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In vivo neurochemical effects of tail pinch
Journal of Neuroscience Methods, 34 (1990) 151-157
151
Elsevier NSM 01118
In vivo neurochemical effects of tail pinch M a r t y n G. Boutelle, T y r a Zetterstr~Sm, Qi Pei, L e n n a r t S v e n s s o n a n d M a r i a n n e Fillenz
University Laboratory of Physiology, Oxford (U.K.) (Received 1 November 1989) (Accepted 28 February 1990)
Key words: Behaviour pattern; Microdialysis; Rat; Tail pinch; Voltammetry Tail pinch in the rat gives rise to a well characterised pattern of behaviour which includes gnawing, licking and eating. We have used both in vivo voltammetryand mierodialysis to monitor neurochemical changes which accompanythe behavioural response to a 5-rain tail pinch. Tail pinch resulted in a increase of extracellular 5-hydroxytryptamineand a smaller and more delayed increase of 5-hydroxyindoleacetic acid in the hippocampus. In the striatum there was a rise of both extracellular dopamine and ascorbate. With a recently developed constant potential voltammetric technique we can continuously monitor changes in extraceUular ascorbate. Using this technique we found a very rapid rise in ascorbate current during a 5-rain tail pinch; the current began to decline as soon as the clip was removed. The high time resolution of the technique also allowed us to record similar ascorbate changes during a 0.5-s tall pinch.
Introduction Tail pinch in the rat gives rise to a well characterized behaviour pattern which consists of gnawing, licking, eating and a general increase in the level of motor activity. Antelman et al. (1975) who first described this behaviour carried out a number of experiments to discover the neuronal pathways which mediated the behaviour. Pharmacological and lesion experiments suggested that the dopaminergic nigrostriatal pathway initiated and maintained the tail pinch-induced behaviour and that serotonergic pathways had a modulatory role. In an effort to gain a better understanding of the neuronal mechanism which mediates the response to tail pinch we have used microdialysis and in vivo voltammetry in unanaesthetized freely
Correspondence: M. Fillenz, University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, U.K.
moving rats to monitor neurochemical changes which are elicited by tail pinch. Previous ex vivo tissue measurements of metabolite to transmitter ratios after tail pinch showed most pronounced increases in dopamine (DA) turnover in the striatum and 5-hydroxytryptamine (5-HT) turnover in the hippocampus (Pei et al., 1988). Our studies therefore concentrated on these two brain regions.
Experimental procedures Tail pinch A mild tail-pinch was performed by placing a paperclip (3.2 cm long) on the rat's tail (approximately 3 cm from the tip) and leaving it there for 5 min. This produced a vigourous gnawing behaviour. The animal chewed intensively and occasionally licked a wooden stick held by the observer, and as long as the stick was present the animal did not attempt to remove the paper-clip.
0165-0270/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
152 The gnawing stopped when the paperclip was removed.
Microdialysis Male Sprague-Dawley rats (200-300 g) were anaesthetized with chloral hydrate (500 mg/kg, i.p.). A dialysis probe (4 mm effective length, 300 # m outer diameter, 5000 M.W. cut-off) was prepared (for details see Sharp et al., 1986), and stereotaxically implanted into the anterior striatum (AP + 1.0 mm (from bregma), L + 2.5 mm, DV - 6 . 5 mm) or ventral hippocampus (AP - 4 . 0 mm (from bregma), L +4.6 mm, DV - 8 . 5 ram) using the coordinates of Paxinos and Watson (1982). Dialysis probes were secured to the skull using dental cement and screws. After surgery the rats were allowed to recover for 24 h in their home cages with free access to food and water. On the day of experiments rats were placed in a circular bowl and secured in a harness connected to a liquid swivel. Dialysis probes were perfused with Ringer solution ([Na + ] = 147 mM, [K + ] = 4 mM, [Ca 2+] = 2 raM, [C1-] = 155 mM, pH = 6.0) at 2 # l / m i n using polyethylene tubing connected to a microinfusion pump (CMA/100, Carnegie Medicin, Solna, Sweden). The perfusates were collected every 10 or 20 min and directly assayed for monoamines and their acidic metabolites using HPLC with electrochemical detection.
Determination of microdialysis sample content Dialysates were directly injected into a HPLC system with electrochemical detection for the measurements of DA, 5-HT, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindolacetic acid (5-HIAA) and ascorbic acid (AA). Indoles were separated on a Microsorb ODS column using a phosphate buffer system as the mobile phase (see Pei et al., 1989). Catechols and ascorbic acid were separated on another system also using a reversed phase column with a phosphate buffer as the mobile phase but with a 10 times higher concentration of the ion pair reagent (for details see Sharp et al., 1986). The effect of tail pinch on DA and 5-HT release in anterior striatum or ventral hippocampus was measured in awake, freely moving rats 24 h after dialysis probe implantation. After at least
6-7 control samples had been taken and when levels of DA and DOPAC in striatum or 5-HT and 5-HIAA in hippocampus were stable, tail pinch was administered for 5 min. During and immediately after the tail pinch 3-4 10-rain sampies were taken. Sampling at 20-min intervals was then resumed.
In vivo voltammetry Male Sprague-Dawley rats (initial weight 300390 g) were stereotaxically implanted, under chloral hydrate anaesthesia (500 mg/kg), with voltammetric electrodes. Carbon paste working electrodes were prepared as described previously (O'Neill et al., 1982). The paste (2.8 g carbon powder to 1 ml silicone oil) was packed into a 1.5-mm deep cavity at one end of a 300-/~m o.d. teflon-coated silver wire (Leico Inc., U.S.A.). Silver wires placed in the cortex were used as auxiliary and reference electrodes. Rats were normally implanted with 2 carbon paste working electrodes placed bilaterally in the corpus striatum. The coordinates used, with the skull leveled between bregma and lambda, were: AP - 0 . 5 mm (from bregma), L + 3.0 mm, DV - 4 . 8 mm (from skull) using the coordinates of Paxinos and Watson (1982). The animals were then placed in the recording cages (25 x 25 x 25 cm), connected to the recording equipment through a flexible screened 6-core cable (Plastics One, U.S.A.) with a swivel connector and left to recover for at least 24 h. All animals were kept in a windowless room under a 12-h light 12-h dark cycle with food and water ad lib. The recording equipment consisted of a novel low-noise, low-damping potentiostat of our own design capable of measuring current of 0.2 pA without the need for filters. To achieve this level of performance we used modern monolithic lasertrimmed operational amplifiers with a separate headstage situated close to the animal's cage. This was then connected to a microcomputer-based data acquisition system (O'Neill et al., 1983) which logged the current, and measured animal motor activity.
Voltammetric recording of ascorbate Constant potential experiments began with a single linear sweep recording. A potential was
153 chosen above the peak potential for ascorbic acid oxidation but below that for the onset of uric acid oxidation and kept constant at this level (for details see Boutelle et al. 1989). The continuous current output from the potentiostat was either sampled sequentially for each electrode and averaged over 30-s intervals or recorded directly b y a digital storage oscilloscope (Gould OS4000). The total motor activity was simultaneously recorded over the same time interval. After completion of the experimental recording the animals were left for 30 rain, after which a second single linear sweep scan was performed to confirm that there had been no change in the position of the oxidation peaks. Statistics All values are quoted as mean 5: standard error. Unpaired Student t-tests between control and tail-pinched animals were used throughout.
Results
Hippocampal changes in 5 - H T Basal levels of 5-HT in samples of hippocampal dialysate were 31 5:6 f m o l / 2 0 - m i n sample of 40 gl. A 5-min tail pinch resulted in an increase in 5-HT in the first 10-rain sample collected after the 300
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Fig. 1. The effect of a 5-rain tail pinch indicated by the black bar on levels of 5-HT. Filled circles are data from the tail pinch group and open circles are data from ~u~uals which were undisturbed. Values are expressed as a percentage of their absolute concentration immediatelybefore tail pinch (time = 0). Each point represents mean + S.E.M. for 7 rats.
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Fig. 2. The effect of a 5-rain tail pinch indicated by the black bar on levels of DA. Filled circles are data from the tail pinch group and open circles are data from animals which were undisturbed. Values are expressed as a percentage of their absolute concentration immediatelybefore tail pinch (time = 0). Each point represents mean+ S.E.M. for 7 rats.
onset of the stimulus and reached a peak of 189 5: 26% ( P < 0.05, n = 9) of control in the next 10-min sample. By the end of 1 h 5-HT concentrations in the dialysates had returned to control level. This is shown in Fig. 1. Basal levels of the metabolite 5-hydroxyindole acetic acid (5HIAA) were 8.7 5:0.3 p m o l / 2 0 - m i n sample of 40 gl. They also increased during tail pinch although the changes were smaller and slower; the m a x i m u m increase was 38 5: 11% ( P < 0.05, n = 9) above control and was reached 30 rain after tail pinch with a return to control levels after 100 min (data not shown). Striatal changes in DA and A A - Dialysis results Basal levels of D A in striatal dialysate were 67 + 7 f m o l / 1 0 - m i n sample of 20 gl. Tail pinch caused a rise in the extracellular concentration of D A followed by a rapid return to baseline values. The mean increase shown in Fig. 2 was to 238 5: 66% of control ( P < 0.05, n --- 7) and occurred in the first 10-min sample after the stimulus. However, there was greater variation between rats in b o t h the percentage increase (indicated by the S.E.M.) and the time of m a x i m u m increase; the increase in the first 10-min sample varying between 5 and 483%, and in the 4 cases with lowest percentage increase the m a x i m u m increase occurred later than the first 10-min sample after the tail pinch.
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Fig. 3. The effect of a 5-rnin tail pinch indicated by the black bar on levels of AA monitored by microdialysis. Filled circles are data from the tail pinch group and open circles are data from animals which were undisturbed. Values are expressed as a percentage of their absolute concentration immediately before tail pinch (time = 0). Each point represents mean + S.E.M. for 7 rats.
Concentrations of the metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) showed a small (16%) non-significant increase in the first 10-rain sample, but homovanillic acid (HVA) levels were unaltered (data not shown). In parallel with the D A increase there was also an increase in the extracellular concentration of ascorbate. Basal levels of ascorbic acid (AA) in striatal dialysates were 129.6 + 41 pmol/20-min sample of 40 #1. Tail pinch caused a significant increase in AA to 131 + 9% of control in the first sample following the pinch ( P < 0.05, n = 7) which was maintained for 2 further 10-min samples before returning to control levels. This is shown in Fig. 3. There was again variability in the magnitude and time-course of response between animals. However, as shown in Fig. 4, the maximum percentage increase in AA at whatever time after the tail pinch was always smaller than that for DA, and showed much less variation. It should of course be remembered that in terms of absolute concentration changes the increases in AA are 1000 times those in DA. Striatal changes in A A - Voltammetric results
Extracellular AA levels can be measured with very high time resolution using constant potential recording at implanted carbon paste electrodes.
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Fig. 4. The maximal percentage increase of DA (dark bars) and AA (light bars) occurring during the 30-rain following tall pinch. Pairs of bars are from 6 individual animals.
Basal ascorbate currents were stable for each animal, but varied between each animal in the range 2.0-4.0 nA. It was not possible to relate these currents to absolute extracellular AA concentrations using in vitro calibrations because the sensitivity of the electrode to AA changed markedly on implantation. However, we have shown that both this basal current and physiologically and pharmacologically-induced changes in this current represent levels of AA alone (Boutelle 4.4
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Fig. 5. The effect of a 5-rain tail pinch indicated by the dark bar on levels of AA monitored by constant potential voltammetric recording. The continuous current obtained from each animal was averaged over 30-s periods. Each point on the graph represents the mean of such data points from 7 animals. The error bar shows the maximum S.E.M. value.
155 3.6
The duration of the pinch was determined using the time base of the oscilloscope, in the case of Fig. 6 it was 0.5 s. The current then fell to prepinch levels within 1.0 s. There is a striking similaxity between the AA time-courses of the 5-rain and 0.5-s tail pinches.
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recording. The trace shows the continuous real time current from a striatal electrodeof a freelymovinganimal.
et al., 1989). On starting a 5-min tail pinch the AA current increased immediately, and this rise continued until the moment when the pinch was stopped. The current then fell rapidly reaching pre-pinch levels within 10 rain. A mean response for 7 animals is shown in Fig. 5, with the error bar indicating the maximum standard error. Constant potential recording, as has already been mentioned, gives a continuous measure of AA levels - each data point in Fig. 5 representing an average of this current over a 30-s period. With a newly designed potentiostat we were able to see much smaller changes in AA. Figure 6 shows the current output of an implanted electrode captured directly by an oscilloscope (N.B., the increased current and time resolution). The baseline is no longer featureless but consists of rapid changes which are not artifactual or electronic noise, and are only present in a live animal. At the black bar padded forceps were used to gently squeeze the animal's tail. When the pressure was sufficient for the animal to respond behaviourally the current increased very rapidly until the pinch was stopped.
We have used two in vivo techniques to monitor the neurochemical changes which accompany the behavioural response to tail pinch in the rat. With dialysis we have demonstrated the involvement of the dopaminergic nigrostriatal pathway and the serotonergic raphe-hippocampal pathway in this behavioural paradigm; with voltammetry we have shown that there are rapid changes in the concentration of ascorbate. In our experiments a 5-min tail pinch produced a maximal increase in 5-HT release in the hippocampus of 89% above control levels, whereas Kalrn et al. (1989) reported a 48% increase after a 15-rain tail pinch. The maximum increase in 5-HT occurred in the second 10-rain sample and in 5HIAA in the third 10-rain sample after tail pinch application. Since the behaviour starts immediately this suggests that 5-HT acts in a modulatory rather than initiatory role. Changes in 5-HIAA, were smaller (38% in our experiments) or absent (Kalrn et al., 1989). The increase in D A in response to tail pinch was greater, faster but very much more variable than the increase in 5-HT. This variability in both magnitude and time-course of DA response has also been found by other workers (Damsma et al., 1989) although in this case the increase varied from 10-30% for a 10-min tail pinch. An important factor here may be the state of the animals before and during tail pinch. There was no change in DOPAC concentration. The increase in D A was paralleled by an increase in striatal ascorbate. This increase showed much less variation in size than the changes in DA although there was some variation in the time of occurrence of the maximum of this increase: in some rats this was in the first sample and in others in the second sample after tail pinch. The reason
156 for this variation in time-course is at present not known. We also monitored tail pinch-related changes in ascorbate using in vivo voltammetry. With the constant potential technique such changes can be followed continuously. With this technique it becomes clear that the ascorbate changes are very rapid indeed; the reason for the very different time-course of the ascorbate change monitored with microdialysis and voltammetry is not clear. It is unlikely that the difference in sampling rates between the two techniques is to blame as the D A response, measured at the same time, shows that dialysis is capable of measuring a faster response than that found with AA. A n o t h e r possible cause is that the dialysis probes in this study are more rostral and ventral than the electrodes. This is currently under investigation. F o r both the 5-min tail pinch shown in Fig. 4 and the 0.5-s tail pinch shown in Fig. 5 the current starts to rise when the pinch is applied, and falls immediately at the end of the pinch. There is also a striking similarity between the time constants for the current fall in the 2 cases. C o m p a r i s o n of the current increases for the 2 durations of pinch (0.16 n A for 0.5 s and 1.1 n A for 5 min) indicates that the longer pinch produces proportionately less current increase. The fast time-course of the ascorbate response resembles that of a neurotransmitter. We have shown that microinjection of ascorbic acid oxidase abolishes the basal current and reduces the tail pinch-induced increase (Boutelle et al., 1989). This provides evidence that the current is dependent on the oxidation of ascorbate alone. Furthermore, other tests such as the separate microinfusion of D A and AA, and systemic injection of a selective D A - D - 2 agonist have allowed us to specifically exclude the participation of D A in this response (unpublished results). It should also be noted at this point that in our dialysis studies the increase in A A is 1000 times that of D A when c o m p a r e d in terms of absolute concentration. We have previously provided evidence which suggests that changes in ascorbate are closely coupled to the release of glutamate (Fillenz et al., 1986). This might account for the extremely rapid changes in ascorbate found in this study.
Acknowledgements We wish to thank Action Research and the U.K. Medical Research Council (M.G.B.), the H e n r y Lester Trust Ltd (Q.P.), the Swedish Medical Research Council (T.Z., L.S.) the Wellcome Trust (T.Z.), the Swedish Institute, the Swedish Council for Planing and Coordination of Research and R ~ d m a n och Fru Ernst Collianders Stiftelse (L.S.).
References Antelman, S.M., Szechtman, H., Chin, P. and Fisher, A.E. (1975) Tail pinch-induced eating, gnawing and licking behavior in rats: dependence on the nigrostriatal dopamine system. Brain Res., 99: 319-337. Boutelle, M.G., Svensson, L. and Fillenz, M. (1989) Rapid changes in striatal ascorbate in response to tail-pinch monitored by constant potential voltammetry. Neuroscience, 30: 11-17. Damsma, G., Yoshida, M., Wenkstern, D., Nomikos, G.G., Philips, A.G. and Fibiger, H.C. (1989) Dopamine transmission in the rat striatum, nucleus accumbens and pre-frontal cortex is differently affected by feeding, taft pinch and immobilisation. J. Neurosci. Methods, 29: 272. Fillenz, M., O'Neill, R.D. and Gnmewald, R.A. (1986) Changes in extracellular brain ascorbate concentration as an index of excitatory amino acid release, in: M.H. Joseph, M. Fillenz, I.A. MacDonald and C.A. Marsden (Eds.), Measuring Peripheral and Central Neurotransmitter Release During Behaviour, Ellis Horwood, Chichester, pp. 144-163. Kal6n, P., Rosengren, E., Lindvall, O. and BjiSrkltmd, A. (1989) Hippocampal noradrenaline and serotonin release over 24 hours measured by the dialysis te~:hnique in freely moving rats: correlation to behavioural activity state, effect of handling and tail-pinch. Eur. J. Neurosci., 3: 181-189. O'Neill, R.D., Grunewald, R.A,, Fillenz, M. and Albery, W.J. (1982) Linear sweep voltammetry with carbon paste electrodes in the rat striatum. Neuroscience, 7: 1945-1954. O'Neill, R.D., Fillenz, M., Albery, W.J. and Goddard, N.J. (1983) The monitoring of ascorbate and monoamine transmitter metabolites in the striatum of unanaesthetised rats using microprocessor-based voltammetry. Neuroscience, 9: 87-93. Paxinos, S. and Watson, C. (1982) The Rat Brain in Stereotaxic Co-ordinates, Academic Press, New York, Sydney. Pei, Q., Zetterstrt~m, T. and Fillenz, M. (1988) The effect of tail pinch on the turnover of 5-HT in the rat hippocampus. Neurochem. Int., 13: 158. Pei, Q., Zetterstrt~m, T. and Fillenz, M. (1989) Both systemic
157 and local administration of benzodiazepine agonists inhibit in vivo release of 5-HT from ventral hippocampus. Neuropharmacology, 28: 1061-1066. Sharp, T., ZetterstrtSra, T. and Ungerstedt, U. (1986) An in vivo study of dopamine release and metabolism in rat brain regions using intracerebral dialysis. J. Neurochem., 47: 113-122.
Sharp, T,, ZetterstrSm, T., Series, H.G., Carlsson, A., Grahame-Smith, D.G. and Ungerstedt, U. (1987) HPLC-EC analysis of catechols and indoles in rat brain dialysis. Life Sci., 41: 869-872.
151
Elsevier NSM 01118
In vivo neurochemical effects of tail pinch M a r t y n G. Boutelle, T y r a Zetterstr~Sm, Qi Pei, L e n n a r t S v e n s s o n a n d M a r i a n n e Fillenz
University Laboratory of Physiology, Oxford (U.K.) (Received 1 November 1989) (Accepted 28 February 1990)
Key words: Behaviour pattern; Microdialysis; Rat; Tail pinch; Voltammetry Tail pinch in the rat gives rise to a well characterised pattern of behaviour which includes gnawing, licking and eating. We have used both in vivo voltammetryand mierodialysis to monitor neurochemical changes which accompanythe behavioural response to a 5-rain tail pinch. Tail pinch resulted in a increase of extracellular 5-hydroxytryptamineand a smaller and more delayed increase of 5-hydroxyindoleacetic acid in the hippocampus. In the striatum there was a rise of both extracellular dopamine and ascorbate. With a recently developed constant potential voltammetric technique we can continuously monitor changes in extraceUular ascorbate. Using this technique we found a very rapid rise in ascorbate current during a 5-rain tail pinch; the current began to decline as soon as the clip was removed. The high time resolution of the technique also allowed us to record similar ascorbate changes during a 0.5-s tall pinch.
Introduction Tail pinch in the rat gives rise to a well characterized behaviour pattern which consists of gnawing, licking, eating and a general increase in the level of motor activity. Antelman et al. (1975) who first described this behaviour carried out a number of experiments to discover the neuronal pathways which mediated the behaviour. Pharmacological and lesion experiments suggested that the dopaminergic nigrostriatal pathway initiated and maintained the tail pinch-induced behaviour and that serotonergic pathways had a modulatory role. In an effort to gain a better understanding of the neuronal mechanism which mediates the response to tail pinch we have used microdialysis and in vivo voltammetry in unanaesthetized freely
Correspondence: M. Fillenz, University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, U.K.
moving rats to monitor neurochemical changes which are elicited by tail pinch. Previous ex vivo tissue measurements of metabolite to transmitter ratios after tail pinch showed most pronounced increases in dopamine (DA) turnover in the striatum and 5-hydroxytryptamine (5-HT) turnover in the hippocampus (Pei et al., 1988). Our studies therefore concentrated on these two brain regions.
Experimental procedures Tail pinch A mild tail-pinch was performed by placing a paperclip (3.2 cm long) on the rat's tail (approximately 3 cm from the tip) and leaving it there for 5 min. This produced a vigourous gnawing behaviour. The animal chewed intensively and occasionally licked a wooden stick held by the observer, and as long as the stick was present the animal did not attempt to remove the paper-clip.
0165-0270/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
152 The gnawing stopped when the paperclip was removed.
Microdialysis Male Sprague-Dawley rats (200-300 g) were anaesthetized with chloral hydrate (500 mg/kg, i.p.). A dialysis probe (4 mm effective length, 300 # m outer diameter, 5000 M.W. cut-off) was prepared (for details see Sharp et al., 1986), and stereotaxically implanted into the anterior striatum (AP + 1.0 mm (from bregma), L + 2.5 mm, DV - 6 . 5 mm) or ventral hippocampus (AP - 4 . 0 mm (from bregma), L +4.6 mm, DV - 8 . 5 ram) using the coordinates of Paxinos and Watson (1982). Dialysis probes were secured to the skull using dental cement and screws. After surgery the rats were allowed to recover for 24 h in their home cages with free access to food and water. On the day of experiments rats were placed in a circular bowl and secured in a harness connected to a liquid swivel. Dialysis probes were perfused with Ringer solution ([Na + ] = 147 mM, [K + ] = 4 mM, [Ca 2+] = 2 raM, [C1-] = 155 mM, pH = 6.0) at 2 # l / m i n using polyethylene tubing connected to a microinfusion pump (CMA/100, Carnegie Medicin, Solna, Sweden). The perfusates were collected every 10 or 20 min and directly assayed for monoamines and their acidic metabolites using HPLC with electrochemical detection.
Determination of microdialysis sample content Dialysates were directly injected into a HPLC system with electrochemical detection for the measurements of DA, 5-HT, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindolacetic acid (5-HIAA) and ascorbic acid (AA). Indoles were separated on a Microsorb ODS column using a phosphate buffer system as the mobile phase (see Pei et al., 1989). Catechols and ascorbic acid were separated on another system also using a reversed phase column with a phosphate buffer as the mobile phase but with a 10 times higher concentration of the ion pair reagent (for details see Sharp et al., 1986). The effect of tail pinch on DA and 5-HT release in anterior striatum or ventral hippocampus was measured in awake, freely moving rats 24 h after dialysis probe implantation. After at least
6-7 control samples had been taken and when levels of DA and DOPAC in striatum or 5-HT and 5-HIAA in hippocampus were stable, tail pinch was administered for 5 min. During and immediately after the tail pinch 3-4 10-rain sampies were taken. Sampling at 20-min intervals was then resumed.
In vivo voltammetry Male Sprague-Dawley rats (initial weight 300390 g) were stereotaxically implanted, under chloral hydrate anaesthesia (500 mg/kg), with voltammetric electrodes. Carbon paste working electrodes were prepared as described previously (O'Neill et al., 1982). The paste (2.8 g carbon powder to 1 ml silicone oil) was packed into a 1.5-mm deep cavity at one end of a 300-/~m o.d. teflon-coated silver wire (Leico Inc., U.S.A.). Silver wires placed in the cortex were used as auxiliary and reference electrodes. Rats were normally implanted with 2 carbon paste working electrodes placed bilaterally in the corpus striatum. The coordinates used, with the skull leveled between bregma and lambda, were: AP - 0 . 5 mm (from bregma), L + 3.0 mm, DV - 4 . 8 mm (from skull) using the coordinates of Paxinos and Watson (1982). The animals were then placed in the recording cages (25 x 25 x 25 cm), connected to the recording equipment through a flexible screened 6-core cable (Plastics One, U.S.A.) with a swivel connector and left to recover for at least 24 h. All animals were kept in a windowless room under a 12-h light 12-h dark cycle with food and water ad lib. The recording equipment consisted of a novel low-noise, low-damping potentiostat of our own design capable of measuring current of 0.2 pA without the need for filters. To achieve this level of performance we used modern monolithic lasertrimmed operational amplifiers with a separate headstage situated close to the animal's cage. This was then connected to a microcomputer-based data acquisition system (O'Neill et al., 1983) which logged the current, and measured animal motor activity.
Voltammetric recording of ascorbate Constant potential experiments began with a single linear sweep recording. A potential was
153 chosen above the peak potential for ascorbic acid oxidation but below that for the onset of uric acid oxidation and kept constant at this level (for details see Boutelle et al. 1989). The continuous current output from the potentiostat was either sampled sequentially for each electrode and averaged over 30-s intervals or recorded directly b y a digital storage oscilloscope (Gould OS4000). The total motor activity was simultaneously recorded over the same time interval. After completion of the experimental recording the animals were left for 30 rain, after which a second single linear sweep scan was performed to confirm that there had been no change in the position of the oxidation peaks. Statistics All values are quoted as mean 5: standard error. Unpaired Student t-tests between control and tail-pinched animals were used throughout.
Results
Hippocampal changes in 5 - H T Basal levels of 5-HT in samples of hippocampal dialysate were 31 5:6 f m o l / 2 0 - m i n sample of 40 gl. A 5-min tail pinch resulted in an increase in 5-HT in the first 10-rain sample collected after the 300
eomn~ 250
----'O'--
taa pl~.~
[-,
~
150
5~-60a
i -40
i -20
i 0
i 20 Time
i 40
i 60
i 80
i 100
(rain)
Fig. 1. The effect of a 5-rain tail pinch indicated by the black bar on levels of 5-HT. Filled circles are data from the tail pinch group and open circles are data from ~u~uals which were undisturbed. Values are expressed as a percentage of their absolute concentration immediatelybefore tail pinch (time = 0). Each point represents mean + S.E.M. for 7 rats.
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Fig. 2. The effect of a 5-rain tail pinch indicated by the black bar on levels of DA. Filled circles are data from the tail pinch group and open circles are data from animals which were undisturbed. Values are expressed as a percentage of their absolute concentration immediatelybefore tail pinch (time = 0). Each point represents mean+ S.E.M. for 7 rats.
onset of the stimulus and reached a peak of 189 5: 26% ( P < 0.05, n = 9) of control in the next 10-min sample. By the end of 1 h 5-HT concentrations in the dialysates had returned to control level. This is shown in Fig. 1. Basal levels of the metabolite 5-hydroxyindole acetic acid (5HIAA) were 8.7 5:0.3 p m o l / 2 0 - m i n sample of 40 gl. They also increased during tail pinch although the changes were smaller and slower; the m a x i m u m increase was 38 5: 11% ( P < 0.05, n = 9) above control and was reached 30 rain after tail pinch with a return to control levels after 100 min (data not shown). Striatal changes in DA and A A - Dialysis results Basal levels of D A in striatal dialysate were 67 + 7 f m o l / 1 0 - m i n sample of 20 gl. Tail pinch caused a rise in the extracellular concentration of D A followed by a rapid return to baseline values. The mean increase shown in Fig. 2 was to 238 5: 66% of control ( P < 0.05, n --- 7) and occurred in the first 10-min sample after the stimulus. However, there was greater variation between rats in b o t h the percentage increase (indicated by the S.E.M.) and the time of m a x i m u m increase; the increase in the first 10-min sample varying between 5 and 483%, and in the 4 cases with lowest percentage increase the m a x i m u m increase occurred later than the first 10-min sample after the tail pinch.
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Fig. 3. The effect of a 5-rnin tail pinch indicated by the black bar on levels of AA monitored by microdialysis. Filled circles are data from the tail pinch group and open circles are data from animals which were undisturbed. Values are expressed as a percentage of their absolute concentration immediately before tail pinch (time = 0). Each point represents mean + S.E.M. for 7 rats.
Concentrations of the metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) showed a small (16%) non-significant increase in the first 10-rain sample, but homovanillic acid (HVA) levels were unaltered (data not shown). In parallel with the D A increase there was also an increase in the extracellular concentration of ascorbate. Basal levels of ascorbic acid (AA) in striatal dialysates were 129.6 + 41 pmol/20-min sample of 40 #1. Tail pinch caused a significant increase in AA to 131 + 9% of control in the first sample following the pinch ( P < 0.05, n = 7) which was maintained for 2 further 10-min samples before returning to control levels. This is shown in Fig. 3. There was again variability in the magnitude and time-course of response between animals. However, as shown in Fig. 4, the maximum percentage increase in AA at whatever time after the tail pinch was always smaller than that for DA, and showed much less variation. It should of course be remembered that in terms of absolute concentration changes the increases in AA are 1000 times those in DA. Striatal changes in A A - Voltammetric results
Extracellular AA levels can be measured with very high time resolution using constant potential recording at implanted carbon paste electrodes.
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Fig. 4. The maximal percentage increase of DA (dark bars) and AA (light bars) occurring during the 30-rain following tall pinch. Pairs of bars are from 6 individual animals.
Basal ascorbate currents were stable for each animal, but varied between each animal in the range 2.0-4.0 nA. It was not possible to relate these currents to absolute extracellular AA concentrations using in vitro calibrations because the sensitivity of the electrode to AA changed markedly on implantation. However, we have shown that both this basal current and physiologically and pharmacologically-induced changes in this current represent levels of AA alone (Boutelle 4.4
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Fig. 5. The effect of a 5-rain tail pinch indicated by the dark bar on levels of AA monitored by constant potential voltammetric recording. The continuous current obtained from each animal was averaged over 30-s periods. Each point on the graph represents the mean of such data points from 7 animals. The error bar shows the maximum S.E.M. value.
155 3.6
The duration of the pinch was determined using the time base of the oscilloscope, in the case of Fig. 6 it was 0.5 s. The current then fell to prepinch levels within 1.0 s. There is a striking similaxity between the AA time-courses of the 5-rain and 0.5-s tail pinches.
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recording. The trace shows the continuous real time current from a striatal electrodeof a freelymovinganimal.
et al., 1989). On starting a 5-min tail pinch the AA current increased immediately, and this rise continued until the moment when the pinch was stopped. The current then fell rapidly reaching pre-pinch levels within 10 rain. A mean response for 7 animals is shown in Fig. 5, with the error bar indicating the maximum standard error. Constant potential recording, as has already been mentioned, gives a continuous measure of AA levels - each data point in Fig. 5 representing an average of this current over a 30-s period. With a newly designed potentiostat we were able to see much smaller changes in AA. Figure 6 shows the current output of an implanted electrode captured directly by an oscilloscope (N.B., the increased current and time resolution). The baseline is no longer featureless but consists of rapid changes which are not artifactual or electronic noise, and are only present in a live animal. At the black bar padded forceps were used to gently squeeze the animal's tail. When the pressure was sufficient for the animal to respond behaviourally the current increased very rapidly until the pinch was stopped.
We have used two in vivo techniques to monitor the neurochemical changes which accompany the behavioural response to tail pinch in the rat. With dialysis we have demonstrated the involvement of the dopaminergic nigrostriatal pathway and the serotonergic raphe-hippocampal pathway in this behavioural paradigm; with voltammetry we have shown that there are rapid changes in the concentration of ascorbate. In our experiments a 5-min tail pinch produced a maximal increase in 5-HT release in the hippocampus of 89% above control levels, whereas Kalrn et al. (1989) reported a 48% increase after a 15-rain tail pinch. The maximum increase in 5-HT occurred in the second 10-rain sample and in 5HIAA in the third 10-rain sample after tail pinch application. Since the behaviour starts immediately this suggests that 5-HT acts in a modulatory rather than initiatory role. Changes in 5-HIAA, were smaller (38% in our experiments) or absent (Kalrn et al., 1989). The increase in D A in response to tail pinch was greater, faster but very much more variable than the increase in 5-HT. This variability in both magnitude and time-course of DA response has also been found by other workers (Damsma et al., 1989) although in this case the increase varied from 10-30% for a 10-min tail pinch. An important factor here may be the state of the animals before and during tail pinch. There was no change in DOPAC concentration. The increase in D A was paralleled by an increase in striatal ascorbate. This increase showed much less variation in size than the changes in DA although there was some variation in the time of occurrence of the maximum of this increase: in some rats this was in the first sample and in others in the second sample after tail pinch. The reason
156 for this variation in time-course is at present not known. We also monitored tail pinch-related changes in ascorbate using in vivo voltammetry. With the constant potential technique such changes can be followed continuously. With this technique it becomes clear that the ascorbate changes are very rapid indeed; the reason for the very different time-course of the ascorbate change monitored with microdialysis and voltammetry is not clear. It is unlikely that the difference in sampling rates between the two techniques is to blame as the D A response, measured at the same time, shows that dialysis is capable of measuring a faster response than that found with AA. A n o t h e r possible cause is that the dialysis probes in this study are more rostral and ventral than the electrodes. This is currently under investigation. F o r both the 5-min tail pinch shown in Fig. 4 and the 0.5-s tail pinch shown in Fig. 5 the current starts to rise when the pinch is applied, and falls immediately at the end of the pinch. There is also a striking similarity between the time constants for the current fall in the 2 cases. C o m p a r i s o n of the current increases for the 2 durations of pinch (0.16 n A for 0.5 s and 1.1 n A for 5 min) indicates that the longer pinch produces proportionately less current increase. The fast time-course of the ascorbate response resembles that of a neurotransmitter. We have shown that microinjection of ascorbic acid oxidase abolishes the basal current and reduces the tail pinch-induced increase (Boutelle et al., 1989). This provides evidence that the current is dependent on the oxidation of ascorbate alone. Furthermore, other tests such as the separate microinfusion of D A and AA, and systemic injection of a selective D A - D - 2 agonist have allowed us to specifically exclude the participation of D A in this response (unpublished results). It should also be noted at this point that in our dialysis studies the increase in A A is 1000 times that of D A when c o m p a r e d in terms of absolute concentration. We have previously provided evidence which suggests that changes in ascorbate are closely coupled to the release of glutamate (Fillenz et al., 1986). This might account for the extremely rapid changes in ascorbate found in this study.
Acknowledgements We wish to thank Action Research and the U.K. Medical Research Council (M.G.B.), the H e n r y Lester Trust Ltd (Q.P.), the Swedish Medical Research Council (T.Z., L.S.) the Wellcome Trust (T.Z.), the Swedish Institute, the Swedish Council for Planing and Coordination of Research and R ~ d m a n och Fru Ernst Collianders Stiftelse (L.S.).
References Antelman, S.M., Szechtman, H., Chin, P. and Fisher, A.E. (1975) Tail pinch-induced eating, gnawing and licking behavior in rats: dependence on the nigrostriatal dopamine system. Brain Res., 99: 319-337. Boutelle, M.G., Svensson, L. and Fillenz, M. (1989) Rapid changes in striatal ascorbate in response to tail-pinch monitored by constant potential voltammetry. Neuroscience, 30: 11-17. Damsma, G., Yoshida, M., Wenkstern, D., Nomikos, G.G., Philips, A.G. and Fibiger, H.C. (1989) Dopamine transmission in the rat striatum, nucleus accumbens and pre-frontal cortex is differently affected by feeding, taft pinch and immobilisation. J. Neurosci. Methods, 29: 272. Fillenz, M., O'Neill, R.D. and Gnmewald, R.A. (1986) Changes in extracellular brain ascorbate concentration as an index of excitatory amino acid release, in: M.H. Joseph, M. Fillenz, I.A. MacDonald and C.A. Marsden (Eds.), Measuring Peripheral and Central Neurotransmitter Release During Behaviour, Ellis Horwood, Chichester, pp. 144-163. Kal6n, P., Rosengren, E., Lindvall, O. and BjiSrkltmd, A. (1989) Hippocampal noradrenaline and serotonin release over 24 hours measured by the dialysis te~:hnique in freely moving rats: correlation to behavioural activity state, effect of handling and tail-pinch. Eur. J. Neurosci., 3: 181-189. O'Neill, R.D., Grunewald, R.A,, Fillenz, M. and Albery, W.J. (1982) Linear sweep voltammetry with carbon paste electrodes in the rat striatum. Neuroscience, 7: 1945-1954. O'Neill, R.D., Fillenz, M., Albery, W.J. and Goddard, N.J. (1983) The monitoring of ascorbate and monoamine transmitter metabolites in the striatum of unanaesthetised rats using microprocessor-based voltammetry. Neuroscience, 9: 87-93. Paxinos, S. and Watson, C. (1982) The Rat Brain in Stereotaxic Co-ordinates, Academic Press, New York, Sydney. Pei, Q., Zetterstrt~m, T. and Fillenz, M. (1988) The effect of tail pinch on the turnover of 5-HT in the rat hippocampus. Neurochem. Int., 13: 158. Pei, Q., Zetterstrt~m, T. and Fillenz, M. (1989) Both systemic
157 and local administration of benzodiazepine agonists inhibit in vivo release of 5-HT from ventral hippocampus. Neuropharmacology, 28: 1061-1066. Sharp, T., ZetterstrtSra, T. and Ungerstedt, U. (1986) An in vivo study of dopamine release and metabolism in rat brain regions using intracerebral dialysis. J. Neurochem., 47: 113-122.
Sharp, T,, ZetterstrSm, T., Series, H.G., Carlsson, A., Grahame-Smith, D.G. and Ungerstedt, U. (1987) HPLC-EC analysis of catechols and indoles in rat brain dialysis. Life Sci., 41: 869-872.