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Dopamine release in the amygdaloid complex of the rat, studied by brain microdialysis
Neuroscience Letters 249 (1998) 49–52
Dopamine release in the amygdaloid complex of the rat, studied by brain microdialysis Andrew M.J. Young*, Katy R. Rees1 Behavioural Neurochemistry Group, Department of Psychology, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK Received 23 February 1998; received in revised form 1 May 1998; accepted 7 May 1998
Abstract The dopaminergic projection from the ventral tegmental area to the amygdaloid complex may be modulatory on the processes of associative learning in the latter region. We measured dopamine in four different amygdaloid subfields in the rat, using brain microdialysis. Extracellular levels of dopamine in two sites in the lateral nucleus were not consistently measurable, even after treatment with amphetamine. However, basal dopamine levels were measurable in more medial locations (basolateral and central nuclei), with higher concentrations in the caudal than in the rostral probe placement, and were increased around 3fold by systemic amphetamine. Similarly, dopamine levels in caudal-medial amygdala were increased by local potassium stimulation and by mild footshock in a calcium-dependent manner, indicating a neurotransmitter origin. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Amphetamine; Amygdala; Aversive stimulus; Calcium-dependent; Dopamine; Microdialysis
Evidence from pharmacological and electrophysiological experiments suggests that the lateral (lAMY) and basolateral (blAMY) amygdaloid nuclei are likely to be important regions for stimulus integration in emotional conditioning in aversive and possibly appetitive situations, whilst the central amygdaloid nucleus (cAMY) is probably more concerned with expression of conditioned responses [3,13,14]. Most of the sensory projections to the amygdaloid complex (AMY) from cortical and thalamic regions terminate in the lAMY and blAMY and are glutamatergic [3,14]. The demonstration of long-term potentiation in AMY [2] and the growing body of evidence that NMDA-receptor antagonists block fear conditioning [5,15] suggest that LTP-like mechanisms may underlie emotional learning in these regions. In addition, dopaminergic projections to AMY, originating in the ventral tegmental area (VTA) [4,6], * Corresponding author. Behavioural Neuroscience Group, Department of Psychology, University of Leicester, University Road, Leicester LE1 7RH, UK. 1 Present address: Addiction Sciences Centre, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK.
have also been shown to influence conditioning [1,9]. However, little is known about dopamine release in AMY, nor about its function in the modulation of such learning. As a prelude to studies addressing questions of function, we have used brain microdialysis to look at (1) the distribution of extracellular dopamine within subfields of AMY and its response to pharmacological and physiological stimulation and (2) the calcium dependence (and therefore neurotransmitter origins) of this dopamine. A preliminary report of these results has been published [18]. All experiments used male Sprague–Dawley rats (B and K Universal, Hull, UK), approximately 280 g at the start of the procedure, and were performed with appropriate licence authority under the Animals (Scientific Procedures) Act, UK, 1986. In the first experiment, twin microdialysis probe assemblies were constructed by gluing two standard dialysis monoprobes [19] together, and bending the stainless-steel shafts, such that they ran parallel and 1 mm apart. One such assembly was implanted into either a medial or a lateral location in AMY of urethane (1.75 g/kg, i.p.) anaesthetised rats. In the lateral location, both probe tips lay in lAMY (Fr, +5.2 and +6.2; Tr, +5.0; V, +1.0: Fig. 1b,d), whereas in the
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A.M.J. Young, K.R. Rees / Neuroscience Letters 249 (1998) 49–52
medial location, both tips lay in blAMY and cAMY (Fig. 1a,c). The sizes of cAMY and blAMY relative to the dimensions of the dialysis probe tip preclude measurements specifically from one nucleus or the other. However the more caudal probe placement was located primarily in blAMY (Fr, +5.7; Tr, +4.5; V, +0.3: Fig. 1c), whilst the more rostral was primarily in cAMY but also sampled a portion of blAMY (Fr, +6.7; Tr, +4.5; V, +0.3: Fig. 1a). All coordinates quoted are relative to the intra-aural line [16]. Simultaneous dialysis perfusion of both probes with artificial cerebrospinal fluid (aCSF, mM: NaCl, 125; KCl, 3.3; MgSO4, 2.4; KH2PO4, 1.25; CaCl2, 1.85), at a flow rate of 3 ml/min, commenced immediately. After 120 min equilibration, consecutive 15-min dialysate samples were collected into glass vials containing 3 ml of 2 N H3PO4 (to minimise oxidation), and placed immediately into a refrigerated (8°C) autosampler (CMA200). All samples were analysed within 5 h of collection, by HPLC with electrochemical detection, as previously described [19], but using a 20-ml injection loop. The detection limit for dopamine was 2.5 fmol/injection (equivalent to 20 ml injection of 0.12 nM). The first four samples (60 min) collected were used to determine basal dopamine levels in the dialysates, after which amphetamine (1 mg/kg, i.p.) was administered, and a further six samples (90 min) were collected. In a second group of animals, after the 60-min basal collection period, the aCSF was replaced by calcium-free aCSF for the remainder of the experiment. Amphetamine (1 mg/kg, i.p.) was injected 60 min later, and dialysis perfusion continued, as before, for a further 90 min. In the second experiment, rats were implanted with stainless-steel guide cannulae [19] aimed at blAMY, under Immobilon anaesthetic (0.15 ml/kg, i.m.). After a recovery period of at least 7 days, dialysis probes [19] were inserted into the guide cannulae, under brief, light halothane anaesthetic, to lie bilaterally in medial-caudal AMY, (mostly blAMY: tip position: Fr, +5.7; Tr, ±4.5; V, +0.3, relative to the intra-aural line [16], Fig. 1c). Twenty-four hours later, animals were connected for microdialysis perfusion in a Skinner box, as previously described [19]. Dialysis perfusion with aCSF commenced immediately (flow rate 3 ml/min). Following 90 min equilibration, consecutive 15-min dialysate samples were collected into glass vials containing 3 ml of H3PO4, for HPLC analysis as described above. The first four samples (60 min) were collected for determination of the basal levels of dopamine, then the animals received five presentations of a mild footshock (1-s train of 6-ms pulses, 0.33 mA, 25 Hz), presented through the grid floor of the Skinner box, at 2-min intervals (first shock delivered 1 min into the sample). Sixty minutes after the end of the footshock presentation, a 30-ml (10 min, starting 1 min into the sample) pulse of high-potassium aCSF (100 mM KCl; NaCl reduced to 28 mM) was applied in the dialysis stream, after which dialysis perfusion continued for a further 45 min (three samples).
In a separate group of animals the calcium-dependence of the responses to footshock and to potassium was assessed. After the initial basal collection period (60 min), the dialysis perfusion medium was replaced by calcium-free aCSF for the remainder of the experiment. Mild footshock (60 min later) and potassium stimulation (60 min after that) were given as before. On completion of the experiments, animals were killed by anaesthetic overdose, and 20-mm cryostat sections were cut to verify the probe locations. In all experiments, the sample collection times were adjusted to take into account the dead volume of the dialysis collection tubes (12 ml in experiment 1; 36 ml in experiment 2). Statistical analysis by two-way analysis of variance with repeated measures was performed using SPSS for Windows (version 6.0). In the more lateral probe positions, corresponding to lAMY (Fig. 1b,d), dialysate levels of dopamine were for the most part undetectable, and amphetamine administration had no consistent measurable effect (data not shown). However basal dopamine was measurable in the two medial probe locations (Fig. 2). Levels were higher in the more caudal location, which measured primarily from blAMY (0.26 ± 0.04 nM) than in the more rostral location, which measured also from cAMY (0.18 ± 0.03 nM: F(1,14) = 4.99; P = 0.042).
Fig. 1. Diagram showing probe locations in relation to amygdaloid nuclei, on sections redrawn from the atlas of Paxinos and Watson [16], the horizontal coordinate is quoted relative to the intra-aural line (IAL). The black bar represents the intended location of the dialysis tip (excluding epoxy-sealing plug) according to the implantation coordinates used. The two lateral sites are shown in (b) and (d), and the two medial sites in (a) and (c). Implantation sites are depicted in the left side only, although in experiment 2, where bilateral probe implantations at (c) were used, probes were also implanted in an equivalent position on the right side of the brain. Histological examination showed that most probes lay close to these positions: those which did not (three animals in experiment 1) were not included in the analyses.
A.M.J. Young, K.R. Rees / Neuroscience Letters 249 (1998) 49–52
Fig. 2. The effect of amphetamine (1 mg/kg, i.p.; Amph) on dialysate concentrations of dopamine (mean ± SEM) in (a) blAMY and (b) cAMY, during perfusion with normal aCSF (dark shading, n = 6) and calcium-free aCSF (light shading, n = 4), in urethane anaesthetised animals. Basal levels of dopamine in the latter group prior to removal of calcium are also shown (mean ± SEM of four samples from each animal). Open bars represent time points where two or more samples fell below the detection limit of the analysis system.
In agreement with a previous report [8], amphetamine caused a significant rise in dopamine in both regions, peaking at 20–30 min after injection (F(7,98) = 26.03; P , 0.001: Fig. 2). However there was no significant difference in the response between the two regions (region × drug interaction, F(7,98) = 1.69; P = 0.120). Removal of calcium from the perfusion stream decreased the levels of dopamine to around 50% in both probe locations (F(1,14) = 8.78; P = 0.01: Fig. 2). In the rostral location the levels became unmeasurable in two out of the four animals. Amphetamine-evoked increases in extracellular dopamine were also reduced in both locations (Fig. 2), although it is not clear whether this was simply due to the reduced basal levels (calcium × amphetamine interaction: F(7,98) = 2.06; P = 0.054). On the basis of the results from experiment 1, dopamine measurements were made bilaterally in the medial-caudal probe location (principally blAMY: Fig. 1c), in conscious, unrestrained animals. Since both basal levels (left, 0.47 ± 0.08 nM; right, 0.44 ± 0.04 nM) and stimulated levels of dopamine, in the presence or absence of calcium were found to be similar between the two sides (F(1,14) =
51
0.37; P = 0.555), data from both were pooled for all subsequent analyses. Both repeated mild footshock and increased potassium evoked a significant increase in extracellular dopamine (F(2,28) = 14.14; P , 0.001, Fig. 3). Removal of calcium from the dialysis perfusion medium caused a reduction in basal extracellular dopamine to 0.26 ± 0.02 nM after 60 min (F(1,14) = 16.86, P = 0.001), and an abolition of the increases seen during footshock and potassium stimulation (calcium × stimulation interaction, F(2,28) = 10.11, P , 0.001, Fig. 3). The very low levels of extracellular dopamine in lAMY, even after amphetamine administration, indicate that there is unlikely to be substantial dopaminergic innervation of lAMY. Levels in the two medial locations, however, were measurable, although still 4- to 5-fold lower than reported in nucleus accumbens [19], indicating a more substantial dopaminergic innervation of blAMY and cAMY. Within the medial probe locations it is difficult to delineate between blAMY and cAMY, although the probes in the caudal site sampled mostly from blAMY, those in the more rostral site sampled from both blAMY and cAMY (Fig. 1a,c). These data provide evidence for dopamine release in blAMY, and also probably in cAMY, but little in lAMY, which corresponds to the reported pattern of innervation [4,6]. On the basis of these results, and in view of our ultimate aim of investigating the role of dopamine in the modulation of the conditioned learning process itself (occurring in lAMY and blAMY [3,13,14]), as opposed to expression of conditioned responding, we chose the caudal probe location for further investigations in conscious animals. Basal levels of extracellular dopamine measured primarily from blAMY in conscious rats were significantly higher than in anaesthetised rats, but were similar to levels recently
Fig. 3. Effect of mild footshock (Fs) and potassium stimulation (K) on dialysate dopamine concentrations (mean ± SEM) in blAMY, during perfusion with normal aCSF (dark shading: combined data from left and right sides of five animals) or calcium-free aCSF (light shading: combined data from left and right sides of four animals), in conscious, unrestrained animals. Basal levels of dopamine in the latter group prior to removal of calcium are also shown (mean ± SEM of four samples from each side of each animal).
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reported in dialysates from conscious rats with probes in cAMY [7] or spanning both blAMY and cAMY [8,12]. The difference may be a direct effect of anaesthesia, or it may be due to the different recovery times after probe implantation in the two situations (i.e. 2 h compared to 24 h). Both chemical (potassium) and behavioural (footshock) stimulation evoked large increases in extracellular dopamine, which were abolished by removal of calcium from the perfusion stream, indicating that the extracellular changes measured here do indeed reflect release of neurotransmitter dopamine. This is an important issue, not addressed in recent reports of microdialysis in AMY [7,8,10,12], since what is measured by microdialysis is the extracellular level of a compound, which does not necessarily represent neurotransmitter release. It should be noted, however, that the response to amphetamine was not abolished by removal of calcium. This is in line with previous findings that dopamine increases after a single injection of amphetamine are largely calcium-independent [11,17]. It is not clear from the present data whether there is a calciumdependent component of the response, since the interaction (calcium × amphetamine), although not significant, was close to it (P = 0.054), and further studies with larger group sizes would be needed to be certain. These studies confirm that dopamine measurements can be made in AMY, and, more importantly, show that both basal levels and increases following potassium stimulation and footshock are calcium-dependent, and therefore reflect neurotransmitter release.
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We thank the Wellcome Trust and the Medical Research Council (UK) for funding this research. KRR was a project student on the part-time MSc (Neuroscience) course. [1] Borowski, T.B. and Kokkinidis, L., Contribution of ventral tegmental area dopamine neurons to expression of conditioned fear: effects of electrical stimulation, excitotoxic lesions and quimperole infusion on potentiated startle in rats, Behav. Neurosci., 110 (1996) 1349–1364. [2] Clugnet, M.-C. and LeDoux, J.E., Synaptic plasticity in fear conditioning circuits: induction of LTP in the lateral nucleus of the amygdala by stimulation of the medial geniculate body, J. Neurosci., 10 (1990) 2818–2824. [3] Davis, M., Falls, W.A., Campeau, S. and Kim, M., Fear potentiated startle: a neural and pharmacological analysis, Behav. Brain Res., 58 (1993) 175–198. [4] Fallon, J.H. and Ciofi, P., Distribution of monoamines within the amygdala. In J.P. Aggleton (Ed.), The Amygdala: Neurobiolo-
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gical Aspects of Emotion, Memory and Mental Dysfunction, Wiley-Liss, New York, 1992, pp. 97–114. Fanselow, M.S. and Kim, J.J., Acquisition of contextual Pavlovian fear conditioning is blocked by application of an NMDA receptor antagonist D,L-2-amino-5-phosphonovaleric acid to the basolateral amygdala, Behav. Neurosci., 108 (1994) 210– 212. Freedman, L.J. and Cassell, M.D., Distribution of dopaminergic fibres in the central division of the extended amygdala of the rat, Brain Res., 633 (1994) 243–252. Hajnal, A. and Lenard, L., Feeding related dopamine release in the amygdala of freely moving rats, NeuroReport, 8 (1997) 2817–2820. Harmer, C.J., Hitchcott, P.K., Morutto, S.L. and Phillips, G.D., Repeated d-ampthetamine enhances stimulated mesoamygdaloid dopamine transmission, Psychopharmacology, 132 (1997) 247–254. Hitchcott, P.K., Bonardi, C.M.T. and Philips, G.D., Enhanced stimulus-reward learning by intra-amygdala administration of a D3 dopamine receptor agonist, Psychopharmacology, 133 (1997) 240–248. Hori, K., Tanaka, J. and Nomura, M., Effects of discrimination learning on the rat amygdala dopamine release: a microdialysis study, Brain Res., 621 (1993) 296–300. Hurd, Y.L. and Ungerstedt, U., Ca+ + dependence of amphetamine, nomifensin and Lu 19–005 effect on in vivo dopamine transmission, Eur. J. Pharmacol., 166 (1989) 261–269. Hurd, Y.L., McGregor, A. and Ponten, M., In vivo amygdala dopamine levels modulate cocaine self-administration behaviour in the rat: D1 dopamine receptor involvement, Eur. J. Neuroscience, 9 (1997) 2541–2548. Killcross, S., Robbins, T.W. and Everitt, B.J., Different types of fear-conditioned behaviour mediated by separate nuclei within amygdala, Nature, 388 (1997) 377–380. LeDoux, J.E., Emotional memory systems in the brain, Behav. Brain Res., 58 (1993) 69–79. Maren, S., Aharonov, G., Stote, D.L. and Fanselow, M.S., Nmethyl-D-aspartate receptors in the basolateral amygdala are required for both acquisition and expression of conditional fear in rats, Behav. Neurosci., 110 (1996) 1365–1374. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Orlando, FL, 1982. Warburton, E.C., Mitchell, S.N. and Joseph, M.H., Calcium dependence of sensitised dopamine release in rat nucleus accumbens following amphetamine challenge: implications for disruption of latent inhibition, Behav. Pharmacol., 7 (1996) 119–129. Young, A.M.J. and Rees, K.R., Dopamine release in the amygdaloid complex of the rat measured by microdialysis. In J.L. Gonzalez-Mora, R. Borgas and M. Mas (Eds.), Monitoring Molecules in the Neurosciences: Proceedings of the 7th International conference on In Vivo Methods, 1996, pp. 77–78. Young, A.M.J., Ahier, R.G., Upton, R.G., Joseph, M.H. and Gray, J.A., Increased extracellular dopamine in the nucleus accumbens of the rat during associative learning of neutral stimuli, Neuroscience, 83 (1998) 1175–1183.
Dopamine release in the amygdaloid complex of the rat, studied by brain microdialysis Andrew M.J. Young*, Katy R. Rees1 Behavioural Neurochemistry Group, Department of Psychology, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK Received 23 February 1998; received in revised form 1 May 1998; accepted 7 May 1998
Abstract The dopaminergic projection from the ventral tegmental area to the amygdaloid complex may be modulatory on the processes of associative learning in the latter region. We measured dopamine in four different amygdaloid subfields in the rat, using brain microdialysis. Extracellular levels of dopamine in two sites in the lateral nucleus were not consistently measurable, even after treatment with amphetamine. However, basal dopamine levels were measurable in more medial locations (basolateral and central nuclei), with higher concentrations in the caudal than in the rostral probe placement, and were increased around 3fold by systemic amphetamine. Similarly, dopamine levels in caudal-medial amygdala were increased by local potassium stimulation and by mild footshock in a calcium-dependent manner, indicating a neurotransmitter origin. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Amphetamine; Amygdala; Aversive stimulus; Calcium-dependent; Dopamine; Microdialysis
Evidence from pharmacological and electrophysiological experiments suggests that the lateral (lAMY) and basolateral (blAMY) amygdaloid nuclei are likely to be important regions for stimulus integration in emotional conditioning in aversive and possibly appetitive situations, whilst the central amygdaloid nucleus (cAMY) is probably more concerned with expression of conditioned responses [3,13,14]. Most of the sensory projections to the amygdaloid complex (AMY) from cortical and thalamic regions terminate in the lAMY and blAMY and are glutamatergic [3,14]. The demonstration of long-term potentiation in AMY [2] and the growing body of evidence that NMDA-receptor antagonists block fear conditioning [5,15] suggest that LTP-like mechanisms may underlie emotional learning in these regions. In addition, dopaminergic projections to AMY, originating in the ventral tegmental area (VTA) [4,6], * Corresponding author. Behavioural Neuroscience Group, Department of Psychology, University of Leicester, University Road, Leicester LE1 7RH, UK. 1 Present address: Addiction Sciences Centre, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK.
have also been shown to influence conditioning [1,9]. However, little is known about dopamine release in AMY, nor about its function in the modulation of such learning. As a prelude to studies addressing questions of function, we have used brain microdialysis to look at (1) the distribution of extracellular dopamine within subfields of AMY and its response to pharmacological and physiological stimulation and (2) the calcium dependence (and therefore neurotransmitter origins) of this dopamine. A preliminary report of these results has been published [18]. All experiments used male Sprague–Dawley rats (B and K Universal, Hull, UK), approximately 280 g at the start of the procedure, and were performed with appropriate licence authority under the Animals (Scientific Procedures) Act, UK, 1986. In the first experiment, twin microdialysis probe assemblies were constructed by gluing two standard dialysis monoprobes [19] together, and bending the stainless-steel shafts, such that they ran parallel and 1 mm apart. One such assembly was implanted into either a medial or a lateral location in AMY of urethane (1.75 g/kg, i.p.) anaesthetised rats. In the lateral location, both probe tips lay in lAMY (Fr, +5.2 and +6.2; Tr, +5.0; V, +1.0: Fig. 1b,d), whereas in the
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medial location, both tips lay in blAMY and cAMY (Fig. 1a,c). The sizes of cAMY and blAMY relative to the dimensions of the dialysis probe tip preclude measurements specifically from one nucleus or the other. However the more caudal probe placement was located primarily in blAMY (Fr, +5.7; Tr, +4.5; V, +0.3: Fig. 1c), whilst the more rostral was primarily in cAMY but also sampled a portion of blAMY (Fr, +6.7; Tr, +4.5; V, +0.3: Fig. 1a). All coordinates quoted are relative to the intra-aural line [16]. Simultaneous dialysis perfusion of both probes with artificial cerebrospinal fluid (aCSF, mM: NaCl, 125; KCl, 3.3; MgSO4, 2.4; KH2PO4, 1.25; CaCl2, 1.85), at a flow rate of 3 ml/min, commenced immediately. After 120 min equilibration, consecutive 15-min dialysate samples were collected into glass vials containing 3 ml of 2 N H3PO4 (to minimise oxidation), and placed immediately into a refrigerated (8°C) autosampler (CMA200). All samples were analysed within 5 h of collection, by HPLC with electrochemical detection, as previously described [19], but using a 20-ml injection loop. The detection limit for dopamine was 2.5 fmol/injection (equivalent to 20 ml injection of 0.12 nM). The first four samples (60 min) collected were used to determine basal dopamine levels in the dialysates, after which amphetamine (1 mg/kg, i.p.) was administered, and a further six samples (90 min) were collected. In a second group of animals, after the 60-min basal collection period, the aCSF was replaced by calcium-free aCSF for the remainder of the experiment. Amphetamine (1 mg/kg, i.p.) was injected 60 min later, and dialysis perfusion continued, as before, for a further 90 min. In the second experiment, rats were implanted with stainless-steel guide cannulae [19] aimed at blAMY, under Immobilon anaesthetic (0.15 ml/kg, i.m.). After a recovery period of at least 7 days, dialysis probes [19] were inserted into the guide cannulae, under brief, light halothane anaesthetic, to lie bilaterally in medial-caudal AMY, (mostly blAMY: tip position: Fr, +5.7; Tr, ±4.5; V, +0.3, relative to the intra-aural line [16], Fig. 1c). Twenty-four hours later, animals were connected for microdialysis perfusion in a Skinner box, as previously described [19]. Dialysis perfusion with aCSF commenced immediately (flow rate 3 ml/min). Following 90 min equilibration, consecutive 15-min dialysate samples were collected into glass vials containing 3 ml of H3PO4, for HPLC analysis as described above. The first four samples (60 min) were collected for determination of the basal levels of dopamine, then the animals received five presentations of a mild footshock (1-s train of 6-ms pulses, 0.33 mA, 25 Hz), presented through the grid floor of the Skinner box, at 2-min intervals (first shock delivered 1 min into the sample). Sixty minutes after the end of the footshock presentation, a 30-ml (10 min, starting 1 min into the sample) pulse of high-potassium aCSF (100 mM KCl; NaCl reduced to 28 mM) was applied in the dialysis stream, after which dialysis perfusion continued for a further 45 min (three samples).
In a separate group of animals the calcium-dependence of the responses to footshock and to potassium was assessed. After the initial basal collection period (60 min), the dialysis perfusion medium was replaced by calcium-free aCSF for the remainder of the experiment. Mild footshock (60 min later) and potassium stimulation (60 min after that) were given as before. On completion of the experiments, animals were killed by anaesthetic overdose, and 20-mm cryostat sections were cut to verify the probe locations. In all experiments, the sample collection times were adjusted to take into account the dead volume of the dialysis collection tubes (12 ml in experiment 1; 36 ml in experiment 2). Statistical analysis by two-way analysis of variance with repeated measures was performed using SPSS for Windows (version 6.0). In the more lateral probe positions, corresponding to lAMY (Fig. 1b,d), dialysate levels of dopamine were for the most part undetectable, and amphetamine administration had no consistent measurable effect (data not shown). However basal dopamine was measurable in the two medial probe locations (Fig. 2). Levels were higher in the more caudal location, which measured primarily from blAMY (0.26 ± 0.04 nM) than in the more rostral location, which measured also from cAMY (0.18 ± 0.03 nM: F(1,14) = 4.99; P = 0.042).
Fig. 1. Diagram showing probe locations in relation to amygdaloid nuclei, on sections redrawn from the atlas of Paxinos and Watson [16], the horizontal coordinate is quoted relative to the intra-aural line (IAL). The black bar represents the intended location of the dialysis tip (excluding epoxy-sealing plug) according to the implantation coordinates used. The two lateral sites are shown in (b) and (d), and the two medial sites in (a) and (c). Implantation sites are depicted in the left side only, although in experiment 2, where bilateral probe implantations at (c) were used, probes were also implanted in an equivalent position on the right side of the brain. Histological examination showed that most probes lay close to these positions: those which did not (three animals in experiment 1) were not included in the analyses.
A.M.J. Young, K.R. Rees / Neuroscience Letters 249 (1998) 49–52
Fig. 2. The effect of amphetamine (1 mg/kg, i.p.; Amph) on dialysate concentrations of dopamine (mean ± SEM) in (a) blAMY and (b) cAMY, during perfusion with normal aCSF (dark shading, n = 6) and calcium-free aCSF (light shading, n = 4), in urethane anaesthetised animals. Basal levels of dopamine in the latter group prior to removal of calcium are also shown (mean ± SEM of four samples from each animal). Open bars represent time points where two or more samples fell below the detection limit of the analysis system.
In agreement with a previous report [8], amphetamine caused a significant rise in dopamine in both regions, peaking at 20–30 min after injection (F(7,98) = 26.03; P , 0.001: Fig. 2). However there was no significant difference in the response between the two regions (region × drug interaction, F(7,98) = 1.69; P = 0.120). Removal of calcium from the perfusion stream decreased the levels of dopamine to around 50% in both probe locations (F(1,14) = 8.78; P = 0.01: Fig. 2). In the rostral location the levels became unmeasurable in two out of the four animals. Amphetamine-evoked increases in extracellular dopamine were also reduced in both locations (Fig. 2), although it is not clear whether this was simply due to the reduced basal levels (calcium × amphetamine interaction: F(7,98) = 2.06; P = 0.054). On the basis of the results from experiment 1, dopamine measurements were made bilaterally in the medial-caudal probe location (principally blAMY: Fig. 1c), in conscious, unrestrained animals. Since both basal levels (left, 0.47 ± 0.08 nM; right, 0.44 ± 0.04 nM) and stimulated levels of dopamine, in the presence or absence of calcium were found to be similar between the two sides (F(1,14) =
51
0.37; P = 0.555), data from both were pooled for all subsequent analyses. Both repeated mild footshock and increased potassium evoked a significant increase in extracellular dopamine (F(2,28) = 14.14; P , 0.001, Fig. 3). Removal of calcium from the dialysis perfusion medium caused a reduction in basal extracellular dopamine to 0.26 ± 0.02 nM after 60 min (F(1,14) = 16.86, P = 0.001), and an abolition of the increases seen during footshock and potassium stimulation (calcium × stimulation interaction, F(2,28) = 10.11, P , 0.001, Fig. 3). The very low levels of extracellular dopamine in lAMY, even after amphetamine administration, indicate that there is unlikely to be substantial dopaminergic innervation of lAMY. Levels in the two medial locations, however, were measurable, although still 4- to 5-fold lower than reported in nucleus accumbens [19], indicating a more substantial dopaminergic innervation of blAMY and cAMY. Within the medial probe locations it is difficult to delineate between blAMY and cAMY, although the probes in the caudal site sampled mostly from blAMY, those in the more rostral site sampled from both blAMY and cAMY (Fig. 1a,c). These data provide evidence for dopamine release in blAMY, and also probably in cAMY, but little in lAMY, which corresponds to the reported pattern of innervation [4,6]. On the basis of these results, and in view of our ultimate aim of investigating the role of dopamine in the modulation of the conditioned learning process itself (occurring in lAMY and blAMY [3,13,14]), as opposed to expression of conditioned responding, we chose the caudal probe location for further investigations in conscious animals. Basal levels of extracellular dopamine measured primarily from blAMY in conscious rats were significantly higher than in anaesthetised rats, but were similar to levels recently
Fig. 3. Effect of mild footshock (Fs) and potassium stimulation (K) on dialysate dopamine concentrations (mean ± SEM) in blAMY, during perfusion with normal aCSF (dark shading: combined data from left and right sides of five animals) or calcium-free aCSF (light shading: combined data from left and right sides of four animals), in conscious, unrestrained animals. Basal levels of dopamine in the latter group prior to removal of calcium are also shown (mean ± SEM of four samples from each side of each animal).
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reported in dialysates from conscious rats with probes in cAMY [7] or spanning both blAMY and cAMY [8,12]. The difference may be a direct effect of anaesthesia, or it may be due to the different recovery times after probe implantation in the two situations (i.e. 2 h compared to 24 h). Both chemical (potassium) and behavioural (footshock) stimulation evoked large increases in extracellular dopamine, which were abolished by removal of calcium from the perfusion stream, indicating that the extracellular changes measured here do indeed reflect release of neurotransmitter dopamine. This is an important issue, not addressed in recent reports of microdialysis in AMY [7,8,10,12], since what is measured by microdialysis is the extracellular level of a compound, which does not necessarily represent neurotransmitter release. It should be noted, however, that the response to amphetamine was not abolished by removal of calcium. This is in line with previous findings that dopamine increases after a single injection of amphetamine are largely calcium-independent [11,17]. It is not clear from the present data whether there is a calciumdependent component of the response, since the interaction (calcium × amphetamine), although not significant, was close to it (P = 0.054), and further studies with larger group sizes would be needed to be certain. These studies confirm that dopamine measurements can be made in AMY, and, more importantly, show that both basal levels and increases following potassium stimulation and footshock are calcium-dependent, and therefore reflect neurotransmitter release.
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