . . . . . . . .
I10mV
1 min
FIG. 6. Effects of (D-Arg 1, D-Pro z, D-Trp 7"9, L e u ' ) - S P (2 x l0 5 M for 2 rain) on the responses of a dorsal horn neuron to dorsal root stimulation (A), SP (B), and vasoactive intestinal polypeptide (C). In each record, the left trace represents control response, the middle trace shows response obtained 1 min after stopping the perfusion with (D-Arg 1, D.Pro s, D.Trpr,9, Leu~l).Sp, and the right trace represents response obtained 9 rain after the removal of the SP analogue. Fourteen-day-old rat, Vm = - 6 4 mV. amplitude
%
100
50
o
s
1o
l"S
2o
FIG. 7. Depressant effect of (D-Arg 1, D-Pro s, D-Trp 7"9, Leull)-SP (2-4x10 -5 M) on the SP-induced depolarization ((3) and on the dorsal root-elicited slow postsynaptic depolarization (A) in 5 dorsal horn neurons. Black horizontal bar indicates application period of 2 min for the analogue (n =5). Standard deviations of the mean are presented with vertical bars. Almost complete recovery of both responses occurred within 10-15 min following the onset of administration of the SP analogue.
min
658
RANDIC E T A L . DORSAL ROOT
VENTRAL ROOT 15 Hz 5s
10 Hz 5s
(D-Arg', D-Pro 2, D-Trpr, g , L e u ' ) - - S P
L
.....
w-
~'l
....
z--AT"-
_L.__
5mV 1 rain
FIG. 8. Slow excitatory synaptic responses recorded from a dorsal horn neuron in response to electrical stimulation of a lumbar dorsal root (15 V; 0.2 msec; 10 Hz; 5 sec) or ventral root (15 V, 0.2 msec; 15 Hz; 5 sec). In each column the upper trace represents the control response, the middle trace, the response obtained 1 rain after stopping the perfnsion with (D-Arg1, D-Pro2, D-Trpr'9, Leu11)-SP (2 :x:10 s M for 2 rain), and the bottom trace represents response obtained 9 min after the removal of the SP antagonist. V,, =-62 mV.
of (D-Arg 1, D-Pro 2, D-Trp 7"0 Leull)-SP on the SP evoked depolarization (Fig. 6B) and the dorsal root-elicited slow depolarization of a dorsal horn neuron (Fig. 6A). The SP analogue significantly depressed both depolarizations, while the VIP-evoked depolarization (Fig. 6C) and the peak amplitude of the fast excitatory synaptic potential (not illustrated) were not affected. There is evidence that some small diameter primary afferent fibers reach the spinal cord via the ventral roots [3]. We have shown that repetitive stimulation of the ventral roots infrequently (about 10--20% of all tested cells) elicits slow postsynaptic depolarization in dorsal horn neurons [28]. As illustrated in Fig. 8 (D-Arg 1, D-Pro 2, D-Trp r'a, Leull)-SP at a concentration level of 2x10 -5 M suppressed both dorsal root- and ventral root-elicited slow depolarizations in a reversible manner. After the removal of the SP analogue 10-15 minutes were required for the responses to recover. DISCUSSION
The present work showed that the SP-induced excitatory responses of cat dorsal horn neurons examined in vivo and rat dorsal horn neurons examined in vitro, are reversibly depressed by (D-Pro 2, D-Phe 7, D-Trpg)-SP, (D-Pro z, D-TrpT,9)-SP and (D-Arg 1, D-Pro 2, D-Trp 7,9, L e u ' ) - S P . In addition, (D-Pro 2, D-Trpr'9)-SP and (D-Arg ~, D-Pro 2, D-Trp r'9, Leull)-SP significantly reduced the dorsal rootelicited slow depolarizing potential of rat dorsal horn neurons. However, potency and the degree of specificity of their antagonistic actions varied considerably between indi-
vidual SP analogues and in different cells. In addition, all three SP analogues tested retained a partial agonism. Weak antagonistic effect of (D-Pro z, D-Phe 7, D-Trpg)-sP observed in our study is in agreement with previously reported data in the mammalian ganglia [7]. Both, in cat and rat dorsal horn neurons (D-Pro 2, D-Trpr'a)-sP depressed SPinduced excitation in about two-thirds of all cells tested. The result obtained in the cat is in a good agreement with the findings observed in the cat locus coeruleus neurons where (D-Pro z, D-TrpT'o)-SP abolished the excitatory action of iontophoretically applied SP [5]. However, Salt et al. [33] showed that (D-Pro z, D-TrpT,a)-SP apparently had no specific antagonistic effect in the rat caudal trigeminal nucleus or in the newborn rat hemisected isolated spinal cord preparation. In the spinal cord slice preparation we found that (D-Arg 1, D-Pro z, D-Trp 7,9, Leull)-SP proved to be the most potent antagonist of SP-induced depolarization and dorsal rootevoked slow depolarization. Similar antagonism for this SP analogue has been observed in the isolated rat spinal cord [38,39] and in guinea pig sympathetic ganglia [14]. The specificity of the antagonistic action of the SP analogues towards SP has been a controversial issue. We have observed in several cat dorsal horn neurons that the excitatory response to L-glutamate was suppressed by (DPro z, D-Trpr'a)-SP. Similar finding was reported by Salt et al. [33] on a greater sample of trigeminal neurons. However, in Fig. 3 of our work and also in the experiment illustrated in Fig. 1 of Salt et al. [33], it does not appear that the depression of L-glutamate-induced excitation is a result of a postsynaptic receptor antagonism. The onset and recovery
SP A N A L O G U E S AND DORSAL HORN N E U R O N S of glutamate response is too rapid and appears to coincide with onset and turning off the current used to eject the SP analogue. Furthermore, a high degree of specificity toward SP for (D-Pro 2, D-TrpT.9)-SP has been observed in locus coeruleus neurons [5]. This result may also be interpreted that the depressant action of (D-Pro 2, D-TrpT"a)-sP in cat dorsal horn neurons is due to a presynaptic action involving the release of inhibitory substances. Surprenant et al. [34] have recently provided evidence that (D-Arg ~, D-Pro z, D-Trp 7,9, LeuH)-SP releases noradrenaline from sympathetic nerves in the submucosal plexus, with a consequent membrane hyperpolarization and a possible presynaptic inhibition of transmitter release. However, the failure of (D-Arg 1, D-Pro 2, D-Trp 7,9, Leun)-SP in our experiments to modify the depolarizing responses of dorsal horn neurons to CCK-8 and VIP demonstrates that in a certain range of dosages this SP analogue exhibits a relatively high degree of specificity towards SP receptors. The specificity of the SP analogues with D-amino acid substitutions has been also questioned since in high doses they can inhibit responses not only to tachykinins but also to bombesin [37] and exert a nonspecific neurosuppressive effect [23] at least in some tissues. In addition, they have also been shown to elicit motor impairment which can be persistent [23,30]. It is also apparent that peptides with increasing purity show the same effects, thereby excluding the possibility that this effect is mediated by some contaminant. Furthermore, it was demonstrated that the ventral parts of the spinal cord are more susceptible and that thyrotropin releasing hormone has a protective effect against the behavioral and histological effects of the SP analogue, spantide
659 [24]. The motor blockade observed in animals was possibly due to the observed local anesthetic effect of the SP analogues [12]. At histopathological examination it was found that rats showing paresis had motoneuronal necrosis [24]. Our observation that (D-Pro 2, D-Trp~'a)-sP and (D-Arg 1, D-Pro 2, D-Trp 7,a, LeuH)-SP produced a depression of the dorsal root-evoked slow depolarization is in agreement with earlier observations [38,39]. Furthermore, our finding that SP analogues depress the slow depolarization to about similar degree as the SPinduced depolarization support the notion that the slow depolarizing potential is possibly mediated by a SP, or a SP-like peptide. Recently, Matsuto et al. [18] showed that (D-Arg ~, D-Pro 2, D-Trp r,9, Leu~I)-SP depressed the responses of the ventral root to neurokinin A and neurokinin B, the two SPrelated peptides shown to occur in the mammalian spinal dorsal horn [11, 13, 17, 22]. Therefore, some of the endogenous tachykinins other than SP may have a physiological role similar to that of SP. To examine whether other tachykinins are also involved in the generation of the dorsal root-evoked slow depolarization further improvement of specificity of SP and other tachykinin antagonists is highly desirable.
ACKNOWLEDGEMENTS This work was supported in part by grants from the National Institute for Neurological and Communicative Disorders and Stroke (NS 17297); National Science Foundation (BNS 8418042); and the United States Department of Agriculture.
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