347
Brain Research, 553 (1991) 347-352 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 0006899391 BRES 24742
Outflow of endogenous aspartate and glutamate from the rat spinal dorsal horn in vitro by activation of low- and high-threshold primary afferent fibers. Modulation by t-opioids Ivan Kangrga and Mirjana Randi6 Department of Veterinary Physiology and Pharmacology, Iowa State University, Ames, IA 50010 (U.S.A.) (Accepted 9 April 1991) Key words: Excitatory synaptic transmission; Glutamate; Aspartate; g-Opioid receptor; Rat; Dorsal horn slice; High-performanceliquid chromatography
Possible correlation of release of endogenous glutamate (Gin) and aspartate (Asp) with stimulation parameters used to activate primary sensory neurons was examined using the rat spinal cord slice - - dorsal root ganglion preparation and high performance liquid chromatography with fluorimetric detection. Selective activation of the low-threshold (Aft) primary afferent fibers resulted in a two-fold increase in the rate of basal outflow of Asp and a smaller increase in the outflow of Glu from the rat spinal dorsal horn slices into the superfusing medium. The activation of both the low (Aft)- and the high-threshold (At$+C) primary afferents elicited also a significant increase in the outflow of Asp and Gin relative to control. Ghi and Asp are released in significant amounts following superfusion of the dorsal root ganglia with capsaicin or resiniferatoxin. DAGO (Tyr-D-Ala-Gly-MePhe-Gly-ol-enkephalin),an agonist at /~-opioid receptors, attenuated the high-intensity stimulation-evoked outflow of Asp and Gin in a naloxone-sensitive manner. Our results have provided further evidence in support of the contention that Glu and Asp act as excitatory synaptic transmitters in the spinal dorsal horn. A role for g-opioid receptors in modulation of spinal processing of somatosensory information is indicated. The excitatory amino acids (EAA), glutamate (Glu) and aspartate (Asp), are the principal excitatory neurotransmitter candidates in the central nervous system 12, 15,18 and spinal cord 4"15,2°. Although Glu has been long favored as a transmitter of the primary afferent fibers, anatomical and physiological evidence has recently emerged suggesting a role for Asp in the primary afferent neurotransmission. Asp has been detected by immunocytochemistry in approximately 15% of the unmyelinated and 4% of the myelinated axons in the rat LA dorsal root (DR) 22, and electrically evoked release of Asp has been demonstrated from the slices of medulla ]2 and the spinal dorsal horn 9-1]. The actions of E A A are mediated by at least three distinct receptor subtypes characterized on the basis of their responsiveness to selective agonists: Nmethyl-D-aspartate (NMDA),a-amino-3-hydroxy-5-meth_ yl-4-isoxazole-propionic acid (AMPA) and kainate (KA). Whereas glutamate activates both NMDA and nonN M D A receptors, aspartate is thought to act preferentially at the N M D A receptors 3,4,15. About 85% of acutely isolated rat spinal dorsal horn neurons are sensitive to N M D A 17 and both non-NMDA and N M D A receptors participate in the fast 2'3'5 and the slow excitatory trans-
mission in the dorsal horn 6. Opioid peptides have been implicated in modulation of spinal segmental transmission 4's'2° and the presence of functional g- and iS-receptors on C primary afferent neurons has been demonstrated 1,7. The objective of this study was to investigate the pattern of outflow of endogenous Glu, Asp and glutamine (Gin) from the superfused dorsal horn slice in response to selective activation of the low-threshold or both the low- and high-threshold primary afferent fibers by electrical stimulation of lumbar dorsal roots. In addition, the possibility of a modulation of the D R stimulation-evoked outflow of Glu and Asp by a/z-opioid receptor agonist D A G O was examined. Some aspects of this work have been reported l°,n. Longitudinal slices (300-400/zm), with dorsal roots (DR) and dorsal root ganglia (L4-L6) attached, were obtained from 27- to 57-day-old Sprague-Dawley rats 9. After 1 h of incubation at 30 _+ 1°C a slice was transferred into one compartment of a 2 compartmentchamber and continuously superfused with oxygenated artificial cerebrospinal fluid at a flow rate of 0.4 ml/min -1. The composition of the solution was in mM: NaC1, 124;
Correspondence: M. Randi6, Department of Veterinary Physiologyand Pharmacology,2008 Vet Med Bldg, Iowa State University, Ames, IA 50010, U.S.A.
348 KC1, 1.9; KHzPO4, 1.2; CaCI2, 2.4; MgSO4, 1.3; N a H C O 3, 26; glucose, 10; p H 7.4. The dorsal roots were led across a leak-proof partition of vaseline into the other compartment which was filled with mineral oil and placed on two pairs of bipolar platinum electrodes. The distal pair was used for stimulation of the D R , and the proximal pair for recording and continuous monitoring of compound action potentials of primary afferent fibers. Each dorsal root having good and stable A and C compound action potentials was subjected to two 3-min periods of electrical stimulation. The stimulation parameters were selected to activate low-threshold, fast conducting (Aft) fibers during the first stimulation period and the additional recruitment of high-threshold, slowly-conducting myelinated (A6) and unmyelinated (C) fibers during the second stimulation period. In a few experiments this stimulation sequence was reversed. Conduction velocities, estimated from the distance between the centers of the two bipolar electrodes and the latency of the first negative deflection of a volley, were: Aa,fl, 22.6 + 1.9 ms -1 and C, 0.32 + 0.02 ms -1, (n = 20). D A G O (Tyr-D-Ala-Gly-MePhe-Gly-ol-enkephalin, Cambridge Research Biochemicals), naloxone (Du Pont), capsaicin (Sigma) and resiniferatoxin (RTX) (Chemsyn Science Laboratories) were bath-applied in known concentrations. In experiments where the effects of D A G O and naioxone on the stimulation-elicited outflow of Glu and/or Asp were examined, the combined application of D A G O + naloxone always followed D A G O application alone in the same slices. Samples of the perfusate were continuously collected at regular intervals (during the entire 3 min periods) prior to, during, and after the
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Fig. 1. A,B: the time course of the outflow of Asp (A) and Glu (B) from the spinal slice in response to dorsal root electrical stimulation. While the selective activation of the low-threshold (Aft) PAF (5-12 V, 20/~s, 3 Hz, 180-300 pulses) produced a significant increase in the basal outflow of Asp and Glu during the first 3-min collection period (A-B, single arrows), the activation of both the low- and the high-threshold (A+C) PAF (25-30 V, 0.4-1.0 ms, 1 Hz, 180-300 pulses) resulted in a somewhat greater but characteristically more prolonged increase in the outflow of both Asp and Glu (A-B, double arrows). The results are presented as mean percent of the basal outflow preceding each stimulation period + S.E.M. for 8 experiments. **P < 0.01; *P < 0.05. The inset in A depicts corresponding A and A + C compound action potentials of the PAF recorded during the low- (left record) and the highintensity (right record) dorsal root electrical stimulation; arrows show the stimulus artifacts. In the right record the Aft action potential was truncated. Bars: left record = 6 mV and 1.5 ms; right record = 0.6 mV and 12 ms. C: the average (n = 6) amount of Asp (solid column) and Glu (hatched column) released in response to activation of Aft or A+C afferents is presented. The basal outflow of Asp and Glu measured prior to each stimulation period has been subtracted. When A+C primary afferents were activated, a marked increase in the absolute amounts of Asp and Glu (702.0% and 216.3%, respectively, of that elicited by Aft stimulation) in the spinal perfusate was observed. 30- to 57-day-old rats.
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349 periods of DR stimulation and/or chemical application, and stored at -80 °C until chemical analysis. Solutions of drugs applied to the slice were tested for amino acid content. Determination of amino acid concentrations in the spinal perfusate was performed using high-performance liquid chromatography (HPLC) with o-phthaldialdehyde (OPA) precolumn derivatization and fluorimetric detection 13. Results are expressed as mean percent + S.E.M. of the basal outflow determined as the average of first 3 samples collected prior to stimulation. The statistical significance was determined by ANOVA: *P < 0.05; **P < 0.01. Basal concentrations of amino acids detected in 3-min samples of spinal superfusate were in the nanomolar range and the absolute values (nM) were 39.4 _+ 8.0 for Asp and 488.6 + 58.6 for Glu (n = 20). During the experiment, there was no tendency for the rate of outflow of the amino acids to show any increase, rather there was a gradual decline in time. In 8 different slices, selective activation of primary afferent A~-fibers (5-12 V, 20 ps, 3 Hz, 180-300 pulses), elicited a significant increase in the rate of basal outflow of Asp (194.2 _+ 15.6%, P < 0.01) and Glu (148.4 + 13.8%, P < 0.05) during the first collection period following the period of electrical stimulation of the dorsal roots (Fig. 1A,B). In the same slices, higher intensity of electrical stimulation (25-30 V, 0.41.0 ms, 0.5-1.0 Hz, 180-300 pulses) that recruited both primary afferent A- and C-fibers also caused a significant increase in the outflow of Asp (231.2 + 17.6%, P < 0.01) and Glu (182.9 _+ 16.5%, P < 0.01). However, the level of glutamine was not significantly changed. In Fig. 1A,B, the increase in the outflow of Asp and Glu is presented as a function of time. Whereas the increase in the basal outflow of Asp and Glu, evoked by a selective low-threshold (Aft) stimulation of primary afferents was present only during the first 3 mincollection period, the outflow elicited by a low- and high-threshold ( A + C ) stimulation was somewhat greater and needed a longer time to recover (Fig. 1A,B). However, when the effects of the A~ and the A + C stimulation were subjected to statistical analysis, there was no statistically significant difference (Asp: P < 0.67; Glu: P < 0.2).
For better assessment of the outflow of Asp and Glu observed in response to the differential stimulation of primary afferents, the net amount of Asp and Glu released in consecutive samples following Aft or A + C stimulation, was calculated for 6 experiments (Fig. 1C). Three results were noted: (1) recruitment of both the low (Aft)- and the low- and high-threshold ( A + C ) primary afferents resulted in a marked increase in the absolute amounts of Asp and Glu (Fig. 1C); (2) although the absolute amount of Glu in the spinal superfusate significantly (P < 0.01) exceeded that of Asp (Fig. 1C), the relative increase in the Aft or A + C stimulation-evoked outflow of Asp was higher (P < 0.05) than that of Glu (Fig. 1A,B); (3) the greater increase in the amount of Asp relative to Glu produced by the electrical stimulation of A + C afferents (720.0% and 216.3%, respectively, of that elicited by Aft-fiber stimulation), was reflected in an increased Asp/Glu outflow ratio. The Asp/Glu outflow ratio was 0.045 after stimulation of Afl-afferents and 0.145 after stimulation of A+C-afferents (P < 0.09). In order to examine whether the selective activation of small diameter primary afferents results in the increased outflow of Glu and/or Asp from the spinal slice, we used capsaicin (8-methyl-N-vanillyl-6-noneamide) and its potent analog, resiniferatoxin (RTX) 14, as tools for selective activation of most of C afferent fibers. When the L4-L6 D R G were perfused with capsaicin (5-10 #M for 10 min, n = 4) or RTX (0.1-1.0 nM for 3 rain, n = 4) an increase in the outflow of Asp and Glu was measured in the spinal slice superfusate (Table I). Superfusion of the spinal slices (n = 4) with a #-opioid receptor agonist D A G O did not have any consistent effect on the basal outflow of Asp and Glu. D A G O (5-10 /zM for 10 rain), however, applied 5 rain prior to and during the period of electrical stimulation of DRs, significantly reduced the outflow of Asp and Glu evoked by A + C stimulation (30 V, 1 ms, 1 Hz for 5 min) of primary afferents in 4 different slices (Fig. 2A,B). In the same slices, the depressant effect of D A G O was effectively reversed by naloxone (5-10/zM for 15-20 min), as illustrated in Fig. 2C. These findings suggest that the effect of D A G O on the stimulation-evoked outflow of Glu and Asp is a true/a-opioid receptor mediated response.
TABLE I Summarized effects of the perfusion of DRG (Lc-L6) with capsaicin and R TX on the ouOqow of endogenous Asp, Glu and Gin from the spinal slices
Results are presented as mean percentages _+S.E.M. of the basal effluxin 30- to 37-day-oldrats.
Capsaicin (5-10/~Mfor 5-10 rain) RTX (0.1-1.0 nM for 3 min) *P < 0.05; **P < 0.01.
n
Aspartate
Glutamate
Glutamine
4 4
229.5 _+45.1" 235.7 + 54.6
161.8 + 19.8" 235.5 + 49.5"*
104.5 + 28.3 191.2 + 57.6
350 The experiments described here demonstrate that the electrical activation of either low- or both low- and high-threshold primary afferent fibers is accompanied by an increase in the amounts of endogenous Glu and Asp released into the spinal slice superfusate 1°,11. An interesting finding is that significant release of physiologically active amino acids occurs at relatively modest rates of stimulation. Although there are good morphological4,21,22 and f~Jnctional indications4,17 that especially Glu, but more recently also Asp, can serve as neurotransmitters of primary afferent fibers, biochemical evidence of the release of these amino acids from defined neuronal elements has been difficult to obtain. Since it is experimentally possible to activate relatively selectively most of primary afferent C-fibers with capsaicin4 we have used this agent, and also a more potent capsaicin analog rcsiniferatoxin14 to investigate whether neuronal release of Gin and Asp in the spinal dorsal horn can be attributed to the activation of a specific population of capsaicin-sensitive primary sensory neurons. Our finding that the acute administration of capsaicin, or resiniferatoxin, caused the enhancement of the basal release of both Glu and Asp suggests that it is likely that a significant proportion of the two amino acids released in the spinal dorsal horn arises as a result of the activation of primary sensory endings sensitive to capsaicin. Similar conqlusion was reached in our earlier work where the neonatal capsaicin treatment attenuated the enhancement of the high-intensity electrical stimulation-evoked release of Glu and Asp 9. However, before it can be concluded that a causal relationship exists between increased Asp and/or Glu release and the activation of a specific category of primary afferent fibers it will be essential to establish in a future work that the increased amounts of the respective amino acids in the spinal slice superfusate reflect an increase in the amount of release from active primary afferent terminals, rather than a decrease in uptake, or release from other neuronal or non-neuronal (glia) sources. The possibility that Glu and/or Asp are transmitters of the primary sensory neurons has been frequently dis.-.>
Fig. 2. D A G O blockade of the DR-stimulation evoked outflow of endogenous Asp and Glu is naloxone-sensitive. Electrical stimulation of a lumbar dorsal rootlet (30 V, 1 ms, 1 Hz for 5 min; second set of arrows) in 4 experiments elicited a significant increase (P < 0.01) in the basal outflow of Asp (A) and Glu (B). The evoked outflow of Asp and Giu was reduced in the presence of D A G O (10 /JM for 10 rain; first set of arrows). 33- to 34-day-old rats. C: in 4 experiments, electrical stimulation of a lumbar dorsal rootlet (30 V, 200 ps, 1 Hz for 5 rain) in the presence of D A G O (5-10/~M for 10 min, application began 5 rain prior to stimulation and lasted for 10 min) elicited only a small increase in the basal outflow of Asp (hatched bars) and Glu (solid bars). However, during the combined administration of D A G O and naloxone (5-10/~M for 15-20 min, application started 5-10 rain prior to DAGO), the dorsal rootelectrical stimulation evoked a marked increase in the outflow of Asp and Glu. 32- to 36-day-old rats.
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Brain Research, 553 (1991) 347-352 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 0006899391 BRES 24742
Outflow of endogenous aspartate and glutamate from the rat spinal dorsal horn in vitro by activation of low- and high-threshold primary afferent fibers. Modulation by t-opioids Ivan Kangrga and Mirjana Randi6 Department of Veterinary Physiology and Pharmacology, Iowa State University, Ames, IA 50010 (U.S.A.) (Accepted 9 April 1991) Key words: Excitatory synaptic transmission; Glutamate; Aspartate; g-Opioid receptor; Rat; Dorsal horn slice; High-performanceliquid chromatography
Possible correlation of release of endogenous glutamate (Gin) and aspartate (Asp) with stimulation parameters used to activate primary sensory neurons was examined using the rat spinal cord slice - - dorsal root ganglion preparation and high performance liquid chromatography with fluorimetric detection. Selective activation of the low-threshold (Aft) primary afferent fibers resulted in a two-fold increase in the rate of basal outflow of Asp and a smaller increase in the outflow of Glu from the rat spinal dorsal horn slices into the superfusing medium. The activation of both the low (Aft)- and the high-threshold (At$+C) primary afferents elicited also a significant increase in the outflow of Asp and Gin relative to control. Ghi and Asp are released in significant amounts following superfusion of the dorsal root ganglia with capsaicin or resiniferatoxin. DAGO (Tyr-D-Ala-Gly-MePhe-Gly-ol-enkephalin),an agonist at /~-opioid receptors, attenuated the high-intensity stimulation-evoked outflow of Asp and Gin in a naloxone-sensitive manner. Our results have provided further evidence in support of the contention that Glu and Asp act as excitatory synaptic transmitters in the spinal dorsal horn. A role for g-opioid receptors in modulation of spinal processing of somatosensory information is indicated. The excitatory amino acids (EAA), glutamate (Glu) and aspartate (Asp), are the principal excitatory neurotransmitter candidates in the central nervous system 12, 15,18 and spinal cord 4"15,2°. Although Glu has been long favored as a transmitter of the primary afferent fibers, anatomical and physiological evidence has recently emerged suggesting a role for Asp in the primary afferent neurotransmission. Asp has been detected by immunocytochemistry in approximately 15% of the unmyelinated and 4% of the myelinated axons in the rat LA dorsal root (DR) 22, and electrically evoked release of Asp has been demonstrated from the slices of medulla ]2 and the spinal dorsal horn 9-1]. The actions of E A A are mediated by at least three distinct receptor subtypes characterized on the basis of their responsiveness to selective agonists: Nmethyl-D-aspartate (NMDA),a-amino-3-hydroxy-5-meth_ yl-4-isoxazole-propionic acid (AMPA) and kainate (KA). Whereas glutamate activates both NMDA and nonN M D A receptors, aspartate is thought to act preferentially at the N M D A receptors 3,4,15. About 85% of acutely isolated rat spinal dorsal horn neurons are sensitive to N M D A 17 and both non-NMDA and N M D A receptors participate in the fast 2'3'5 and the slow excitatory trans-
mission in the dorsal horn 6. Opioid peptides have been implicated in modulation of spinal segmental transmission 4's'2° and the presence of functional g- and iS-receptors on C primary afferent neurons has been demonstrated 1,7. The objective of this study was to investigate the pattern of outflow of endogenous Glu, Asp and glutamine (Gin) from the superfused dorsal horn slice in response to selective activation of the low-threshold or both the low- and high-threshold primary afferent fibers by electrical stimulation of lumbar dorsal roots. In addition, the possibility of a modulation of the D R stimulation-evoked outflow of Glu and Asp by a/z-opioid receptor agonist D A G O was examined. Some aspects of this work have been reported l°,n. Longitudinal slices (300-400/zm), with dorsal roots (DR) and dorsal root ganglia (L4-L6) attached, were obtained from 27- to 57-day-old Sprague-Dawley rats 9. After 1 h of incubation at 30 _+ 1°C a slice was transferred into one compartment of a 2 compartmentchamber and continuously superfused with oxygenated artificial cerebrospinal fluid at a flow rate of 0.4 ml/min -1. The composition of the solution was in mM: NaC1, 124;
Correspondence: M. Randi6, Department of Veterinary Physiologyand Pharmacology,2008 Vet Med Bldg, Iowa State University, Ames, IA 50010, U.S.A.
348 KC1, 1.9; KHzPO4, 1.2; CaCI2, 2.4; MgSO4, 1.3; N a H C O 3, 26; glucose, 10; p H 7.4. The dorsal roots were led across a leak-proof partition of vaseline into the other compartment which was filled with mineral oil and placed on two pairs of bipolar platinum electrodes. The distal pair was used for stimulation of the D R , and the proximal pair for recording and continuous monitoring of compound action potentials of primary afferent fibers. Each dorsal root having good and stable A and C compound action potentials was subjected to two 3-min periods of electrical stimulation. The stimulation parameters were selected to activate low-threshold, fast conducting (Aft) fibers during the first stimulation period and the additional recruitment of high-threshold, slowly-conducting myelinated (A6) and unmyelinated (C) fibers during the second stimulation period. In a few experiments this stimulation sequence was reversed. Conduction velocities, estimated from the distance between the centers of the two bipolar electrodes and the latency of the first negative deflection of a volley, were: Aa,fl, 22.6 + 1.9 ms -1 and C, 0.32 + 0.02 ms -1, (n = 20). D A G O (Tyr-D-Ala-Gly-MePhe-Gly-ol-enkephalin, Cambridge Research Biochemicals), naloxone (Du Pont), capsaicin (Sigma) and resiniferatoxin (RTX) (Chemsyn Science Laboratories) were bath-applied in known concentrations. In experiments where the effects of D A G O and naioxone on the stimulation-elicited outflow of Glu and/or Asp were examined, the combined application of D A G O + naloxone always followed D A G O application alone in the same slices. Samples of the perfusate were continuously collected at regular intervals (during the entire 3 min periods) prior to, during, and after the
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Fig. 1. A,B: the time course of the outflow of Asp (A) and Glu (B) from the spinal slice in response to dorsal root electrical stimulation. While the selective activation of the low-threshold (Aft) PAF (5-12 V, 20/~s, 3 Hz, 180-300 pulses) produced a significant increase in the basal outflow of Asp and Glu during the first 3-min collection period (A-B, single arrows), the activation of both the low- and the high-threshold (A+C) PAF (25-30 V, 0.4-1.0 ms, 1 Hz, 180-300 pulses) resulted in a somewhat greater but characteristically more prolonged increase in the outflow of both Asp and Glu (A-B, double arrows). The results are presented as mean percent of the basal outflow preceding each stimulation period + S.E.M. for 8 experiments. **P < 0.01; *P < 0.05. The inset in A depicts corresponding A and A + C compound action potentials of the PAF recorded during the low- (left record) and the highintensity (right record) dorsal root electrical stimulation; arrows show the stimulus artifacts. In the right record the Aft action potential was truncated. Bars: left record = 6 mV and 1.5 ms; right record = 0.6 mV and 12 ms. C: the average (n = 6) amount of Asp (solid column) and Glu (hatched column) released in response to activation of Aft or A+C afferents is presented. The basal outflow of Asp and Glu measured prior to each stimulation period has been subtracted. When A+C primary afferents were activated, a marked increase in the absolute amounts of Asp and Glu (702.0% and 216.3%, respectively, of that elicited by Aft stimulation) in the spinal perfusate was observed. 30- to 57-day-old rats.
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349 periods of DR stimulation and/or chemical application, and stored at -80 °C until chemical analysis. Solutions of drugs applied to the slice were tested for amino acid content. Determination of amino acid concentrations in the spinal perfusate was performed using high-performance liquid chromatography (HPLC) with o-phthaldialdehyde (OPA) precolumn derivatization and fluorimetric detection 13. Results are expressed as mean percent + S.E.M. of the basal outflow determined as the average of first 3 samples collected prior to stimulation. The statistical significance was determined by ANOVA: *P < 0.05; **P < 0.01. Basal concentrations of amino acids detected in 3-min samples of spinal superfusate were in the nanomolar range and the absolute values (nM) were 39.4 _+ 8.0 for Asp and 488.6 + 58.6 for Glu (n = 20). During the experiment, there was no tendency for the rate of outflow of the amino acids to show any increase, rather there was a gradual decline in time. In 8 different slices, selective activation of primary afferent A~-fibers (5-12 V, 20 ps, 3 Hz, 180-300 pulses), elicited a significant increase in the rate of basal outflow of Asp (194.2 _+ 15.6%, P < 0.01) and Glu (148.4 + 13.8%, P < 0.05) during the first collection period following the period of electrical stimulation of the dorsal roots (Fig. 1A,B). In the same slices, higher intensity of electrical stimulation (25-30 V, 0.41.0 ms, 0.5-1.0 Hz, 180-300 pulses) that recruited both primary afferent A- and C-fibers also caused a significant increase in the outflow of Asp (231.2 + 17.6%, P < 0.01) and Glu (182.9 _+ 16.5%, P < 0.01). However, the level of glutamine was not significantly changed. In Fig. 1A,B, the increase in the outflow of Asp and Glu is presented as a function of time. Whereas the increase in the basal outflow of Asp and Glu, evoked by a selective low-threshold (Aft) stimulation of primary afferents was present only during the first 3 mincollection period, the outflow elicited by a low- and high-threshold ( A + C ) stimulation was somewhat greater and needed a longer time to recover (Fig. 1A,B). However, when the effects of the A~ and the A + C stimulation were subjected to statistical analysis, there was no statistically significant difference (Asp: P < 0.67; Glu: P < 0.2).
For better assessment of the outflow of Asp and Glu observed in response to the differential stimulation of primary afferents, the net amount of Asp and Glu released in consecutive samples following Aft or A + C stimulation, was calculated for 6 experiments (Fig. 1C). Three results were noted: (1) recruitment of both the low (Aft)- and the low- and high-threshold ( A + C ) primary afferents resulted in a marked increase in the absolute amounts of Asp and Glu (Fig. 1C); (2) although the absolute amount of Glu in the spinal superfusate significantly (P < 0.01) exceeded that of Asp (Fig. 1C), the relative increase in the Aft or A + C stimulation-evoked outflow of Asp was higher (P < 0.05) than that of Glu (Fig. 1A,B); (3) the greater increase in the amount of Asp relative to Glu produced by the electrical stimulation of A + C afferents (720.0% and 216.3%, respectively, of that elicited by Aft-fiber stimulation), was reflected in an increased Asp/Glu outflow ratio. The Asp/Glu outflow ratio was 0.045 after stimulation of Afl-afferents and 0.145 after stimulation of A+C-afferents (P < 0.09). In order to examine whether the selective activation of small diameter primary afferents results in the increased outflow of Glu and/or Asp from the spinal slice, we used capsaicin (8-methyl-N-vanillyl-6-noneamide) and its potent analog, resiniferatoxin (RTX) 14, as tools for selective activation of most of C afferent fibers. When the L4-L6 D R G were perfused with capsaicin (5-10 #M for 10 min, n = 4) or RTX (0.1-1.0 nM for 3 rain, n = 4) an increase in the outflow of Asp and Glu was measured in the spinal slice superfusate (Table I). Superfusion of the spinal slices (n = 4) with a #-opioid receptor agonist D A G O did not have any consistent effect on the basal outflow of Asp and Glu. D A G O (5-10 /zM for 10 rain), however, applied 5 rain prior to and during the period of electrical stimulation of DRs, significantly reduced the outflow of Asp and Glu evoked by A + C stimulation (30 V, 1 ms, 1 Hz for 5 min) of primary afferents in 4 different slices (Fig. 2A,B). In the same slices, the depressant effect of D A G O was effectively reversed by naloxone (5-10/zM for 15-20 min), as illustrated in Fig. 2C. These findings suggest that the effect of D A G O on the stimulation-evoked outflow of Glu and Asp is a true/a-opioid receptor mediated response.
TABLE I Summarized effects of the perfusion of DRG (Lc-L6) with capsaicin and R TX on the ouOqow of endogenous Asp, Glu and Gin from the spinal slices
Results are presented as mean percentages _+S.E.M. of the basal effluxin 30- to 37-day-oldrats.
Capsaicin (5-10/~Mfor 5-10 rain) RTX (0.1-1.0 nM for 3 min) *P < 0.05; **P < 0.01.
n
Aspartate
Glutamate
Glutamine
4 4
229.5 _+45.1" 235.7 + 54.6
161.8 + 19.8" 235.5 + 49.5"*
104.5 + 28.3 191.2 + 57.6
350 The experiments described here demonstrate that the electrical activation of either low- or both low- and high-threshold primary afferent fibers is accompanied by an increase in the amounts of endogenous Glu and Asp released into the spinal slice superfusate 1°,11. An interesting finding is that significant release of physiologically active amino acids occurs at relatively modest rates of stimulation. Although there are good morphological4,21,22 and f~Jnctional indications4,17 that especially Glu, but more recently also Asp, can serve as neurotransmitters of primary afferent fibers, biochemical evidence of the release of these amino acids from defined neuronal elements has been difficult to obtain. Since it is experimentally possible to activate relatively selectively most of primary afferent C-fibers with capsaicin4 we have used this agent, and also a more potent capsaicin analog rcsiniferatoxin14 to investigate whether neuronal release of Gin and Asp in the spinal dorsal horn can be attributed to the activation of a specific population of capsaicin-sensitive primary sensory neurons. Our finding that the acute administration of capsaicin, or resiniferatoxin, caused the enhancement of the basal release of both Glu and Asp suggests that it is likely that a significant proportion of the two amino acids released in the spinal dorsal horn arises as a result of the activation of primary sensory endings sensitive to capsaicin. Similar conqlusion was reached in our earlier work where the neonatal capsaicin treatment attenuated the enhancement of the high-intensity electrical stimulation-evoked release of Glu and Asp 9. However, before it can be concluded that a causal relationship exists between increased Asp and/or Glu release and the activation of a specific category of primary afferent fibers it will be essential to establish in a future work that the increased amounts of the respective amino acids in the spinal slice superfusate reflect an increase in the amount of release from active primary afferent terminals, rather than a decrease in uptake, or release from other neuronal or non-neuronal (glia) sources. The possibility that Glu and/or Asp are transmitters of the primary sensory neurons has been frequently dis.-.>
Fig. 2. D A G O blockade of the DR-stimulation evoked outflow of endogenous Asp and Glu is naloxone-sensitive. Electrical stimulation of a lumbar dorsal rootlet (30 V, 1 ms, 1 Hz for 5 min; second set of arrows) in 4 experiments elicited a significant increase (P < 0.01) in the basal outflow of Asp (A) and Glu (B). The evoked outflow of Asp and Giu was reduced in the presence of D A G O (10 /JM for 10 rain; first set of arrows). 33- to 34-day-old rats. C: in 4 experiments, electrical stimulation of a lumbar dorsal rootlet (30 V, 200 ps, 1 Hz for 5 rain) in the presence of D A G O (5-10/~M for 10 min, application began 5 rain prior to stimulation and lasted for 10 min) elicited only a small increase in the basal outflow of Asp (hatched bars) and Glu (solid bars). However, during the combined administration of D A G O and naloxone (5-10/~M for 15-20 min, application started 5-10 rain prior to DAGO), the dorsal rootelectrical stimulation evoked a marked increase in the outflow of Asp and Glu. 32- to 36-day-old rats.
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