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Purine receptors and kynurenic acid modulate the somatosensory evoked potential in rat cerebral cortex
Electroencephalograph)' and clinical Neurophysiologv, 1988, 69: 186-189
186
Elsevier Scientific Publishers Ireland, Ltd. EEG02059
Short communication
Purine receptors and kyn~nrenic acid modulate the somatosensory evoked potential in rat cerebral cortex Jonas I. Addae 1 and Trevor W. Stone Department of Physiology, St. George's Hospital Medwal School, Cranmer Terrace, London S WI 7 ORE ( L~K.) (Accepted for publication: 6 October 1987)
Summary
Adenosine and analogues, and antagonists to adenosine and putauve excitatory amino acid transmitters were topically applied to the cerebral cortex of urethane-anaesthetised rats and their effects on the somatosensory evoked potentials (SEPs) examined. 2-Chloro-adenosine decreased the amplitude of the SEPs whereas adenosine did not. Both L-( )N6-phenyl-isopropyladenosine (L-PIA) and 5'-N-ethylcarboxamide adenosine (NECA) depressed the SEPs: the effect of L-PIA was more marked than that of NECA. 8-p-Sulphophenyl theophylline increased the amplitude of the SEPs and also inhibited the effects of 2-chloro-adenosine and L-PIA. Kynurenic acid decreased the amplitude of the SEPs. The results suggest that the initial component of the SEP is a post-synaptic event and that endogenous adenosine probably modulates thalamo-cortical synaptic transmission
Key words: A m i n o acids: Evoked potentials; Cerebral cortex; Purines
Recordings of the somatosensory evoked potential from the cortical surface usually result in an initial positive followed by a negative wave. A recording from more than 0.4 m m below the cortical surface gives a negative-positive wave form, the time-course of which indicates a mirror image of the simultaneous surface recording (Amassian et al. 1964: Schlag 1973; Bindman and Lippold 1981). The initial negative wave from the deep recording (N1) is perhaps the best understood of the components of the SEP (Schlag 1973; Angel 1977) but its origin is still not known with certainty. It m a y for example represent either the pre-synaptic volley in the thalamo-cortical fibres, or the post-synaptic activity of cell somata located in, or dendrites coursing through, cortical layer IV (Angel 1977: Angel et al. 1980). The actions of purine derivatives in the central nervous system have been well documented. These actions include the alteration of neuronal firing rate (Phillis et al. 1979: Stone and Perkins 1979: Stone 1982) and the modulation of neurotransminer release from nerve terminals in the peripheral and
] Present address: Department of Physiology, Eric Williams Medical Sciences Complex, C a m p Fleurs, Trinidad and Tobago.
Correspondence to: Trevor W. Stone, Department of Physiology, St. George's Hospital Medical School, Cranmer Terrace, London SW17 ORE (U.K.).
central nervous systems (see Stone 1981). At many of these presynaptic sites N6-substituted analogues of adenosine such as N6-phenylisopropyladenosine (PIA) are more potent than compounds substituted at the 5' position. In the present study therefore such analogues were used to assess whether a similar receptor existed in the neocortex and also to determine the origin of the early negative N1 wave recorded within the grey matter. The amino acid antagonist kynurenic acid (Perkins and Stone 1982: Stone and Connick 1985) was also tested.
Method The experimental method used in this study has already been described in detail (Addae and Stone 1986). Briefly, male Wistar rats weighing 180-300 g were anaesthetised with urethane (l.3-1.7 g / k g ) and the contralateral forepaw representation on the cerebral cortex exposed. A well of paraffin wax was constructed to hold 0.5 ml of fluid over the exposed cortex. Solutions of various compounds were made in an artificial cerebro-spinal fluid (ACSF) and used to form a static pool over the exposed cortex at room temperature. The ACSF contained (in mM) K H 2 P O 4, 2.2: MgSO 4, 1.2: KC1, 2.0: glucose, 10.0: NaHCO3, 25; NaC1, 115 and CaCI 2, 1.25. Monopolar recordings of the SEPs from forepaw stimulation were made with a glass microelectrode containing 3 M NaCI, at 0.4-0.8 m m below the cortical surface. The potentials were amplified and displayed on oscilloscopes one of which, a Gould 4020 digital oscilloscope, was arranged to plot each
0013-4649/88/$03.50 C 1988 Elsevier Scientific Publishers Ireland, Ltd.
P U R I N E RECEPTORS
187
potential immediately onto a chart recorder. The potentials were elicited by a pair of needle electrodes inserted into the contralateral forepaw. Stimuli were applied every 1-2 sec (to allow the digital plotting of the potential over a 1 sec period), using parameters of 1 msec duration and 50-500 /~A amplitude applied via a constant current stimulus isolator. The size of the stimulus varied from animal to animal depending on the exact location of the stimulating electrodes. Compounds were applied to the cortex for variable times (5-120 min) but always long enough to cause sustained changes in the amplitude of the SEPs. When a compound, e.g., L-PIA or NECA, was applied for over 15 min, the solution in the cup was replenished every 15 min to minimise stagnation and calcium precipitation. Following the application of a compound, the cortex was washed with pre-gassed (95% 02, 5% CO2) ACSF by continuous superfusion. The effect of the compound was assessed by expressing the change in amplitude of the initial negative wave of the SEP (N1) as a percentage of the control value.
Results
Application of 2-chloro-adenosine to the cortical surface, 0.1 mM for 5 min, caused a 75 + 5% ( m e a n + S.E.M.) decrease in the amplitude of N1 (n = 8) and at 0.01 mM for 5 min
0.1mM Adenosine
,
1
2my
1 rain
~lrnMC~Adenosine
Fig. 1. Effects of adenosine and 2-chloroadenosine (Cl-adenosine) on the SEPs. Cl-adenosine but not adenosine decreased all components of the SEPs. The small increase in the amplitude of N1 and P2 seen on the slow record at the onset of both drugs is an artefact resulting from the introduction of a solution to the cortex. There was recovery of the SEPs to the control level 20 rain after the removal of Cl-adenosine. The labelled graduation bars apply to only the slow record. Representative averaged responses show the wave forms of the SEPs at the times on the slow record indicated by the downward arrows. The horizontal bar at the top right of the figure represents 20 msec and applies to only the averaged responses.
]2mV I rain 5raM
K YA
~ t t t t
Fig. 2. Effect of kynurenic acid (KYA) on the SEPs showing depression of all the components. Upward arrows indicate 5 min breaks in the recording.
decreased N1 by 18+5% (n = 8 ) (Fig. 1). There was always complete recovery of the SEPs 20-40 min after withdrawal of 2-chloro-adenosine from the cup and washing the cortex with pre-gassed ACSF. Adenosine at 0.1 and 1.0 mM for 5 rain did not change the amplitude of the components of the SEP (Fig. 1) presumably due to its rapid uptake and metabolism. The adenosine antagonist 8-p-sulphophenyl theophylline (8-PST), 0.1 mM for 10 min increased the amplitude of N1 by 27 +7% (n = 5). When 0.1 mM 8-PST was applied for 10 min prior to and together with 2-chloro-adenosine (0.1 mM for 5 min) the latter now decreased N1 by only 10+3% (n = 5). The reduction in the effect of 2-chloro-adenosine by 8-PST was highly significant ( P < 0.001, Mann-Whitney test). L-PIA had a slow effect, e.g., when applied at 1 btM the onset of the decrease in the SEPs occurred after 20-30 min and there was a maximal depression after 90-120 min of application. The mean decrease in N1 was 49+ 3% (n = 4). There was recovery of the SEPs to the control values when the cortex was washed for at least 120 min. 8-PST prevented the effects of L-PIA when applied at 10 ~tM for 15 min prior to and together with 1 /~M L-PIA for 120 min (n = 3). A lower concentration of 0.1 /tM L-PIA had no observable effect after 120 min. However, at 5 /tM a maximal depression of 82+8% ( n = 3) was achieved. NECA had a weaker effect than L-PIA. At 1 /~M NECA decreased N1 by 9+6% (n = 4) with the maximum effect observed after 40-70 min of application. There was recovery of the SEPs after washing the cortex for at least 60 min. Again a lower concentration of 0.1 /tM had no apparent effect on the cortex, while 5 /zM produced a maximal depression of 27 + 6% (n = 4).
Kvnurenic acid Kynurenic acid has been shown to block excitatory amino acid-mediated synaptic transmission (Perkins and Stone 1982, 1985; Brookes et al. 1986). Application of kynurenate here caused a dose- and time-dependent decrease in all components of the SEPs. When applied for 5 min, 5 mM kynurenate decreased N1 by 79+4% (n = 8) (Fig. 2) and 1 mM kynurenate decreased N1 by 40+6% (n = 4). There was recovery_ of the SEPs to the control level after washing the cortex for at least 20 min.
188 Discussion
Adenosine is known to block synaptic transmission by inhibiting the release of transmitters from pre-synaptic terminals (Phillis et al. 1979: Stone 198l). Adenosine has an efficient inactivation mechanism resulting from both an enzymatic breakdown (Clark et al. 1952; Rockwell and Maguire 1966) and an uptake system (Huang and Daly 1974: Hertz 1978) although the former is probably more important in vivo (Phillis and Edstrom 1976). 2-Chloro-adenosine selectively acts on the adenosine receptor (Huang and Daly 1974) but is not a substrate for adenosine deaminase (Clark et al. 1952; Rockwell and Maguire 1966). Thus, 2-chloro-adenosine could have diffused from the cortical cup into the deep cortical layers (e.g,, layers Ill and IV) much more readily than adenosine to inhibit synaptic transmission. This probably explains why there was an observable effect with 2-chloro-adenosine but not adenosine. Although the N1 wave is the best understood component of the SEP, it is yet to be established whether N1 is due to the pre-synaptic volley in the thalamo-cortical fibres or the postsynaptic activity in layer IV (Angel 1977: Angel et al. 1980). The decrease in N1 (and subsequent components of the SEP) caused by 2-chloro-adenosine could suggest that N1 is mainly due to a post-synaptic event. This result agrees with the finding that topical application of MgC12 (to block synaptic transmission) greatly attenuated the amplitude of the N1 wave (Bindm a n and Milne 1977; Bindman et al. 1979). 8-p-Sulphophenyl theophylline (8-PST) significantly inhibited the effects of 2-chloro-adenosine as has been reported in previous studies (e.g., Phillis et al. 1979). The 27% increase in N1 caused by 8-PST could be explained by an antagonism of endogenously released adenosine which normally depressed the thalamo-cortical synaptic transmission. This conclusion would be in accord with other studies in which increases of cell excitability have been noted upon treatment with xanthines, both in vivo (Phillis et al. 1979) and in vitro (Dunwiddie 1980; Ault et al. 1986). The very slow time course of responses to L-PIA and N E C A in vivo made any attempt at quantification very difficult. We have therefore compared the activity of these two substances at 3 concentrations. At 0.1 # M no response was seen to either, but at the higher concentrations of 1 and 5 ~M, L-PIA was more effective than NECA. Since the responses were measured at their maximum, equilibrium point, it is unlikely that these activities are as greatly affected by lipid solubility as studies using brief microiontophoretic applications t Phillis 1982; Stone 1982). The present study therefore supports the conclusion of Dunwiddie et al. (1984) that the depression of synaptically evoked activity is mediated by a receptor similar to that which produces a depression of transmitter release in other systems. This conclusion in turn might support the view that the NI wave is due to synaptic transmission rather than being the afferent fibre volley potential. Kynurenic acid has been shown to block excitatory amino acid-mediated synaptic transmission at various parts of the CNS by what appears to be a purely post-synaptic action (Perkins and Stone 1982; G a n o n g et al. 1983: Stone and
J.I. ADDAE, T,W. STONE Connick 1985: Brooks et al. 1986): there is no evidence of a pre-synaptic action. The attenuation of N1 by kynurenate, therefore, also supports the view that N1 is mainly a post-synaptic event and further suggests that the thalamo-cortical afferents act on excitatory amino acid receptors. However, since kynurenate exhibits poor selectivity between the excitant receptor types (Perkins and Stone 1982: G a n o n g et al. 1983), it is not possible to deduce from the present data which of the receptor types is primarily involved. However. we have previously reported failure to block the SEP using the selective N-methyl-aspartate antagonist, 2-amino-5-phosphonovaleric acid (Addae and Stone 1986). The results from D-aspartate transport studies as well as microiontophoretic experiments also suggest that excitatory amino acids may be involved in thalamo-cortical synaptic transmission (Hicks and Guedes 1983; Ottersen et al. 1983; Tsumoto et al. 1086). We are grateful to the Wellcome Trust, Action Research for the Crippled Child, and Association of Commonwealth Universities for support.
References
Addae, J,I. and Stone, T.W. Effects of topically applied excitatory amino acids on evoked potentials and single cell activity in rat cerebral cortex. Europ. J. Pharmacol., 1986, 121: 337-343. Amassian, V.E., Waller, H.J. and Macy, J. Neural mechanism of the primary somatosensory evoked potential. Ann. N.Y. Acad. Sci., 1964, 112: 5-32. Angel, A, Processing of sensory information. Prog. Neurobiol., 1977, 9: 1-122. Angel, A,, Graftom D.A., Halsey, M.J. and Wardley-Smith, B. Pressure reversal of the effect of urethane on the evoked somatosensory cortical response in the rat. Brit. J. Pharmacol., 1980, 70: 241-247. Ault, B., Joyner, J.L., Olney, M.A. and Wang, C.M. Endogenous adenosine modulates neuronal activity in hippocampal area CA 3. Brit. J. Pharmacol., 1986, 88: 461P. Bindman. L. and Lippold, O. An analysis of possible neural mechanisms involved in the cortical response to somatic stimulation. In: The Neurophysiology of the Cerebral Cortex. Edward Arnold, London, 1981: 76-94. Bindman, L.J. and Milne, A.R. The reversible blocking action of topically applied magnesium solutions on neuronal activity in the cerebral cortex of the anaesthetised rat. J. Physiol. (Lond.), 1977, 269: 34-35P. Bindman, LJ., Lippold, O.C.J. and Milne. A.R. Prolonged changes in excitability of pyramidal tract neurones in the cat: a post-synaptic mechanism. J. Physiol. (Lond.), 1979, 286: 457-477. Brooks, P.A., Smith, D.A.S., Stone, T.W. and Kelly, J.S. Postsynaptic actions of kynurenic acid in the rat dentate gyrus. Neurosci. Lett,, 1986, 66: 96-100. Clark, R., Davoll, J., Phillips, F.S. and Brown, G.B. Enzymatic deamination and vasodepressor effects of adenosine analogs. J. Pharmacol. exp. Ther., 1952, 106:291 302.
PURINE RECEPTORS Dunwiddie, T.V. Endogenously released adenosine regulates excitability in the in vitro hippocampus. Epilepsia, 1980, 21:541-548. Dunwiddie, T.V., Basile, A.S. and Palmer, M.R. Electrophysiological responses to adenosine analogues in rat hippocampus and cerebellum. Evidence for mediation by adenosine receptors of the A1 subtype. Life Sci., 1984, 34: 34-47. Ganong, A.H., Lanthorn, T.H. and C o t m a m C.W. Kynurenic acid inhibits synaptic and acidic amino acid-induced responses in the rat hippocampus and spinal cord. Brain Res., 1983, 273: 170-174. Hertz, L. Kinetics of adenosine uptake into astrocytes. J. Neurochem., 1978, 31: 55-62. Hicks, T.P. and Guedes, R.C.A. Neuropharmacological properties of electrophysiologically identified visually responsive neurones of the posterior lateral suprasylvian area. Exp. Brain Res., 1983, 49: 157-173. Huang, M. and Daly, J.W. Adenosine-elicited accumulation of cyclic A M P in brain slices: potentiation by agents which inhibit uptake of adenosine. Life Sci., 1974, 14: 489-503. Ottersen, O.P., Fischer, B.O. and Storm-Mathisen, J. Retrograde transport of D-J3 H]aspartate in thalamo-cortical neurones. Neurosci. Lett., 1983, 42: 19-24. Perkins, M.N. and Stone, T.W. An iontophoretic investigation of convulsant kynurenines and their interaction with the endogenous excitant quinolinic acid, Brain Res., 1982, 247: 184-187. Perkins, M.N. and Stone, T.W. Actions of kynurenic acid in the rat hippocampus in vivo. Exp. Neurol., 1985, 88: 570-579. Phillis, J.W. Evidence for an A2-1ike adenosine receptor on
189 cerebral cortical neurons. J. Pharm. Pharmacol., 1982. 34: 453-454. Phillis, J.W. and Edstrom, J.P. Effects of adenosine analysis on rat cerebral cortical neurones. Life Sci., 1976, 19: 1041-1054. Phillis, J.W., Edstrom, J.P., Kostopoulos, G.K. and Kirkpatrick, J.R. Effects of adenosine and adenine nucleotides on synaptic transmission in the cerebral cortex. Canad. J. Physiol. Pharmacol., 1979, 57:1289 1312. Ribeiro, J.A. Purinergic modulation of transmitter release. J. theor. Biol., 1979, 80: 259-270. Rockwell, M. and Maguire, M.H. Studies on adenosine deaminase. I. Purification and properties of ox heart adenosine deaminase. Molec. Pharmacol., 1966.2: 574-584. Schlag, J. Generation of brain evoked potentials. In: R.F. T h o m p s o n and M.M. Patterson (Eds.). Bioelectric Recording Techniques, Part A. Academic Press, London, 1973: 273-316. Stone, T.W. Physiological roles for adenosine and adenosine 5'-triphosphate in the nervous system. Neuroscience, 1981~ 6: 523-555. Stone, T.W. Purine receptors involved in the depression of neuronal firing in cerebral cortex. Brain Res., 1982, 248: 367-370. Stone, T.W. and Connick, J.H. Quinolinic acid and other kynurenines in the CNS. Neuroscience, 1985, 15: 597-617. Stone, T.W. and Perkins, M.N. Is adenosine the mediator of opiate action on neuronal firing rate?. Nature (Lond./1979, 281: 227-228. Tsumoto, T., Masui, H. and Sato, H. Excitatory amino acid transmitters in neuronal circuits of the cat visual cortex. J. Neurophysiol., 1986, 55: 469-483.
186
Elsevier Scientific Publishers Ireland, Ltd. EEG02059
Short communication
Purine receptors and kyn~nrenic acid modulate the somatosensory evoked potential in rat cerebral cortex Jonas I. Addae 1 and Trevor W. Stone Department of Physiology, St. George's Hospital Medwal School, Cranmer Terrace, London S WI 7 ORE ( L~K.) (Accepted for publication: 6 October 1987)
Summary
Adenosine and analogues, and antagonists to adenosine and putauve excitatory amino acid transmitters were topically applied to the cerebral cortex of urethane-anaesthetised rats and their effects on the somatosensory evoked potentials (SEPs) examined. 2-Chloro-adenosine decreased the amplitude of the SEPs whereas adenosine did not. Both L-( )N6-phenyl-isopropyladenosine (L-PIA) and 5'-N-ethylcarboxamide adenosine (NECA) depressed the SEPs: the effect of L-PIA was more marked than that of NECA. 8-p-Sulphophenyl theophylline increased the amplitude of the SEPs and also inhibited the effects of 2-chloro-adenosine and L-PIA. Kynurenic acid decreased the amplitude of the SEPs. The results suggest that the initial component of the SEP is a post-synaptic event and that endogenous adenosine probably modulates thalamo-cortical synaptic transmission
Key words: A m i n o acids: Evoked potentials; Cerebral cortex; Purines
Recordings of the somatosensory evoked potential from the cortical surface usually result in an initial positive followed by a negative wave. A recording from more than 0.4 m m below the cortical surface gives a negative-positive wave form, the time-course of which indicates a mirror image of the simultaneous surface recording (Amassian et al. 1964: Schlag 1973; Bindman and Lippold 1981). The initial negative wave from the deep recording (N1) is perhaps the best understood of the components of the SEP (Schlag 1973; Angel 1977) but its origin is still not known with certainty. It m a y for example represent either the pre-synaptic volley in the thalamo-cortical fibres, or the post-synaptic activity of cell somata located in, or dendrites coursing through, cortical layer IV (Angel 1977: Angel et al. 1980). The actions of purine derivatives in the central nervous system have been well documented. These actions include the alteration of neuronal firing rate (Phillis et al. 1979: Stone and Perkins 1979: Stone 1982) and the modulation of neurotransminer release from nerve terminals in the peripheral and
] Present address: Department of Physiology, Eric Williams Medical Sciences Complex, C a m p Fleurs, Trinidad and Tobago.
Correspondence to: Trevor W. Stone, Department of Physiology, St. George's Hospital Medical School, Cranmer Terrace, London SW17 ORE (U.K.).
central nervous systems (see Stone 1981). At many of these presynaptic sites N6-substituted analogues of adenosine such as N6-phenylisopropyladenosine (PIA) are more potent than compounds substituted at the 5' position. In the present study therefore such analogues were used to assess whether a similar receptor existed in the neocortex and also to determine the origin of the early negative N1 wave recorded within the grey matter. The amino acid antagonist kynurenic acid (Perkins and Stone 1982: Stone and Connick 1985) was also tested.
Method The experimental method used in this study has already been described in detail (Addae and Stone 1986). Briefly, male Wistar rats weighing 180-300 g were anaesthetised with urethane (l.3-1.7 g / k g ) and the contralateral forepaw representation on the cerebral cortex exposed. A well of paraffin wax was constructed to hold 0.5 ml of fluid over the exposed cortex. Solutions of various compounds were made in an artificial cerebro-spinal fluid (ACSF) and used to form a static pool over the exposed cortex at room temperature. The ACSF contained (in mM) K H 2 P O 4, 2.2: MgSO 4, 1.2: KC1, 2.0: glucose, 10.0: NaHCO3, 25; NaC1, 115 and CaCI 2, 1.25. Monopolar recordings of the SEPs from forepaw stimulation were made with a glass microelectrode containing 3 M NaCI, at 0.4-0.8 m m below the cortical surface. The potentials were amplified and displayed on oscilloscopes one of which, a Gould 4020 digital oscilloscope, was arranged to plot each
0013-4649/88/$03.50 C 1988 Elsevier Scientific Publishers Ireland, Ltd.
P U R I N E RECEPTORS
187
potential immediately onto a chart recorder. The potentials were elicited by a pair of needle electrodes inserted into the contralateral forepaw. Stimuli were applied every 1-2 sec (to allow the digital plotting of the potential over a 1 sec period), using parameters of 1 msec duration and 50-500 /~A amplitude applied via a constant current stimulus isolator. The size of the stimulus varied from animal to animal depending on the exact location of the stimulating electrodes. Compounds were applied to the cortex for variable times (5-120 min) but always long enough to cause sustained changes in the amplitude of the SEPs. When a compound, e.g., L-PIA or NECA, was applied for over 15 min, the solution in the cup was replenished every 15 min to minimise stagnation and calcium precipitation. Following the application of a compound, the cortex was washed with pre-gassed (95% 02, 5% CO2) ACSF by continuous superfusion. The effect of the compound was assessed by expressing the change in amplitude of the initial negative wave of the SEP (N1) as a percentage of the control value.
Results
Application of 2-chloro-adenosine to the cortical surface, 0.1 mM for 5 min, caused a 75 + 5% ( m e a n + S.E.M.) decrease in the amplitude of N1 (n = 8) and at 0.01 mM for 5 min
0.1mM Adenosine
,
1
2my
1 rain
~lrnMC~Adenosine
Fig. 1. Effects of adenosine and 2-chloroadenosine (Cl-adenosine) on the SEPs. Cl-adenosine but not adenosine decreased all components of the SEPs. The small increase in the amplitude of N1 and P2 seen on the slow record at the onset of both drugs is an artefact resulting from the introduction of a solution to the cortex. There was recovery of the SEPs to the control level 20 rain after the removal of Cl-adenosine. The labelled graduation bars apply to only the slow record. Representative averaged responses show the wave forms of the SEPs at the times on the slow record indicated by the downward arrows. The horizontal bar at the top right of the figure represents 20 msec and applies to only the averaged responses.
]2mV I rain 5raM
K YA
~ t t t t
Fig. 2. Effect of kynurenic acid (KYA) on the SEPs showing depression of all the components. Upward arrows indicate 5 min breaks in the recording.
decreased N1 by 18+5% (n = 8 ) (Fig. 1). There was always complete recovery of the SEPs 20-40 min after withdrawal of 2-chloro-adenosine from the cup and washing the cortex with pre-gassed ACSF. Adenosine at 0.1 and 1.0 mM for 5 rain did not change the amplitude of the components of the SEP (Fig. 1) presumably due to its rapid uptake and metabolism. The adenosine antagonist 8-p-sulphophenyl theophylline (8-PST), 0.1 mM for 10 min increased the amplitude of N1 by 27 +7% (n = 5). When 0.1 mM 8-PST was applied for 10 min prior to and together with 2-chloro-adenosine (0.1 mM for 5 min) the latter now decreased N1 by only 10+3% (n = 5). The reduction in the effect of 2-chloro-adenosine by 8-PST was highly significant ( P < 0.001, Mann-Whitney test). L-PIA had a slow effect, e.g., when applied at 1 btM the onset of the decrease in the SEPs occurred after 20-30 min and there was a maximal depression after 90-120 min of application. The mean decrease in N1 was 49+ 3% (n = 4). There was recovery of the SEPs to the control values when the cortex was washed for at least 120 min. 8-PST prevented the effects of L-PIA when applied at 10 ~tM for 15 min prior to and together with 1 /~M L-PIA for 120 min (n = 3). A lower concentration of 0.1 /tM L-PIA had no observable effect after 120 min. However, at 5 /tM a maximal depression of 82+8% ( n = 3) was achieved. NECA had a weaker effect than L-PIA. At 1 /~M NECA decreased N1 by 9+6% (n = 4) with the maximum effect observed after 40-70 min of application. There was recovery of the SEPs after washing the cortex for at least 60 min. Again a lower concentration of 0.1 /tM had no apparent effect on the cortex, while 5 /zM produced a maximal depression of 27 + 6% (n = 4).
Kvnurenic acid Kynurenic acid has been shown to block excitatory amino acid-mediated synaptic transmission (Perkins and Stone 1982, 1985; Brookes et al. 1986). Application of kynurenate here caused a dose- and time-dependent decrease in all components of the SEPs. When applied for 5 min, 5 mM kynurenate decreased N1 by 79+4% (n = 8) (Fig. 2) and 1 mM kynurenate decreased N1 by 40+6% (n = 4). There was recovery_ of the SEPs to the control level after washing the cortex for at least 20 min.
188 Discussion
Adenosine is known to block synaptic transmission by inhibiting the release of transmitters from pre-synaptic terminals (Phillis et al. 1979: Stone 198l). Adenosine has an efficient inactivation mechanism resulting from both an enzymatic breakdown (Clark et al. 1952; Rockwell and Maguire 1966) and an uptake system (Huang and Daly 1974: Hertz 1978) although the former is probably more important in vivo (Phillis and Edstrom 1976). 2-Chloro-adenosine selectively acts on the adenosine receptor (Huang and Daly 1974) but is not a substrate for adenosine deaminase (Clark et al. 1952; Rockwell and Maguire 1966). Thus, 2-chloro-adenosine could have diffused from the cortical cup into the deep cortical layers (e.g,, layers Ill and IV) much more readily than adenosine to inhibit synaptic transmission. This probably explains why there was an observable effect with 2-chloro-adenosine but not adenosine. Although the N1 wave is the best understood component of the SEP, it is yet to be established whether N1 is due to the pre-synaptic volley in the thalamo-cortical fibres or the postsynaptic activity in layer IV (Angel 1977: Angel et al. 1980). The decrease in N1 (and subsequent components of the SEP) caused by 2-chloro-adenosine could suggest that N1 is mainly due to a post-synaptic event. This result agrees with the finding that topical application of MgC12 (to block synaptic transmission) greatly attenuated the amplitude of the N1 wave (Bindm a n and Milne 1977; Bindman et al. 1979). 8-p-Sulphophenyl theophylline (8-PST) significantly inhibited the effects of 2-chloro-adenosine as has been reported in previous studies (e.g., Phillis et al. 1979). The 27% increase in N1 caused by 8-PST could be explained by an antagonism of endogenously released adenosine which normally depressed the thalamo-cortical synaptic transmission. This conclusion would be in accord with other studies in which increases of cell excitability have been noted upon treatment with xanthines, both in vivo (Phillis et al. 1979) and in vitro (Dunwiddie 1980; Ault et al. 1986). The very slow time course of responses to L-PIA and N E C A in vivo made any attempt at quantification very difficult. We have therefore compared the activity of these two substances at 3 concentrations. At 0.1 # M no response was seen to either, but at the higher concentrations of 1 and 5 ~M, L-PIA was more effective than NECA. Since the responses were measured at their maximum, equilibrium point, it is unlikely that these activities are as greatly affected by lipid solubility as studies using brief microiontophoretic applications t Phillis 1982; Stone 1982). The present study therefore supports the conclusion of Dunwiddie et al. (1984) that the depression of synaptically evoked activity is mediated by a receptor similar to that which produces a depression of transmitter release in other systems. This conclusion in turn might support the view that the NI wave is due to synaptic transmission rather than being the afferent fibre volley potential. Kynurenic acid has been shown to block excitatory amino acid-mediated synaptic transmission at various parts of the CNS by what appears to be a purely post-synaptic action (Perkins and Stone 1982; G a n o n g et al. 1983: Stone and
J.I. ADDAE, T,W. STONE Connick 1985: Brooks et al. 1986): there is no evidence of a pre-synaptic action. The attenuation of N1 by kynurenate, therefore, also supports the view that N1 is mainly a post-synaptic event and further suggests that the thalamo-cortical afferents act on excitatory amino acid receptors. However, since kynurenate exhibits poor selectivity between the excitant receptor types (Perkins and Stone 1982: G a n o n g et al. 1983), it is not possible to deduce from the present data which of the receptor types is primarily involved. However. we have previously reported failure to block the SEP using the selective N-methyl-aspartate antagonist, 2-amino-5-phosphonovaleric acid (Addae and Stone 1986). The results from D-aspartate transport studies as well as microiontophoretic experiments also suggest that excitatory amino acids may be involved in thalamo-cortical synaptic transmission (Hicks and Guedes 1983; Ottersen et al. 1983; Tsumoto et al. 1086). We are grateful to the Wellcome Trust, Action Research for the Crippled Child, and Association of Commonwealth Universities for support.
References
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