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Inhibitory effects of trace amines on rat midbrain dopaminergic neurons
Neuropharmacology 46 (2004) 807–814 www.elsevier.com/locate/neuropharm
Inhibitory effects of trace amines on rat midbrain dopaminergic neurons Raffaella Geracitano a,1, Mauro Federici a,1, Simonetta Prisco a, Giorgio Bernardi a,b, Nicola B. Mercuri a,b, a
Department of Experimental Neurology, Fondazione Santa Lucia—IRCCS, Via Ardeatina 306, 00179 Rome, Italy b Clinica Neurologica Universita` ‘‘Tor Vergata’’, Rome, Italy Received 4 September 2003; received in revised form 19 November 2003; accepted 25 November 2003
Abstract Trace amines are biological compounds that are still awaiting identification of their role in neuronal function. Using intracellular electrophysiological recordings, we investigated the depressant action of two trace amines (b-phenylethylamine and tyramine) on the firing activity of dopaminergic neurons of the substantia nigra pars compacta and ventral tegmental area. This inhibition was due to a membrane hyperpolarisation that was blocked by the D2 dopamine receptor antagonist sulpiride and was not potentiated by the dopamine-uptake blocker, cocaine. Inhibition of the dopamine transporter did not mediate the effects of trace amines, because unlike cocaine, trace amines did not potentiate the inhibitory responses to exogenously applied dopamine. The inhibitory actions of b-phenylethylamine and tyramine were present in reserpine-treated animals but were abolished when the dopamine-synthesis inhibitor carbidopa was applied. Our data suggest that trace amines cause an indirect activation of dopamine autoreceptors, by an increased efflux of newly synthesised dopamine. The inhibition of dopaminergic activity by trace amines may relate to their involvement in neuronal processes linked to drug addiction, schizophrenia, attention deficit hyperactive disorders and Parkinson’s disease. # 2003 Elsevier Ltd. All rights reserved. Keywords: DAT; Trafficking; Microelectrode; Brain slice; Synaptic release
1. Introduction Trace amines are present in low concentrations in neuronal tissue where they are packaged and released along with traditional amines (Boulton, 1976; Durden and Phillips, 1980; Parker and Cubeddu, 1988). They have been considered to be ‘‘false transmitters’’, which displace active biogenic amines from their stores and are also believed to act on transporters in an amphetamine-like manner (Janssen et al., 1999; Mundorf et al., 1999; Parker and Cubeddu, 1988; Raiteri et al., 1978), thus, increasing the extracellular concentration of dopamine (DA) (Bailey et al., 1987). In addition, they
Corresponding author. Tel.: +39-06-5150-1383; fax: +39-065150-1384. E-mail address: [email protected] (N.B. Mercuri). 1 The two first authors equally contributed to this work.
0028-3908/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2003.11.031
have been thought as ‘‘modulators’’ of the neurotransmission mediated by catecholamines and serotonin (Boulton, 1991). The recent identification of a novel family of G-protein coupled trace amine receptors linked to cAMP production in expression systems (Borowsky et al., 2001; Bunzow et al., 2001), has revealed new insights into the mechanism by which trace amines function. It is known that the blockade of catecholamine catabolism leads to the accumulation of trace amines in the central nervous system (Boulton, 1991; Dyck et al., 1982; Holschneider et al., 2001). Moreover, antipsychotics, antidepressants, psychostimulants and drugs interfering with amine storage, alter the concentration of trace amines in the brain (Boulton et al., 1977, 1991; Juorio, 1983; Juorio and Danielson, 1978; Juorio et al., 1991a; Greenshaw et al., 1985; Sardar and Juorio, 1987). The role of trace amines in the dopaminergic
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midbrain is of great interest due to the established role of these neurons in schizophrenia, drug abuse, attention deficit hyperactive disorders (ADHD) and Parkinson’s disease. Some lines of evidence have already suggested that trace amines affect mental and motor activity by interacting with the dopaminergic system. Previous extracellular electrophysiological studies in vivo (b-PEA) (Barroso and Rodriguez, 1996) and in vitro (TYR) (Pinnock, 1983) have reported an inhibitory effect of two trace amines, b-phenylethylamine (b-PEA) and tyramine (TYR), on the firing rate of the dopaminergic neurons. However, nothing is known of the mechanism of this action of trace amines. Trace amines colocalise with dopamine in dopaminergic cells (Juorio et al., 1991b), can be released with DA (Jones et al., 1983), increase the release of DA (Raiteri et al., 1978) and induce homolateral turning in rats with a unilateral 6hydroxydopamine lesion of the nigrostriatal dopamine system (Barroso and Rodriguez, 1996). Since trace amines appear to be co-released with DA and may further stimulate DA release, we hypothesised that exogenous trace amines inhibit dopamine neurons by increasing DA release and the activation of the well characterised inhibitory D2 autoreceptors. We further hypothesised that stimulation of DA release by trace amines was a result of activation of the dopamine transporter (DAT). We have thus examined the inhibitory effects of two trace amines on the spontaneous discharge and membrane potential of dopaminergic neurons in ventral midbrain slices. We investigated the sensitivity of this response to antagonism of the D2 autoreceptor, blockade of DAT and depletion of vesicular dopamine. A preliminary account of this work has been previously presented in abstract form (Mercuri et al., 2002).
2. Materials and methods 2.1. Brain slice preparation and electrophysiological recordings Intracellular recordings with sharp microelectrodes were made from midbrain substantia nigra (SN) and ventral tegmental area (VTA) dopaminergic neurons in horizontal slices (250–300 lm thick) prepared from 21to 35-day-old male Wistar rats (150–300 g) (as described in Mercuri et al., 1995). Animals used in this study were treated in strict accordance with the approved experimental procedures of the Comitato Etico of the Tor Vergata University. Briefly, the animals were anaesthetised with halothane and decapitated. The brain was rapidly removed from the skull and horizontal
slices of the ventral midbrain were cut with a Vibratome 1000 (Pelco International, Redding, CA). For recordings, single slices were transferred to a recording chamber, immobilised with a titanium grid and superfused at a rate of 2.5 ml/min, with a solution v maintained at 35 C and equilibrated with a mixture of 95% O2 and 5% CO2. This standard extracellular solution contained (mM): NaCl, 126; KCl, 2.5; MgCl2, 1.2; NaH2PO4, 1.2; CaCl2, 2.4; Glucose 11, NaHCO3, 20; (pH 7.4). Intracellular recordings were made using borosilicate microelectrodes (tip resistance of 30–80 MX) prepared on a Sutter P97 puller (Sutter Instruments, Novato, CA) and filled with KCl (2 M). Recordings were made with an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA) using Axoscope software and a Digidata 1200B connected to a PC. Electrodes tips were guided into the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) under a dissecting microscope. The neurons were identified by their electrophysiological properties, including (i) the presence of regular spontaneous firing activity, (ii) a relaxation in hyperpolarising electrotonic potentials (sag) caused by the activation of Ih and (iii) hyperpolarisation to dopamine (10–30 lM) (Lacey et al., 1987; Johnson and North, 1992; Mercuri et al., 1995). Only neurons meeting these criteria were studied. 2.2. Drugs Drugs were dissolved as stock solutions and bathapplied at known concentrations by switching the perfusion from standard extracellular solution to drugcontaining solution, via a three-way tap. A complete exchange of the solution in the recording chamber occurred within about 1 min. The following drugs were used: sulpiride, (from Tocris Cookson, UK) bphenylethylamine, p-tyramine, reserpine, carbidopa, dopamine hydrochloride, cocaine hydrochloride, amphetamine hydrochloride from Sigma, tetrodotoxin (TTX) (from Alomone-labs, Israel). During experiments studying the interaction between trace amines, dopamine and cocaine, the membrane potential was manually clamped at 60 mV by injecting constantdepolarising current (50–120 pA). A group of rats was treated with reserpine 5–8 mg/kg injected subcutaneously 15–20 h before the electrophysiological experiments. These dosages produced profound hypokinesia and ptosis. 2.3. Data analysis Numerical data were expressed as mean standard error. Student’s t-test for paired observations was used to compare data. A p< 0:05 was considered significant. Since no differences were apparent in the responses to trace amines of dopaminergic neurons of
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the SNc and VTA, the data from both regions were pooled. The firing rates of neurons following drug application were normalised to the control firing rate in the same neuron. Concentration–response curves were fitted using a logistic equation: y ¼ A2 þ ðA1 A2 Þ= ð1 þ ðx=IC50 ÞpÞ: where y represents the normalised response, x represents the concentration, A1 represents the control firing rate, A2 represents the maximum inhibition, IC50 represents the concentration causing half maximal inhibition and p represents the power.
3. Results Data in this study were obtained from 55 intracellularly recorded ‘‘principal’’ neurons in the SNc and 22 in the VTA that were identified as dopaminergic according to electrophysiological and pharmacological criteria
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(see Methods). All neurons fired spontaneous action potentials at a mean rate of 1.2 Hz (range 0.9–2.8 Hz) and had a relatively long lasting spike (>1.2 ms). 3.1. Trace amines inhibit the activity of dopaminergic neurons Bath application of b-PEA (10–100 lM) (n¼ 10) and TYR (10–100 lM) (n¼ 12) caused a reversible inhibition of the spontaneous firing and a hyperpolarisation of the membrane (Fig. 1a and b). The inhibitory effects of b-PEA and TYR on spontaneous activity were concentration-dependent. The IC50 for b-PEA was 43:2 13:2 lM, (n¼ 5), while the IC50 for TYR was 39:6 6:7 lM, (n¼ 4) (Fig. 1c and d). b-PEA (100 lM) and TYR (100 lM) hyperpolarised the neurons by 3:5 2 mV (n¼ 10) and 6 2 mV (n¼ 10), respectively. The membrane hyperpolarisation caused by both
Fig. 1. TYR and b-PEA inhibit dopaminergic cells. Bath application of (a) TYR (100 lM) and (b) b-PEA (100 lM) produce a reversible hyperpolarisation and inhibition of spontaneous firing activity. The bars above the traces indicate the period of drugs application. In this and the following figures the arrows indicate the membrane potential level. Dose–response curves for the effects of (c) TYR and (d) b-PEA on spontaneous firing rate are shown. Points show mean effects and vertical lines indicate s.e. of the mean for n¼ 4 (TYR) and n¼ 5 (b-PEA) neurons.
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trace amines persisted in the presence of TTX (n¼ 4 cells for each compounds) (Fig. 2a). Under these conditions the membrane potential of the cells was constant between 57 and 60 mV. A drop (20%) of the apparent input resistance caused by both amines (100 lM) was also observed in three cells. 3.2. Sulpiride blocks the inhibition of dopaminergic neurons by trace amines Since trace amines are known to increase DA release (Raiteri et al., 1978) we wished to test if the inhibitory effects of these compounds were mediated by activation of the inhibitory D2 autoreceptor on dopamine neurons. The D2 receptor antagonist, sulpiride (1–3 lM) (Lacey et al., 1987) blocked the hyperpolarising effects of b-PEA and TYR in TTX-treated cells (n¼ 3, Fig. 2b). In the presence of sulpiride trace amines induced a small depolarisation (1–3 mV, n¼ 8) and a
Fig. 2. The inhibitory effects of TYR and b-PEA persist in TTX but not in sulpiride. (a) TYR (100 lM) and b-PEA (100 lM) hyperpolarise neurons in the presence of tetrodotoxin (1lM). (b) Sulpiride (1 lM) antagonises the membrane hyperpolarisation produced by TYR (100 lM) and b-PEA (100 lM) in this dopaminergic cell treated with TTX.
small but significant increase in the spontaneous firing rate (p¼ 0:01 (TYR), p¼ 0:023 (b-PEA) Student’s t-test) in eight neurons (not shown). The mean rate was 1:2 0:3 Hz in sulpiride and 1:7 0:3 Hz in the presence of sulpiride and b-PEA. The mean rate was 1:4 0:2 and 1:7 0:3 Hz in the presence of sulpiride and TYR. Since we have concentrated our attention on the inhibitory effects of trace amines, we did not further study the mechanisms of the excitation. 3.3. Cocaine has no effect on the trace amine-induced responses The membrane hyperpolarisation caused by both b-PEA and TYR was not depressed by the pharmacological inhibition of the DAT. The DAT blocker,
Fig. 3. TYR and b-PEA do not affect the DA transporter. (a) The membrane hyperpolarisation caused by TYR (100 lM) and b-PEA (100 lM) are not potentiated by cocaine (10 lM) in this neuron. Note that cocaine increases the membrane hyperpolarisation caused by DA (30 lM). The cell was manually camped at 60 mV. (b) In a different neuron, TYR (100 lM) and b-PEA (100 lM) produce a slight reduction of DA responses (10 lM). Note that the membrane changes caused by the amines were corrected by injecting constant depolarising current.
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cocaine (10 lM) (Lacey et al., 1990; Mercuri et al., 1991), did not change significantly the membrane hyperpolarisation caused by the superfusion of b-PEA (100 lM) (4 2 vs. 3:5 2 mV, p> 0:05, Student’s t-test, n¼ 4) or TYR (100 lM) (5:2 2 vs. 6 2 mV, p> 0:05, Student’s t-test, n¼ 5) (Fig. 3a). Moreover, b-PEA and TYR (30–100 lM) did not significantly affect the hyperpolarisation induced by exogenous DA application (3–30 lM) (n¼ 4, p> 0:05 Student’s t-test) (Fig. 3b). 3.4. Responses to trace amines after reserpine treatment and carbidopa Because trace amines have been thought to act as false neurotransmitters displacing catecholamines packaged in stores, we then tested whether a depletion of the intracellular stores containing DA affects the cellular responses to b-PEA and TYR. Therefore, we depleted intracellular dopamine by pretreating rats with reserpine 5–8 mg/kg. In midbrain slices obtained
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from such rats, b-PEA (100 lM) and TYR (100 lM) still induced an inhibition of firing associated with a clear hyperpolarisation in dopaminergic cells (not shown). Therefore, the membrane hyperpolarisation caused by b-PEA was 3:5 2 and 3:7 2 mV, and that caused by TYR was 6 2 and 5:8 2 mV, in control condition and after reserpine treatment, respectively. These effects were not significantly modified (p> 0:05, Student’s t-test). Interestingly, carbidopa (300 lM, 30 min), a dopa decarboxylase inhibitor (Mercuri et al., 1990), abolished the neuronal responses to trace amine application when superfused onto midbrain slices from reserpinetreated rats (n¼ 4, for each compound, Fig. 4a and b). Furthermore, the inhibition of firing and membrane hyperpolarisation known to be induced by amphetamine (10 lM, n¼ 3, Lacey, 1990) were also absent in dopamine neurons from reserpine-treated rats and carbidopa treated slices (Fig. 4c), but the extracellular superfusion of DA (10–30 lM) still caused the typical
Fig. 4. Carbidopa blocks the responses to trace amines but not those induced by DA in slices from a rat treated with reserpine. In a neuron from a reserpine treated rat, carbidopa (300 lM for 30 min) abolishes the cellular hyperpolarisation and membrane inhibition caused by (a) TYR (100 lM), (b) b-PEA (100 lM) and (c) amphetamine (10 lM). Note that the treatment with reserpine and carbidopa does not affect the response to (d) DA (10 lM) in the same neuron.
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D2-autoreceptor mediated response (Fig. 4d) well characterised elsewhere (Lacey et al., 1987).
4. Discussion Our intracellular experiments confirm the findings of previous extracellular studies (Rodriguez and Barroso, 1995; Pinnock, 1983) showing an inhibitory action of b-PEA and TYR on the DAergic cells in the ventral mesencephalon. The main finding reported here is that this action of trace amines is mediated by indirect activation of the D2 autoreceptors caused by an increase in dopamine release. The electrophysiological effects of stimulating the recently cloned trace amine receptors has not yet been identified. Despite this, we have shown that the ability of b-PEA and TYR to inhibit firing is related to an indirect stimulation of D2 like autoreceptors by an enhanced efflux of DA. This was evidenced by the fact that the inhibitory effects of trace amines were abolished (a) in the presence of the D2-selective antagonist sulpiride and (b) when presynaptic dopamine depletion in reserpine-treated rats was combined with the inhibition of DA synthesis by carbidopa. Interestingly, the membrane hyperpolarisation induced by trace amines persisted in TTX suggesting that trace amines-induced DA release is independent of neuronal and network activity. Notwithstanding the clear-cut inhibitory effects of b-PEA and TYR on DAergic neurons, trace amines are reported to have only a weak effect on neuronal discharge and to enhance the effects of exogenous catecholamines in in vivo electrophysiological studies of cortical and striatal neurons (Jones and Boulton, 1980). In contrast, we did not observe a potentiation of the electrophysiological effects of exogenous DA application, as would be expected if DAT on the dopaminergic cells was inhibited by b-PEA and TYR (Lacey et al., 1990; Mercuri, 1991). The observation that the membrane changes caused by trace amines were not potentiated by cocaine also suggests that b-PEA and TYR partially stimulate, in an amphetamine-like manner, a carrier-mediated efflux of DA from the dendrites of the dopaminergic cells (Raiteri et al., 1978). Studies from our group and others have previously shown that the pharmacological blockade of DAT reduces amphetamine’s effects on the dopaminergic cells (Scarponi et al., 1999; Paladini et al., 2001). However, because the electrophysiological effects of trace amines as well as those induced by amphetamine (Scarponi et al., 1999) were not abolished by reserpine, it is still possible that the trace amines also mobilise newly synthesised DA from the intracellular to the extracellular space in a manner such as that activated by the DAT independent effects of amphetamine
(Janssen et al., 1999; Parker and Cubeddu, 1988; Mundorf et al., 1999). Therefore, our results support the idea that trace amines regulate the activity of the dopaminergic cells by stimulating the efflux of newly synthesised DA from reserpine-insensitive pools via a carrier-dependent and independent mechanism. The indirect stimulation of DA receptors which accounts for our electrophysiological data is also in line with behavioural observations reporting that b-PEA induces ipsilateral rotations in rats having 6-hydroxydopamine lesion of the nigrostriatal dopaminergic system (Barroso and Rodriguez, 1996). The concentrations of trace amines that induce neuronal inhibition appear to be higher than those previously detected in the brain. These substances are mainly acting intracellularly to induce the release of DA. For this reason, in spite of the fact that their extracellular concentration might below, they could be still effective in inducing an efflux of catecholamines. However, it has to be pointed out that the major extracellular metabolites of classical catecholamines produced by catechol-O-methyl transferase or the product of l-amino acid decarboxylation are b-PEA (from phenylalanine) and TYR (from tyrosine) (Juorio, 1983; Tallman et al., 1976). Thus, changes in the activity of these enzymes might increase their levels in the brain. In addition, treatment with monoamine oxidase (MAO) inhibitors would slow the rapid turnover of trace amines and thus increase their brain concentration (Dyck et al., 1982). 4.1. Functional implications The immediate information arising from the present work is the clear-cut modulation of the firing rate of the dopaminergic neurons caused by trace amines, which is dependent on the release of newly synthesised DA from dendrites. This phenomenon of enhanced DA release could also occur in the dopaminergic terminal fields thus acting to increase the levels of extracellular DA in target areas. Therefore, either the depression of firing activity of the dopaminergic cells or the increased release of DA from the dopaminergic terminals caused by trace amines could be of crucial importance in the functional processes of the mesolimbic and mesostriatal system. This suggests a feasible role for trace amines in the physiopathology of drug addiction, schizophrenia, attention-deficit hyperactive disorders and Parkinson’s disease (Bonci et al., 2003; Branchek and Blackburn, 2003; Grace, 1991; Mercuri et al., 1997; O’Reilly and Davis, 1994; Shannon and Degregorio, 1982).
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Acknowledgements We thank Dr. C. Peter Bengtson for his comments on the manuscript. This work was supported by CNR (Biomolecole Per La Salute Umana) and MURST (Cofin) grants to N. B. Mercuri.
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Inhibitory effects of trace amines on rat midbrain dopaminergic neurons Raffaella Geracitano a,1, Mauro Federici a,1, Simonetta Prisco a, Giorgio Bernardi a,b, Nicola B. Mercuri a,b, a
Department of Experimental Neurology, Fondazione Santa Lucia—IRCCS, Via Ardeatina 306, 00179 Rome, Italy b Clinica Neurologica Universita` ‘‘Tor Vergata’’, Rome, Italy Received 4 September 2003; received in revised form 19 November 2003; accepted 25 November 2003
Abstract Trace amines are biological compounds that are still awaiting identification of their role in neuronal function. Using intracellular electrophysiological recordings, we investigated the depressant action of two trace amines (b-phenylethylamine and tyramine) on the firing activity of dopaminergic neurons of the substantia nigra pars compacta and ventral tegmental area. This inhibition was due to a membrane hyperpolarisation that was blocked by the D2 dopamine receptor antagonist sulpiride and was not potentiated by the dopamine-uptake blocker, cocaine. Inhibition of the dopamine transporter did not mediate the effects of trace amines, because unlike cocaine, trace amines did not potentiate the inhibitory responses to exogenously applied dopamine. The inhibitory actions of b-phenylethylamine and tyramine were present in reserpine-treated animals but were abolished when the dopamine-synthesis inhibitor carbidopa was applied. Our data suggest that trace amines cause an indirect activation of dopamine autoreceptors, by an increased efflux of newly synthesised dopamine. The inhibition of dopaminergic activity by trace amines may relate to their involvement in neuronal processes linked to drug addiction, schizophrenia, attention deficit hyperactive disorders and Parkinson’s disease. # 2003 Elsevier Ltd. All rights reserved. Keywords: DAT; Trafficking; Microelectrode; Brain slice; Synaptic release
1. Introduction Trace amines are present in low concentrations in neuronal tissue where they are packaged and released along with traditional amines (Boulton, 1976; Durden and Phillips, 1980; Parker and Cubeddu, 1988). They have been considered to be ‘‘false transmitters’’, which displace active biogenic amines from their stores and are also believed to act on transporters in an amphetamine-like manner (Janssen et al., 1999; Mundorf et al., 1999; Parker and Cubeddu, 1988; Raiteri et al., 1978), thus, increasing the extracellular concentration of dopamine (DA) (Bailey et al., 1987). In addition, they
Corresponding author. Tel.: +39-06-5150-1383; fax: +39-065150-1384. E-mail address: [email protected] (N.B. Mercuri). 1 The two first authors equally contributed to this work.
0028-3908/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2003.11.031
have been thought as ‘‘modulators’’ of the neurotransmission mediated by catecholamines and serotonin (Boulton, 1991). The recent identification of a novel family of G-protein coupled trace amine receptors linked to cAMP production in expression systems (Borowsky et al., 2001; Bunzow et al., 2001), has revealed new insights into the mechanism by which trace amines function. It is known that the blockade of catecholamine catabolism leads to the accumulation of trace amines in the central nervous system (Boulton, 1991; Dyck et al., 1982; Holschneider et al., 2001). Moreover, antipsychotics, antidepressants, psychostimulants and drugs interfering with amine storage, alter the concentration of trace amines in the brain (Boulton et al., 1977, 1991; Juorio, 1983; Juorio and Danielson, 1978; Juorio et al., 1991a; Greenshaw et al., 1985; Sardar and Juorio, 1987). The role of trace amines in the dopaminergic
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midbrain is of great interest due to the established role of these neurons in schizophrenia, drug abuse, attention deficit hyperactive disorders (ADHD) and Parkinson’s disease. Some lines of evidence have already suggested that trace amines affect mental and motor activity by interacting with the dopaminergic system. Previous extracellular electrophysiological studies in vivo (b-PEA) (Barroso and Rodriguez, 1996) and in vitro (TYR) (Pinnock, 1983) have reported an inhibitory effect of two trace amines, b-phenylethylamine (b-PEA) and tyramine (TYR), on the firing rate of the dopaminergic neurons. However, nothing is known of the mechanism of this action of trace amines. Trace amines colocalise with dopamine in dopaminergic cells (Juorio et al., 1991b), can be released with DA (Jones et al., 1983), increase the release of DA (Raiteri et al., 1978) and induce homolateral turning in rats with a unilateral 6hydroxydopamine lesion of the nigrostriatal dopamine system (Barroso and Rodriguez, 1996). Since trace amines appear to be co-released with DA and may further stimulate DA release, we hypothesised that exogenous trace amines inhibit dopamine neurons by increasing DA release and the activation of the well characterised inhibitory D2 autoreceptors. We further hypothesised that stimulation of DA release by trace amines was a result of activation of the dopamine transporter (DAT). We have thus examined the inhibitory effects of two trace amines on the spontaneous discharge and membrane potential of dopaminergic neurons in ventral midbrain slices. We investigated the sensitivity of this response to antagonism of the D2 autoreceptor, blockade of DAT and depletion of vesicular dopamine. A preliminary account of this work has been previously presented in abstract form (Mercuri et al., 2002).
2. Materials and methods 2.1. Brain slice preparation and electrophysiological recordings Intracellular recordings with sharp microelectrodes were made from midbrain substantia nigra (SN) and ventral tegmental area (VTA) dopaminergic neurons in horizontal slices (250–300 lm thick) prepared from 21to 35-day-old male Wistar rats (150–300 g) (as described in Mercuri et al., 1995). Animals used in this study were treated in strict accordance with the approved experimental procedures of the Comitato Etico of the Tor Vergata University. Briefly, the animals were anaesthetised with halothane and decapitated. The brain was rapidly removed from the skull and horizontal
slices of the ventral midbrain were cut with a Vibratome 1000 (Pelco International, Redding, CA). For recordings, single slices were transferred to a recording chamber, immobilised with a titanium grid and superfused at a rate of 2.5 ml/min, with a solution v maintained at 35 C and equilibrated with a mixture of 95% O2 and 5% CO2. This standard extracellular solution contained (mM): NaCl, 126; KCl, 2.5; MgCl2, 1.2; NaH2PO4, 1.2; CaCl2, 2.4; Glucose 11, NaHCO3, 20; (pH 7.4). Intracellular recordings were made using borosilicate microelectrodes (tip resistance of 30–80 MX) prepared on a Sutter P97 puller (Sutter Instruments, Novato, CA) and filled with KCl (2 M). Recordings were made with an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA) using Axoscope software and a Digidata 1200B connected to a PC. Electrodes tips were guided into the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) under a dissecting microscope. The neurons were identified by their electrophysiological properties, including (i) the presence of regular spontaneous firing activity, (ii) a relaxation in hyperpolarising electrotonic potentials (sag) caused by the activation of Ih and (iii) hyperpolarisation to dopamine (10–30 lM) (Lacey et al., 1987; Johnson and North, 1992; Mercuri et al., 1995). Only neurons meeting these criteria were studied. 2.2. Drugs Drugs were dissolved as stock solutions and bathapplied at known concentrations by switching the perfusion from standard extracellular solution to drugcontaining solution, via a three-way tap. A complete exchange of the solution in the recording chamber occurred within about 1 min. The following drugs were used: sulpiride, (from Tocris Cookson, UK) bphenylethylamine, p-tyramine, reserpine, carbidopa, dopamine hydrochloride, cocaine hydrochloride, amphetamine hydrochloride from Sigma, tetrodotoxin (TTX) (from Alomone-labs, Israel). During experiments studying the interaction between trace amines, dopamine and cocaine, the membrane potential was manually clamped at 60 mV by injecting constantdepolarising current (50–120 pA). A group of rats was treated with reserpine 5–8 mg/kg injected subcutaneously 15–20 h before the electrophysiological experiments. These dosages produced profound hypokinesia and ptosis. 2.3. Data analysis Numerical data were expressed as mean standard error. Student’s t-test for paired observations was used to compare data. A p< 0:05 was considered significant. Since no differences were apparent in the responses to trace amines of dopaminergic neurons of
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the SNc and VTA, the data from both regions were pooled. The firing rates of neurons following drug application were normalised to the control firing rate in the same neuron. Concentration–response curves were fitted using a logistic equation: y ¼ A2 þ ðA1 A2 Þ= ð1 þ ðx=IC50 ÞpÞ: where y represents the normalised response, x represents the concentration, A1 represents the control firing rate, A2 represents the maximum inhibition, IC50 represents the concentration causing half maximal inhibition and p represents the power.
3. Results Data in this study were obtained from 55 intracellularly recorded ‘‘principal’’ neurons in the SNc and 22 in the VTA that were identified as dopaminergic according to electrophysiological and pharmacological criteria
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(see Methods). All neurons fired spontaneous action potentials at a mean rate of 1.2 Hz (range 0.9–2.8 Hz) and had a relatively long lasting spike (>1.2 ms). 3.1. Trace amines inhibit the activity of dopaminergic neurons Bath application of b-PEA (10–100 lM) (n¼ 10) and TYR (10–100 lM) (n¼ 12) caused a reversible inhibition of the spontaneous firing and a hyperpolarisation of the membrane (Fig. 1a and b). The inhibitory effects of b-PEA and TYR on spontaneous activity were concentration-dependent. The IC50 for b-PEA was 43:2 13:2 lM, (n¼ 5), while the IC50 for TYR was 39:6 6:7 lM, (n¼ 4) (Fig. 1c and d). b-PEA (100 lM) and TYR (100 lM) hyperpolarised the neurons by 3:5 2 mV (n¼ 10) and 6 2 mV (n¼ 10), respectively. The membrane hyperpolarisation caused by both
Fig. 1. TYR and b-PEA inhibit dopaminergic cells. Bath application of (a) TYR (100 lM) and (b) b-PEA (100 lM) produce a reversible hyperpolarisation and inhibition of spontaneous firing activity. The bars above the traces indicate the period of drugs application. In this and the following figures the arrows indicate the membrane potential level. Dose–response curves for the effects of (c) TYR and (d) b-PEA on spontaneous firing rate are shown. Points show mean effects and vertical lines indicate s.e. of the mean for n¼ 4 (TYR) and n¼ 5 (b-PEA) neurons.
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trace amines persisted in the presence of TTX (n¼ 4 cells for each compounds) (Fig. 2a). Under these conditions the membrane potential of the cells was constant between 57 and 60 mV. A drop (20%) of the apparent input resistance caused by both amines (100 lM) was also observed in three cells. 3.2. Sulpiride blocks the inhibition of dopaminergic neurons by trace amines Since trace amines are known to increase DA release (Raiteri et al., 1978) we wished to test if the inhibitory effects of these compounds were mediated by activation of the inhibitory D2 autoreceptor on dopamine neurons. The D2 receptor antagonist, sulpiride (1–3 lM) (Lacey et al., 1987) blocked the hyperpolarising effects of b-PEA and TYR in TTX-treated cells (n¼ 3, Fig. 2b). In the presence of sulpiride trace amines induced a small depolarisation (1–3 mV, n¼ 8) and a
Fig. 2. The inhibitory effects of TYR and b-PEA persist in TTX but not in sulpiride. (a) TYR (100 lM) and b-PEA (100 lM) hyperpolarise neurons in the presence of tetrodotoxin (1lM). (b) Sulpiride (1 lM) antagonises the membrane hyperpolarisation produced by TYR (100 lM) and b-PEA (100 lM) in this dopaminergic cell treated with TTX.
small but significant increase in the spontaneous firing rate (p¼ 0:01 (TYR), p¼ 0:023 (b-PEA) Student’s t-test) in eight neurons (not shown). The mean rate was 1:2 0:3 Hz in sulpiride and 1:7 0:3 Hz in the presence of sulpiride and b-PEA. The mean rate was 1:4 0:2 and 1:7 0:3 Hz in the presence of sulpiride and TYR. Since we have concentrated our attention on the inhibitory effects of trace amines, we did not further study the mechanisms of the excitation. 3.3. Cocaine has no effect on the trace amine-induced responses The membrane hyperpolarisation caused by both b-PEA and TYR was not depressed by the pharmacological inhibition of the DAT. The DAT blocker,
Fig. 3. TYR and b-PEA do not affect the DA transporter. (a) The membrane hyperpolarisation caused by TYR (100 lM) and b-PEA (100 lM) are not potentiated by cocaine (10 lM) in this neuron. Note that cocaine increases the membrane hyperpolarisation caused by DA (30 lM). The cell was manually camped at 60 mV. (b) In a different neuron, TYR (100 lM) and b-PEA (100 lM) produce a slight reduction of DA responses (10 lM). Note that the membrane changes caused by the amines were corrected by injecting constant depolarising current.
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cocaine (10 lM) (Lacey et al., 1990; Mercuri et al., 1991), did not change significantly the membrane hyperpolarisation caused by the superfusion of b-PEA (100 lM) (4 2 vs. 3:5 2 mV, p> 0:05, Student’s t-test, n¼ 4) or TYR (100 lM) (5:2 2 vs. 6 2 mV, p> 0:05, Student’s t-test, n¼ 5) (Fig. 3a). Moreover, b-PEA and TYR (30–100 lM) did not significantly affect the hyperpolarisation induced by exogenous DA application (3–30 lM) (n¼ 4, p> 0:05 Student’s t-test) (Fig. 3b). 3.4. Responses to trace amines after reserpine treatment and carbidopa Because trace amines have been thought to act as false neurotransmitters displacing catecholamines packaged in stores, we then tested whether a depletion of the intracellular stores containing DA affects the cellular responses to b-PEA and TYR. Therefore, we depleted intracellular dopamine by pretreating rats with reserpine 5–8 mg/kg. In midbrain slices obtained
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from such rats, b-PEA (100 lM) and TYR (100 lM) still induced an inhibition of firing associated with a clear hyperpolarisation in dopaminergic cells (not shown). Therefore, the membrane hyperpolarisation caused by b-PEA was 3:5 2 and 3:7 2 mV, and that caused by TYR was 6 2 and 5:8 2 mV, in control condition and after reserpine treatment, respectively. These effects were not significantly modified (p> 0:05, Student’s t-test). Interestingly, carbidopa (300 lM, 30 min), a dopa decarboxylase inhibitor (Mercuri et al., 1990), abolished the neuronal responses to trace amine application when superfused onto midbrain slices from reserpinetreated rats (n¼ 4, for each compound, Fig. 4a and b). Furthermore, the inhibition of firing and membrane hyperpolarisation known to be induced by amphetamine (10 lM, n¼ 3, Lacey, 1990) were also absent in dopamine neurons from reserpine-treated rats and carbidopa treated slices (Fig. 4c), but the extracellular superfusion of DA (10–30 lM) still caused the typical
Fig. 4. Carbidopa blocks the responses to trace amines but not those induced by DA in slices from a rat treated with reserpine. In a neuron from a reserpine treated rat, carbidopa (300 lM for 30 min) abolishes the cellular hyperpolarisation and membrane inhibition caused by (a) TYR (100 lM), (b) b-PEA (100 lM) and (c) amphetamine (10 lM). Note that the treatment with reserpine and carbidopa does not affect the response to (d) DA (10 lM) in the same neuron.
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D2-autoreceptor mediated response (Fig. 4d) well characterised elsewhere (Lacey et al., 1987).
4. Discussion Our intracellular experiments confirm the findings of previous extracellular studies (Rodriguez and Barroso, 1995; Pinnock, 1983) showing an inhibitory action of b-PEA and TYR on the DAergic cells in the ventral mesencephalon. The main finding reported here is that this action of trace amines is mediated by indirect activation of the D2 autoreceptors caused by an increase in dopamine release. The electrophysiological effects of stimulating the recently cloned trace amine receptors has not yet been identified. Despite this, we have shown that the ability of b-PEA and TYR to inhibit firing is related to an indirect stimulation of D2 like autoreceptors by an enhanced efflux of DA. This was evidenced by the fact that the inhibitory effects of trace amines were abolished (a) in the presence of the D2-selective antagonist sulpiride and (b) when presynaptic dopamine depletion in reserpine-treated rats was combined with the inhibition of DA synthesis by carbidopa. Interestingly, the membrane hyperpolarisation induced by trace amines persisted in TTX suggesting that trace amines-induced DA release is independent of neuronal and network activity. Notwithstanding the clear-cut inhibitory effects of b-PEA and TYR on DAergic neurons, trace amines are reported to have only a weak effect on neuronal discharge and to enhance the effects of exogenous catecholamines in in vivo electrophysiological studies of cortical and striatal neurons (Jones and Boulton, 1980). In contrast, we did not observe a potentiation of the electrophysiological effects of exogenous DA application, as would be expected if DAT on the dopaminergic cells was inhibited by b-PEA and TYR (Lacey et al., 1990; Mercuri, 1991). The observation that the membrane changes caused by trace amines were not potentiated by cocaine also suggests that b-PEA and TYR partially stimulate, in an amphetamine-like manner, a carrier-mediated efflux of DA from the dendrites of the dopaminergic cells (Raiteri et al., 1978). Studies from our group and others have previously shown that the pharmacological blockade of DAT reduces amphetamine’s effects on the dopaminergic cells (Scarponi et al., 1999; Paladini et al., 2001). However, because the electrophysiological effects of trace amines as well as those induced by amphetamine (Scarponi et al., 1999) were not abolished by reserpine, it is still possible that the trace amines also mobilise newly synthesised DA from the intracellular to the extracellular space in a manner such as that activated by the DAT independent effects of amphetamine
(Janssen et al., 1999; Parker and Cubeddu, 1988; Mundorf et al., 1999). Therefore, our results support the idea that trace amines regulate the activity of the dopaminergic cells by stimulating the efflux of newly synthesised DA from reserpine-insensitive pools via a carrier-dependent and independent mechanism. The indirect stimulation of DA receptors which accounts for our electrophysiological data is also in line with behavioural observations reporting that b-PEA induces ipsilateral rotations in rats having 6-hydroxydopamine lesion of the nigrostriatal dopaminergic system (Barroso and Rodriguez, 1996). The concentrations of trace amines that induce neuronal inhibition appear to be higher than those previously detected in the brain. These substances are mainly acting intracellularly to induce the release of DA. For this reason, in spite of the fact that their extracellular concentration might below, they could be still effective in inducing an efflux of catecholamines. However, it has to be pointed out that the major extracellular metabolites of classical catecholamines produced by catechol-O-methyl transferase or the product of l-amino acid decarboxylation are b-PEA (from phenylalanine) and TYR (from tyrosine) (Juorio, 1983; Tallman et al., 1976). Thus, changes in the activity of these enzymes might increase their levels in the brain. In addition, treatment with monoamine oxidase (MAO) inhibitors would slow the rapid turnover of trace amines and thus increase their brain concentration (Dyck et al., 1982). 4.1. Functional implications The immediate information arising from the present work is the clear-cut modulation of the firing rate of the dopaminergic neurons caused by trace amines, which is dependent on the release of newly synthesised DA from dendrites. This phenomenon of enhanced DA release could also occur in the dopaminergic terminal fields thus acting to increase the levels of extracellular DA in target areas. Therefore, either the depression of firing activity of the dopaminergic cells or the increased release of DA from the dopaminergic terminals caused by trace amines could be of crucial importance in the functional processes of the mesolimbic and mesostriatal system. This suggests a feasible role for trace amines in the physiopathology of drug addiction, schizophrenia, attention-deficit hyperactive disorders and Parkinson’s disease (Bonci et al., 2003; Branchek and Blackburn, 2003; Grace, 1991; Mercuri et al., 1997; O’Reilly and Davis, 1994; Shannon and Degregorio, 1982).
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Acknowledgements We thank Dr. C. Peter Bengtson for his comments on the manuscript. This work was supported by CNR (Biomolecole Per La Salute Umana) and MURST (Cofin) grants to N. B. Mercuri.
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