Nicola B. Mercuri⁽¹⁾, Pernilla Grillner^(2)
(1)IRCCS Santa Lucia, University of Rome Tor Vergata - Rome. Italy
⁽²⁾Dept Physiology and Pharmacology. Karolinska Institutet - Stockholm. Sweden

	[Cell Biology & Cytology]	[Neuroscience]

INTRODUCTION

A. Dopaminergic systems of the midbrain

1. Anatomy

Dopamine was discovered as an independent neurotransmitter in the 1950´s (Carlsson et al. 1958). The description of the neuronal populations containing dopamine and their anatomical distribution was subsequently made possible by the introduction of the histochemical formaldehyde fluorescence technique by Falck and Hillarp (Falck et al. 1962) by which the monoaminergic neurones in the brain could be visualised. Dahlström and Fuxe (1964) subsequently provided a more detailed description of the dopaminergic systems in the rat brain. The dopamine neurones of the midbrain are mainly located in three distinct areas, the retrorubral field (A8), the substantia nigra pars compacta (SNPC; A9) and the ventral tegmental area (VTA; A10), which contain 70-75% of the dopamine neurones in the brain. They are organised in two principal dopaminergic systems that are named according to their respective projection areas (see for review Ungerstedt 1971, Björklund and Lindvall 1984). The nigrostriatal system thus originates in the SNPC and projects to the dorsal striatum, i.e. caudate and putamen (Andén et al. 1964), and the mesolimbocortical system projects from the VTA to the limbic areas of the ventral striatum (i.e. nucleus accumbens), amygdala and olfactory tubercle, as well as limbic cortices such as the medial prefrontal, cingulate and entorhinal (Andén et al. 1966, Ungerstedt 1971, Björklund and Lindvall 1984). The dopamine neurones of the retrorubral field (A8) also project to the dorsal striatum and can be viewed as a caudal extension of SNPC (Ungerstedt 1971, Nauta 1978).

 

2. Physiological characteristics of the midbrain dopamine neurones

The dopaminergic neurones of the SNPC and VTA have been well characterised by the use of electrophysiological methods both in vivo and in vitro combined with immunohistochemical techniques (Aghajanian and Bunney 1973, Grace and Bunney 1983, Grace and Onn 1989, Lacey et al. 1989, Johnson and North 1992b). The dopamine neurones described in vivo by intracellular or extracellular recordings in anaesthetised rats have a typical firing pattern that is irregular and usually consists of bursts of action potentials riding on a depolarising wave. The action potential has a characteristic shape with a long duration of the depolarising phase (>2 ms) (Aghajanian and Bunney 1973, Grace and Bunney 1983, 1984). The burst firing activity of dopamine neurones recorded in vivo has been shown to be more efficient in increasing the dopamine efflux in the terminal areas than regular firing activity (Gonon and Buda 1985, Gonon 1988, Bean and Roth 1991, Chergui et al. 1996, 1997). In vitro studies from neurones of the SNPC and VTA in a rat brain slice preparation has revealed the existence of two neuronal types with distinct electrophysiological characteristics (Lacey et al. 1989, Johnson and North 1992b). One class, called principal neurones, consists of neurones with immunoreactivity for the dopamine synthesising enzyme tyrosine hydroxylase and is thus regarded as dopaminergic (Grace and Onn 1989). The other class, named secondary neurones, is considered to be g-aminobutyric acid (GABA)ergic (Mugnaini and Oertel 1985) and/or enkephalinergic (Finley et al. 1981) interneurones. The dopaminergic neurones in vitro display a spontaneous, relatively slow and highly regular firing activity of 0.5-4 Hz. They have action potentials with a long duration (>2ms), a pronounced afterhyperpolarisation (AHP), show an inward current in response to hyperpolarising pulses, and respond with hyperpolarisation to dopamine application (Grace and Onn 1989, Johnson and North 1992b; Mercuri et al., 1995). When recorded in vitro the dopamine neurones can display burst activity if NMDA is applied together with the Ca²⁺-dependent K⁺ channel (K_Ca) blocker apamine (see Seutin et al. 1993). The dopamine cells are readily distinguished from the presumed GABAergic interneurones, which in contrast display an irregular or silent firing activity, with action potentials of shorter duration (<1.5 ms) and show no inhibitory response to dopamine but are inhibited by opiates (Lacey et al. 1989, Johnson and North 1992b). The dopamine neurones in the two different areas have been shown to markedly differ in vitro neither in their physiological properties nor in their response to various pharmacological manipulations. However, in vivo a difference in sensitivity to pharmacological agents is often present and may be explained by the absence of active afferent inputs in vitro.

3. Afferent input to the midbrain dopamine neurones

The SNPC and VTA receive glutamatergic, GABAergic, cholinergic, serotoninergic and noradrenergic afferent inputs.

The glutamatergic afferents to midbrain dopamine neurones arise mainly from the prefrontal cortex, the subthalamic, laterodorsal- (LDTg) and pedunculopontine (PPTg) tegmental nuclei. The glutamatergic input is believed to regulate the firing activity, in particular burst firing, seen in vivo (Grenhoff et al. 1988, Svensson and Tung 1989, Charléty et al. 1991) particularly through activation of ionotropic glutamate receptors of the N-methyl-D-aspartate (NMDA)-type (Chergui et al. 1993, 1994). By electrical stimulation of the afferents in a brain slice preparation, synaptic potentials can be elicited in dopamine neurones that are mediated through activation of predominantly a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors and to a smaller extent through NMDA receptor activation (Mereu et al. 1991, Johnson and North 1992b). Metabotropic glutamate receptors (mGluRs) have also been shown to mediate a slow excitatory (Shen and Johnson 1997) as well as an inhibitory synaptic potential (Fiorillo and Williams 1998). Bath application of a metabotropic agonist induces an excitatory current in the dopamine neurones (Mercuri et al.1993). The presence of glutamate in the dopamine neurones themselves has recently been reported (Sulzer et al. 1998). It was shown that cultured midbrain dopamine neurones form functional glutamatergic synapses, so-called autapses.

The GABAergic input to the midbrain dopamine neurones originates in the striatum and pallidum (Fonnum et al. 1978, Bolam and Smith 1990, Smith and Bolam 1990) and from intrinsic GABAergic interneurones of the midbrain (Di Chiara et al. 1979, Grofova et al. 1982, Stanford and Lacey 1996). Both GABA_A- and GABA_B-mediated synaptic potentials can be evoked by focal stimulation of the slice preparation (Johnson and North 1992b). Bath application of GABA induces a hyperpolarisation mediated through both GABA_A and GABA_B receptor activation. The GABA_B-mediated responses are G-protein mediated causing an increase in K⁺ conductance (Lacey et al. 1988).

The cholinergic input to the dopamine neurones arises in the PPTg and in the adjacently located LDTg (Beninato and Spencer 1987, Clarke et al. 1987, Bolam et al. 1991). The LDTg predominantly projects to VTA while PPTg projects to SNPC (Oakman et al. 1995). These pontine nuclei thus provide both glutamatergic and cholinergic innervation to the dopamine neurones as revealed by immunocytochemical labelling for choline-acetyltransferase and glutamate combined with anterograde tracing techniques (Lavoie and Parent 1994a, c). The PPTg sends an extensive projection to the SNPC, where the terminals closely surround the soma and proximal dendrites of the dopamine neurones (Lavoie and Parent 1994b). Both muscarinic and nicotinic receptors are present on dopamine neurones (Clarke et al. 1985, Cortès and Palacios 1986, Schwartz 1986, Wada et al. 1989) and pharmacological activation of these receptors leads to excitation of the dopamine neurones in vitro (Calabresi et al. 1989, Lacey et al. 1990). Nicotine, administered systemically in vivo, increases the burst firing activity of VTA dopamine neurones (Grenhoff et al. 1986, Mereu et al. 1987) associated with an increased release of dopamine in the nucleus accumbens (Imperato et al. 1986, Damsma et al. 1989), that is blocked by local application into the VTA, but not into the nucleus accumbens, of the nicotinic receptor antagonist mecamylamine (Nisell et al. 1994). Furthermore, chemical stimulation of the PPTg excites the dopamine neurones of SNPC, an effect that is blocked by local application of mecamylamine in the SNPC (Clarke et al. 1987). However, synaptic potentials mediated through activation of cholinergic receptors have, as yet, not been convincingly demonstrated in dopamine neurones.

Also the raphe nuclei and the locus coeruleus provide serotoninergic (Dray et al. 1976, Fibiger and Miller 1977, Phillipson 1979, Hervé et al. 1987, van Bockstaele et al. 1994) and noradrenergic (Phillipson 1979, Grenhoff et al. 1993, 1995) afferent inputs to dopamine neurones.

 

4. Functional role of the midbrain dopamine neurones

The dopamine neurones of the midbrain are implicated in motor tasks related to normal movements, motivational as well as reward-related behaviour and cognitive functions (see for review Le Moal and Simon 1991, Schultz 1997, 1998, Koob et al. 1998).

The nigrostriatal dopamine neurones are critically involved in the pathophysiology of Parkinson´s disease (Carlsson et al. 1959, Ehringer and Hornykiewicz 1960), and degeneration of the dopamine neurones in the ventrolateral part of SNPC and to a lesser degree also in the VTA is responsible for the typical symptoms of Parkinson´s disease, namely, bradykinesia, rigidity and tremor. The degeneration of the dopamine neurones leads to a reduced dopamine output to neostriatum (e.g. caudate-putamen), increasing the activity in the indirect loop to globus pallidus internal and substantia nigra pars reticulata (SNPR) of the basal ganglia leading to an increased inhibition of motor output from thalamus (see for review DeLong 1990). As proposed by Carlsson (1959) the dopamine precursor L-DOPA was shown to provide the first effective treatment for the disease by Birkmayer and Hornykiewicz in 1961, and is still the most effective treatment of Parkinson´s disease despite severe side effects affecting up to 75% of the patients after five years treatment.

The mesolimbocortical dopamine neurones are profoundly implicated in reward-related behaviour (see for review Koob et al. 1998, Robbins and Everitt 1999) and many drugs of abuse including cocaine, amphetamine, opiates, ethanol and nicotine are known to directly or indirectly cause an increased release of dopamine in terminal areas (di Chiara and Imperato 1988, see for review Di Chiara 1995). Moreover, specific lesions of nucleus accumbens projecting dopamine neurones reduce self-administration of nicotine (see Corrigall et al. 1992), cocaine and amphetamine (see Roberts et al. 1980, Wise and Bozarth 1987). Rats can also learn to electrically self-stimulate the dopaminergic neurones of the VTA (Corbett and Wise 1980, Wise and Rompre 1989, Wolske et al. 1993, Garris et al. 1999). The cholinergic input to the dopamine neurones has been shown to be implicated in mediating rewarding brain stimulation of the lateral hypothalamus (Yeomans et al. 1985, 1993, 1997). In several in vitro studies nicotine has been shown to excite dopamine neurones (Calabresi et al. 1989, Pidiplicko et al. 1997, Picciotto et al. 1998) whereas opiates have been shown to inhibit the GABAergic input to dopamine neurones, leading to a disinhibition of the dopamine neurones (Johnson and North 1992a).

Studies in the monkey (Schultz et al. 1993, 1998) have further analysed the role of the midbrain dopamine neurones in reward-related tasks and demonstrated that the dopamine neurones are, indeed, activated by a primary reward and especially if it is unexpected. If the reward is coupled to a conditioning signal (visual or auditory), the increased firing of the dopamine neurones will switch from the moment of appearance of the reward to the instant when the conditioning stimulus appears, and not when the actual reward is delivered. Moreover, if the conditioning stimulus is not followed by the expected reward the dopamine neurones decrease their firing. The dopamine neurones can thus detect deviations from the expected reward so that a positive signal is delivered to the brain if the reward is better than expected, no change if the reward is just as expected, and negative if less than expected.

Therefore, it has been suggested that the midbrain dopamine neurones serve two functions: ⁽¹⁾ to provide a tonic, generally enabling input on postsynaptic neurones in terminal fields, and ⁽²⁾ to send a phasic, global reinforcement or teaching signal to the brain, which helps to adapt behaviour and motor acts according to the motivational value of environmental stimuli (see for review Schultz 1997, 1998, Schultz et al. 1997, 1998, Redgrave et al. 1999).

The prefrontal projection of the mesocortical dopamine neurones from VTA are considered to modulate neuronal activity in the prefrontal cortex related to cognitive functions such as working memory, planning and execution of behaviour, inhibitory response control and maintenance of focused attention (see Le Moal and Simon 1991). Also reward-related stimuli increase dopaminergic activity in this region (Taber and Fibiger 1997). In particular, D₁ receptor activation has been demonstrated to be required for the adequate performance of working memory tasks (Sawaguchi and Goldman-Rakic 1994).

In addition to their implication in reward-related behaviour and cognitive functioning, the mesolimbocortical dopamine neurones are considered to be involved in the symptomatology of schizophrenia (see Carlsson 1988). The `dopamine hypothesis of schizophrenia´ was based on the fact that: ⁽¹⁾ all effective neuroleptic drugs used for treatment of schizophrenia share antidopaminergic activity and ⁽²⁾ drugs which directly or indirectly facilitates dopaminergic neurotransmission in brain, e.g. amphetamine or L-DOPA, can precipitate or aggravate psychosis. However, this classical hypothesis has been challenged by the relative lack of effect of the typical neuroleptics on negative symptoms, by the difficulties to find unequivocal evidence for dopaminergic hyperactivity in the brains of schizophrenic patients and by the fact that the atypical antipsychotic drug clozapine, despite its lower occupancy of D₂ receptors, has a significantly higher efficacy and a better effect on negative symptoms (Farde et al. 1988, 1992, Kane et al. 1988, Nordström et al. 1995). Clozapine is not only a D₂ receptor antagonist but has also affinity for D_1,4, a_1,2-adrenergic, 5-HT_2A,6,7, muscarinic and histaminergic receptors (see for review Seeman 1990, Roth et al. 1998), and in contrast to classical neuroleptic drugs it increases dopamine efflux in the prefrontal cortex (Moghaddam and Bunney 1990, Nomikos et al. 1994). This is of particular interest in view of the role of prefrontal dopamine in cognitive functioning and may have bearing on its effectiveness against cognitive and negative symptoms (Hertel et al. 1999). Consequently, the original dopamine hypothesis of schizophrenia, proposing a global hyperactivity of brain dopaminergic systems needs to be revised. A differential dysfunctioning of the dopaminergic systems with hyperactivity or hyperreactivity in some areas and hypofunction in others (Weinberger et al. 1986, Svensson et al. 1993) seems more likely. Such a dysfunction of brain dopamine systems may, in turn, rather be related to alterations in the input neurones to the dopamine neurones (Nauta 1976, Svensson et al. 1995).

 

B. Synaptic transmission and its modulation

The neuronal activity of individual neurones and the accompanying release of neurotransmitter are determined by the intrinsic firing activity and by the afferent control of the neurone. Synaptic transmission can be either fast or slow. In fast synaptic transmission the released neurotransmitter activates ligand-gated ion channels leading to a direct opening of the channel and a flux of ions across the cell membrane. In slow synaptic transmission the transmitter released activates G-protein-coupled receptors that induce opening or closure of ion channels. Glutamate and GABA are the predominant neurotransmitters of the brain and can mediate both fast and slow transmission. Acetylcholine (ACh) and serotonin (5-HT) can also mediate both fast and slow neurotransmission but only few examples of fast neurotransmission have been reported for these transmitters in the brain (see Roerig et al. 1997, Ullian et al. 1997, Alkondon et al. 1998). Noradrenalin and dopamine act exclusively on G-protein-coupled receptors. In addition to their postsynaptic effects these neurotransmitters can also modulate synaptic transmission through activation of various receptors located at the presynaptic terminal.

1. Involvement of voltage-gated Ca²⁺ channels in synaptic transmission

The synaptic release of neurotransmitters from their vesicles in the nerve terminal to the synaptic cleft requires an increase in Ca²⁺ concentration in the terminal. The opening of voltage-gated Ca²⁺ channels provides this Ca²⁺ increase as the action potential invades the presynaptic terminal, leading to Ca²⁺ influx from the extracellular space.

The voltage-gated Ca²⁺ channels are divided into two groups, low voltage activated (LVA) channels that are activated by small membrane depolarisations and high voltage activated (HVA) channels that require a larger depolarisation for their activation. The different types of channels are named after their characteristic properties (Nowycky et al. 1985, Llinás et al. 1992, see for review Tsien et al. 1988, Snutch and Reiner 1992, Hille 1992). LVA channels also called T-type channels, inactivate rapidly and induce a transient current. HVA channels consists of the L-type channels that are large and long lasting, are abundant in all excitable tissue (the heart, smooth muscle and in the nervous system) and are sensitive to 1,4-dihydropyridines (DHPs), N-type channels that were described first in neuronal tissue and are blocked by w-conotoxin GVIA (w-CTX GVIA), P/Q-type channels which are predominant in cerebellar Purkinje cells and have a slow inactivation and are blocked by w-Agatoxin IVA (w-Aga IVA), and finally R-type channels which conduct the residual current remaining after blockade of all other HVA Ca²⁺ channels. The different Ca²⁺ channels can thus be identified using specific pharmacological tools (Nowycky et al. 1985, Fox et al 1987, Tsien et al 1988, Llinás et al 1992, Mintz et al. 1992, Pearson et al. 1995, Randall and Tsien 1995).

The voltage-gated Ca²⁺ channels preferentially involved in synaptic transmission in the mammalian brain are the N- and P/Q-type channels (Takahashi and Momiyama 1993, Regehr and Mintz 1994, Wheeler et al. 1994, Wu and Sagau 1994, see for review Dunlap et al. 1995, Reuter 1996), since blockade of L-type channels does not affect synaptic transmission in the hippocampus, nucleus accumbens, cerebellum and striatum (Kamiya et al. 1988, Horne and Kemp 1991, Regehr and Mintz 1994, Turner et al. 1993, Wheeler et al. 1994). However, L-type channels have been shown to regulate release of catecholamines from chromaffin cells, neuropeptides from the neurohypophysis and hippocampal granule cells and excitatory amino acids in the retina (Lemos and Nowycky 1989, Takibana et al. 1993, Lopez et al. 1994, Simmons et al. 1995, von Gersdorff and Matthews 1996). DHPs have also been shown to inhibit the spontaneous firing of dopamine neurones in vitro (Mercuri et al. 1994).

 

2. Glutamate-mediated synaptic transmission

Glutamate can activate both ligand-gated ion channels, i.e. ionotropic glutamate receptors, and G-protein-coupled receptors, i.e metabotropic glutamate receptors (mGluRs) and synaptic transmission can be mediated through both types.

Ionotropic glutamate receptors are mainly of three types: AMPA, kainate and NMDA receptors. AMPA and kainate receptors are permeable mainly to Na⁺ and K⁺, whereas NMDA receptors, in addition, are permeable to Ca²⁺ and have a particular characteristic of being both ligand-gated and voltage-dependent. Mg²⁺ that blocks the NMDA channel pore only at a more negative potential controls the voltage dependence. In addition, the receptor is also allosterically modulated by glycine. NMDA receptor activation is critical for the induction of long term potentiation (LTP) - a plasticity phenomenon in many systems which has been implicated in memory formation (Bliss and Lomo 1973, see for review Bliss and Collingridge 1993). Whereas AMPA-mediated synaptic transmission is of shorter duration, NMDA receptors mediate a slower synaptic event. Until quite recently the physiological role of kainate receptors was unclear, but it has recently been reported that they can mediate a slow synaptic potential in the mossy fibre pathway of the hippocampus (Castillo et al. 1997, Vignes and Collingridge 1997). At about the same time it was shown that kainate receptors could mediate presynaptic inhibition of GABA release also in the hippocampal formation (Clarke et al. 1997). Moreover, the belief that the AMPA and kainate act exclusively on ionotropic receptors had to be modified, since it has recently been shown that they may also act through G-proteins and affect second-messenger pathways (Wang et al. 1997, Rodriguez-Moreno and Lerma 1998).

The mGluRs are divided into three groups (I, II and III) based on their molecular sequence homology, transduction mechanism and pharmacology. Group I mGluRs (mGluR1 and 5) are coupled to phospholipase C, whereas group II (mGluR 2 and 3) and III (mGluR4, 6, 7 and 8) are negatively coupled to adenylate cyclase. The different groups can be distinguished from each other using specific pharmacological tools. The functional role of mGluRs described so far is to presynaptically modulate GABA and glutamate release and to be involved in synaptic plasticity, for example LTP in hippocampus (see for review Pin and Bockaert, 1995, Pin and Duvoisin 1995, Pin 1998).

In midbrain dopamine neurones application of the mGluR agonist trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD) exerts an excitatory action inducing an inward current (Mercuri et al. 1993). It has also been demonstrated that slow excitatory synaptic currents can be evoked by a repetitive stimulation of afferents to dopamine neurones (Shen and Johnson 1997). This effect was suggested to be mediated through group I mGluR activation. In the same preparation also an inhibitory synaptic current mediated through mGluRs can be elicited by a shorter repetitive stimulation (Fiorillo and Williams 1998). This effect is also due to activation of group I mGluRs, which instead of inducing an excitation increases the release of Ca²⁺ from internal stores leading to activation of K_Ca, and thereby hyperpolarising the membrane potential.

 

3. GABAergic neurotransmission

GABAergic neurotransmission is mediated through activation of the ionotropic GABA_A receptor that is permeable to Cl^- or through the GABA_B receptor that is a G-protein-coupled receptor activating K⁺ channels. The GABA_B receptor consists of two different subunits that form a heteromer. Without co-expression of the two different subunits functional receptors can not be formed (Jones et al. 1998, White et al. 1998, Kaupman et al. 1998, Kuner et al. 1999). In dopamine neurones synaptic potentials mediated through both types of receptors can be evoked, the GABA_A with a single electrical pulse and the GABA_B with a short train of repetitive stimulating pulses (Johnson and North 1992b). The two types of GABA mediated synaptic transmission to midbrain dopamine neurones have proved to be differentially modulated and their afferents might be distinct from each other (Johnson et al. 1992, Cameron and Williams 1993, Wu et al. 1995).

4. Cholinergic neurotransmission

Cholinergic neurotransmission is mediated through nicotinic and muscarinic receptors. Nicotinic acetylcholine receptors (nAChR) are ligand-gated ion channels formed by the assembly of 5 subunits. The subunits found in the central nervous system are of the a- or b- subtype. There are 8 different a-subunits (a_2-9) and 4 b-subunits (b_2-5) described in the CNS of vertebrates (Boulter et al. 1986, Sargent 1993). Functional nAChR can be formed by combinations of various a-and b-subunits, forming heteropentamers, or by the a_7-9 alone, forming homopentamers (Galzi and Changeux 1994). The nAChRs of the nervous system differ somewhat from the muscular types in respect to ion permeability. Neuronal nAChRs are permeable to Na⁺ as the muscular type but also to Ca²⁺ (see for review Vidal and Changeux 1996, Changeux et al. 1998).

Nicotinic receptors have only in a few cases been shown to mediate fast synaptic transmission in the brain (Roering et al. 1997, Ullian et al.1997, Alkondon et al. 1998, see for review Vidal and Changeux 1996). This is surprising since nicotine application clearly can induce excitation of many neuronal types in various regions of the brain (neocortex, VTA, SNPC, medial habenula, hippocampus etc; Calabresi et al. 1989, see for review Vidal and Changeux 1996). In recent years, attention has been focused on the role of nAChRs located presynaptically that are capable of facilitating synaptic release of both glutamate and GABA (Léna et al. 1993, Vidal and Changeux, 1993, McGehee et al. 1995, Gray et al. 1996). It was even hypothesised that the major role of nAChRs in the brain is that of a presynaptic modulator (McGehee et al. 1995).

In the VTA nicotine has been shown to excite dopamine neurones through a direct activation of nAChR on dopamine neurones. In addition, some evidence for a functional role of nicotinic receptors on glutamate afferents to the dopamine cells has recently been obtained in vivo by Schilström et al. (1998a).

Muscarinic receptors are all G-protein-coupled receptors. Five different subtypes have been cloned and are named m_1-5 and these correspond to the M_1-5 that can be distinguished using pharmacological tools (Bonner et al. 1987, Hulme et al. 1990). The m_1,3,5 are coupled to the inositol triphosphate (IP₃) system, whereas the m_2,4 are negatively coupled to adenylate cyclase (Hulme et al. 1990, Bonner et al. 1992). Muscarinic receptors can mediate slow synaptic transmission but also exert presynaptic modulation of synaptic transmission in various areas of the brain (Sugita et al. 1991, Hasselmo and Bower 1992, Hsu et al. 1995, Sim and Griffith 1996). In the midbrain dopamine neurones muscarine has previously been shown to exert an excitatory effect suggested to be M₁-mediated and the ionic mechanisms involved were suggested to be Ca²⁺- rather than K⁺-dependent (Lacey et al. 1990).

MATERIAL & METHODS

Preparation of the tissue

Albino Wistar and Sprague-Dawley rats ^{(100-250 g)} (Morini - Reggio Emilia, Italy and BK Universal – Sollentuna, Sweden) were anaesthetised with halothane and killed with a blow to the chest or by decapitation. The brain was rapidly removed from the skull and horizontal slices (300 µm) of the ventral mesencephalon were cut using a vibratome. The slices containing the VTA and SNPC were transferred to a recording chamber continuously perfused with Ringer solution (mM): NaCl 126, KCl 2.5, MgCl2 1.2, NaH2PO4 1.2, CaCl2 2.4, glucose 11, NaHCO3 19, warmed to 35 °C and saturated with 95 % O2, 5 % CO2 (pH = 7.4). The SNPC and VTA were identified as the region medial (VTA), rostral and caudal (SNPC) to the medial terminal nucleus of the accessory optic tract.

Intracellular recordings

Intracellular recordings were performed using thick wall borosilicate glass electrodes (Clark), pulled on a Brown-Flaming (Sutter Instruments) electrode puller. The electrodes were filled with KAc (2 M) or KCl (2M) and had a resistance of 60-110 MW. The signal was obtained with an amplifier (Axoclamp 2A, Axon instrument) in bridge mode and displayed, averaged and stored on a computer using the pClamp software (Axon Instruments). The membrane potential was recorded at resting membrane potential when the spontaneous firing activity was studied, whereas negative current was injected to suppress firing activity when synaptic potentials were recorded.

Whole-cell patch clamp recordings of synaptic currents

Individual neurones of the SNPC and VTA were visualised using infrared video microscopy (Hamamatsu, Japan). Recording electrodes were pulled (3–5 M using a vertical puller PP-83 (Narishige, Japan) and filled with a solution containing (mM): KCl 120; MgCl2 2; CaCl2 1; EGTA 11; HEPES 10 (pH 7.3, with KOH). In experiments aiming at the study of glutamatergic synaptic responses, K-gluconate (130 mM) was used instead of KCl. Membrane current and potential were monitored using an Axopatch 1D patch clamp amplifier (Axon Instruments). Usually neurones were recorded at a holding potential of -60 mV. Series resistance ranged from 10 to 20 M and was not compensated in order to maintain the highest possible signal-to-noise ratio. However, cells where series resistance changed by more than 10% during drug application were discarded from the analysis. Data were filtered at 1 kHz, digitised at 10 kHz with a Digidata 1200 hardware and acquired with pClamp software (Axon Instruments).

Recordings of synaptic events

The postsynaptic potentials or currents (PSP/Cs) were evoked by a local electrical stimulus (0.03 ms, 10-25 V), delivered at a frequency of 0.1-0.3 Hz and generated by a stimulator (Grass Instruments) through a bipolar tungsten electrode placed close to the recording site in the VTA and the SNPC. The stimulus intensity was adjusted subthreshold for action potential initiation. To evoke slow excitatory PSP/Cs (EPSP/Cs) or inhibitory PSP/Cs (IPSP/Cs) short trains (40-400ms, 100-300 Hz) of repetitive stimulating pulses were used. Fast EPSP/Cs were pharmacologically isolated by the bath application of picrotoxin (PTX; 100 µM) or bicuculline methiodide (30 µM) to block the fast GABAA-mediated IPSP/C. During the recording of fast IPSP/Cs, CNQX (10 µM) and AP5 (50 µM) were present to block the EPSP/C and in some cases also the GABAB antagonist saclofen (200 µM) to block the slow IPSP/C. Recordings of the slow IPSP/C were performed in the presence of PTX (100 µM) or bicuculline methiodide (30 µM), CNQX (10 µM) and AP5 (50 µM).

Data analysis

The peak amplitude of the PSP/Cs was measured on averages of 4-10 traces in each different experimental condition. The peak baseline was measured as the average membrane potential during the 100 ms preceding the stimulus artefact. The peak amplitude was defined as the maximal amplitude difference from baseline following the stimulus artefact. Numerical data were expressed as mean ± S.E.M. Statistical differences were evaluated by paired t-test.

Spontaneous synaptic currents were detected using the Mini Analysis Program (Synaptosoft Inc., USA). The cumulative amplitude and interval distributions of the miniature IPSCs (mIPSCs) or spontaneous EPSCs (sEPSCs) were compared using the non-parametric Kolmogorov-Smirnov test.

Application of drugs

Drugs were made in stock solutions and bath applied at known concentrations via a three-way tap system. A complete exchange of the solution in the recording chamber occurred in about 1 min. To avoid desensitisation, nicotine was also applied by local pressure by using a glass pipette containing nicotine with the tip placed close to the recording site connected to a Picosprizer II (General Valve Corporation).

The following substances were used: 4-aminopyridine (4-AP), atropine sulphate, bicuculline methiodide, carbachol, dicyclomine, dopamine hydrochloride, glutamate, ketamine, mecamylamine, muscarine chloride, N-ethylmaleimide (NEM), nicotine di-(+)-tartrate, physostigmine, picrotoxin, tetrodotoxin (TTX) (all from Sigma), 2-amino-5-phosphonopentanoic acid (AP5), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), dihydro-b-erythroidine (DHBE), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), (4-hydroxy-2-butynyl)-1-trimethanylammonium-m-chloro-carbanilate chloride (McN-A-343), methoctramine tetrahydrochloride, methyllycaconitine (MLA), pirenzepine dihydrochloride, saclofen (all from RBI), (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate ((1S,3R)-ACPD), (S)-2-amino-2-methyl-4-phosphonobutanoic acid/(a-methyl-AP4) (MAP4), L-2-amino-4-phos-phonobutyric acid (L-AP4), (RS)-4-carboxy-3-hydroxyphenylglycine (4C3HPG), (2S,1’S,2’S)-2-(carboxycyclopropyl)glycine (L-CCGI), (RS)-3,5-dihydroxyphenylglycine (3,5-DHPG), (±)-a-methyl-4-carboxyphenylglycine (MCPG), L-serine-O-phosphono-butanoate (L-SOP) (all from Tocris Cookson), w-conotoxin GVIA (w-CgTx GVIA), w-agatoxin IVA (w-AgTx), w-conotoxin MVIIC (w-CgTx MVIIC) (all from Almone Labs), CGP35348 (Ciba-Geigy), Isradipine (Sandoz), Nimodipine and Bay-K 8644 (Bayer).

RESULTS

A. A slow NMDA-mediated excitatory postsynaptic potential involving L-type Ca2+ channel activation.

The glutamatergic synaptic input to midbrain dopamine neurones previously described is predominantly mediated through AMPA/kainate receptors and only to a smaller extent through NMDA receptors. However, the dopamine neurones are sensitive to NMDA application (Mercuri et al. 1992, Overton and Clark 1992, Seutin et al. 1994, Christofferson and Meltzer 1995) and NMDA receptor activation is considered to be responsible for the burst firing activity seen in vivo (Grenhoff et al. 1988, Svensson and Tung 1989, Charléty et al. 1991, Chergui et al. 1993).

Changes in the pattern of electrical stimulation from a single pulse that evokes the fast AMPA/kainate-mediated EPSP to repetitive pulses evoked a synaptic potential mediated through NMDA receptor activation. In fact, local stimulation with a short train of repetitive stimulating pulses (40 ms, 100-400 Hz) elicited a slow EPSP that was not affected by the application of the AMPA/kainate receptor antagonist CNQX, and enhanced by the application of the GABAA antagonist, picrotoxin (PTX), and the GABAB antagonist saclofen. This slow EPSP was dose-dependently inhibited by the application of the NMDA receptor antagonists AP5 (10-100 µM) and ketamine (30 µM), and was enhanced by Mg2+ removal from the extracellular solution, demonstrating that the slow potential is indeed mediated by activation of NMDA receptors. The application of the Na+ channel blocker tetrodotoxin (TTX) completely abolished the potential, demonstrating that it is synaptically mediated and not due to an unspecific direct stimulation of the dopamine neurones. (Mercuri et al., 1996)

The characteristics of the NMDA-mediated synaptic transmission were studied using the whole-cell patch clamp technique. We investigated the role of voltage-dependent Ca2+ channels of the L-type in mediating the slow EPSC. The L-type Ca2+ channel blockers nifedipine (1-100 µM), nimodipide (1-100 µM) and isradipine (0.03-100 µM) were able to depress the amplitude of the slow EPSC by 30-40 %, when evoked by a stimulating train with a duration of 80 ms or more. Moreover, the L-type channel agonist Bay-K 88644 enhanced the amplitude of the slow EPSC by 20 %. Nifedipine did not affect neither the fast EPSC nor the fast and slow IPSC. The change of the postsynaptic membrane holding potential from –100 to +30 mV did not affect the degree of depression of the slow EPSC by nifedipine. Furthermore, the postsynaptic response to glutamate was not affected by the presence of nifedipine, indicating that the effect of the DHPs is presynaptic (Bonci et al., 1998).

These two papers demonstrate that NMDA receptors contribute substantially to excitatory synaptic transmission to the midbrain dopamine neurones during high frequency stimulation of the glutamatergic afferents, that possibly correspond to bursting activity of the input neurones in vivo. This finding is in accordance with studies showing an important role of NMDA receptors in the bursting activity of the dopamine neurones in vivo (Grenhoff et al. 1988, Svensson and Tung 1989, Charléty et al. 1991, Chergui et al. 1993). The involvement of L-type Ca2+ channels in the slow EPSP/C is consistent with high frequency stimulation or a bursting activity, since the biophysical properties of these channels require high threshold activation and conduct large currents with a long activation time. This can provide a larger Ca2+ influx leading to a larger release of glutamate from the nerve terminal. This higher concentration of glutamate in the synaptic cleft might be required for the activation of NMDA receptors while the AMPA receptors that desensitise rapidly (Trussel et al. 1993) do not contribute to the slow EPSP. L-type channels have previously not been considered to play a major role in synaptic transmission, but this study shows that under certain circumstances the L-type channels contribute to the release of glutamate. Interestingly, they are not involved in the slow GABAB-mediated IPSP that is evoked by the same stimulating parameters. This suggests that L-type channels might be present exclusively on the excitatory glutamatergic input to the dopamine neurones.

In conclusion, we describe in paper I an NMDA-mediated slow EPSP/C that is evoked by a repetitive stimulating pulse, and in paper II, that it involves activation of presynaptic L-type Ca2+ channels, in contrast to the fast EPSC and the fast and slow IPSCs recorded in midbrain dopamine neurones which mainly involve N and P/Q-type Ca2+ channels.

B. Metabotropic glutamate receptors mediate presynaptic modulation of synaptic input to dopamine neurones

Activation of metabotropic glutamate receptors (mGluRs) has previously been shown to induce an inward current in midbrain dopamine neurones (Mercuri et al. 1993). To test if mGluRs also mediate presynaptic modulation of the synaptic input to dopamine neurones the effects of specific agonists and antagonists to the mGluRs of group I, II and III were tested on the fast EPSC as well as on the fast and slow IPSC.

The group III agonists L-AP4 and L-SOP depressed all three types of synaptic potentials recorded. The specific group III antagonist MAP4 blocked this depression. The fast EPSC was depressed to a larger degree than the IPSCs. The group II agonist L-CCGI also depressed all the three types of synaptic events to a similar degree as the group III agonists. The group I agonist 3,5-DHPG, on the other hand, reduced the GABAA- and GABAB-mediated synaptic currents to a larger extent than the excitatory synaptic currents. In addition, L-CCGI and 3,5-DHPG both induced an inward current. The effect of L-CCGI and 3,5-DHPG on the synaptic events were partially blocked by the broad spectrum antagonist MCPG, but not by MAP4. To determine if the effects were presynaptic, a paired-pulse protocol was used (DelCastillo and Katz 1954, Rausche et al. 1988, Zucker 1989, Mennerick and Zorumski 1995). Two stimuli of identical intensity were applied with 50 ms interval. The EPSC showed

paired-pulse depression while the IPSC displayed paired-pulse facilitation. During the application of agonists of the different mGluRs the ratio between the second and first pulse (p2/p1) was increased for both the fast EPSC and IPSC, indicating that the depression of these synaptic events is mediated by presynaptic mechanisms (Bonci et al., 1997).

The present work demonstrates, that in addition to the direct postsynaptic excitatory effect on the dopamine neurones (Mercuri et al. 1993, Shen and Johnson 1997), mGluRs also mediate presynaptic inhibition of excitatory and inhibitory inputs to these cells. mGluR-mediated presynaptic inhibition has previously been demonstrated in several regions of the CNS including the cortex, hippocampus, striatum, and thalamus (Baskys and Malenka 1991, Calabresi et al. 1993, Burke and Hablitz 1994, Stefani et al. 1994, Lovinger and McCool 1995, Salt and Eaton 1995). The group II and III mGluRs are most efficient in depressing synaptic events and glutamatergic synaptic events seems more sensitive than the GABA-mediated events. The presynaptic locus of action is indicated by the paired-pulse experiments. The functional role of the mGluRs in VTA and SNPC could be to act as autoreceptors on the glutamatergic input to the dopamine neurones to prevent excessive release of glutamate, but still maintaining some excitability of the dopamine cells by directly stimulating the postsynaptic receptors and, at the same time, presynaptically inhibiting the release of GABA.

C. Muscarinic presynaptic modulation of synaptic input to dopamine neurones

Muscarine has previously been shown to excite midbrain dopamine neurones in vitro through activation of M1-like receptors (Lacey et al. 1990). Since muscarinic receptors are also known to mediate presynaptic modulation of transmitter release in several regions of the mammalian nervous system (Sugita et al. 1991, Hasselmo and Bower 1992, Hsu et al. 1995, Sim and Griffith 1996) we were interested in studying the possible existence of such a modulation of synaptic input to the midbrain dopamine neurones.

We investigated the effects of muscarine and carbachol on the evoked fast EPSP recorded in midbrain dopamine neurones. The EPSP was depressed by bath application of carbachol (0.1–30 µM) in a dose-dependent manner and by muscarine (30 µM). To elucidate the receptor subtype involved four different antagonists were used. Only the M3/M1 antagonist 4-DAMP (1 µM) was able to completely block the effect of carbachol and muscarine on the EPSP. The M1 specific antagonist pirenzepine (1 µM) could not significantly prevent the depression of the EPSP. To demonstrate that the suppressing effect on the EPSP was not due to a change in sensitivity of the glutamate receptors at the postsynaptic membrane, the depolarising response to a short application of glutamate (1 mM; in the presence of 0.5 µM TTX) was compared during control conditions and in the presence of muscarine (30 µM). The depolarisation was of the same amplitude both in control and in the presence of muscarine (30 µM). A paired-pulse protocol (described above) was also used to further confirm the presynaptic site of action of muscarine. A paired-pulse stimulus caused a depression of the second EPSP and application of muscarine depressed the first EPSP to a larger extent than the second, thereby increasing significantly the ratio (p2/p1) (Grillner et al., 1999a). These results thus indicate that M3 receptor activation mediates depression of glutamatergic transmission through a presynaptic mechanism of action. Moreover, the input resistance of the dopamine neurones was not changed by the application of muscarine excluding the possibility that electrical shunting could account for the depression of the EPSP. By the use of physostigmine, an inhibitor of the ACh degrading enzyme ACh-esterase, we could show that ACh, endogenously released from the slice, has the same effect as muscarine. The depression of the EPSP by physostigmine was also blocked by 4-DAMP. To investigate the second messenger pathways involved we used N-ethylmaleimide (NEM) a blocker of the pertussis-toxin-sensitive Gi/o-protein. The preincubation with NEM did not affect the muscarine-induced depression of the EPSP. The presynaptic inhibition was not mediated through Ca2+ channels of N- or P/Q-type since specific antagonists to these channels did not affect the presynaptic inhibition by muscarine of the excitatory potential. Also the involvement of K+ channels were studied by blocking these channels with 4-AP or barium. The blockade of K+ channels did not change the response to muscarine.

We also investigated the muscarinic modulation of GABAA-mediated synaptic transmission to the dopamine neurones (Grillner et al., 1999b). Firstly, we studied the effect of bath applied muscarine on the evoked fast GABAA-mediated IPSP recorded in midbrain dopamine neurones. Muscarine at a concentration of 30 µM depressed the IPSP amplitude. To characterise the receptor subtype involved, the M3/M1 antagonist 4-DAMP (100 nM) and the M1 antagonist pirenzepine (1 µM) were used. The antagonists were applied 15 min prior to the application of muscarine to enable the tissue to be well incubated. Only 4-DAMP could completely block the effect of muscarine, while pirenzepine failed to significantly affect the muscarinic depression of the IPSP. To exclude the possibility that the depressant effect of muscarine was due to a change in sensitivity of the postsynaptic GABAA receptors the hyperpolarisation induced by bath applied GABA (200 µM; in the presence of TTX (0.5 µM), CNQX (10 µM), AP5 (100 µM) and saclofen (200 µM)) was studied under control conditions and in the presence of muscarine (30 µM). The amplitude of the hyperpolarisation was the same in control and in the presence of muscarine. The effect of muscarine on mIPSCs was analysed in the presence of pirenzepine to block the postsynaptic receptors (Lacey et al. 1990). Under these conditions muscarine (30 µM) significantly and reversibly decreased the frequency of mIPSCs without affecting their amplitude.

These results demonstrate that M3 muscarinic receptors depresses the evoked fast glutamate-mediated EPSP and the fast GABAA-mediated IPSP to the midbrain dopaminergic neurones. The M3 receptor involvement is suggested by the counteraction of muscarinic receptor-mediated depression of synaptic transmission by 4-DAMP but not by pirenzepine. This is consistent with autoradiography studies showing a relative enrichment of M3 receptors in relation to M1 and M2 receptors in the substantia nigra (Zubieta and Frey 1993). Furthermore, we demonstrate that the endogenously released transmitter can mediate this presynaptic inhibition. The cerebral cortex and the subthalamic nucleus that both send excitatory afferents to SNPC and VTA have been reported to contain mRNA for m3 receptors (Weiner et al. 1990). However, the presence of m3 receptor expression has not been demonstrated in any of the areas sending GABAergic input to the VTA and SNPC (Weiner et al. 1990). The previously reported postsynaptic action of muscarine is confirmed in these studies since muscarine application gives rise to a depolarisation or an inward current that is prevented by pirenzepine. The role of muscarinic receptors in the SNPC and VTA is complex since activation of muscarinic receptors leads to a direct postsynaptic excitatory action mediated through M1 receptors as well as a presynaptic inhibition of both the GABAergic and the glutamatergic inputs to the dopamine neurones. The functional significance of this dual action on the dopamine neurones might be to enhance excitation of the dopamine neurones when glutamatergic afferents are inactivated, but to prevent excessive excitation of the dopamine neurones when both cholinergic and glutamatergic afferents are active simultaneously.

D. Nicotinic excitation of dopamine neurones

Nicotine is known to excite dopamine neurones in vitro by a direct action on nicotinic receptors on the cell membrane (Calabresi et al. 1989, Pidiplichko et al. 1997, Picciotto et al. 1998). However, in several brain regions nicotine has been shown to facilitate synaptic release of glutamate and GABA through activation of presynaptic nicotinic receptors (Léna et al. 1993, Vidal and Changeux, 1993, McGehee et al. 1995, Gray et al. 1996). In this study, we investigated a possible additional presynaptic action of nicotinic receptors on glutamatergic terminals besides their direct effect on the midbrain dopamine neurones.

Thus, we characterised the effect of nicotine on the dopamine neurones (Grillner and Svensson 1999). Nicotine (10 µM) depolarised the membrane potential by 5 mV and reduced the postspike afterhyperpolarisation (AHP) leading to an increase in firing frequency. The effects of nicotine on the membrane potential, the AHP and the firing frequency were blocked by the nicotinic antagonists dihydro-b-erythroidine (DHBE; 50 µM) and mecamylamine (10 µM), but not by methyllycaconitine (MLA; 10 nM). To investigate whether part of the excitatory role of nicotine is mediated through ionotropic glutamate receptor activation, antagonists to NMDA and AMPA/kainate receptors were used to find out whether the presence of these antagonists would affect the nicotine-induced excitation. Both the NMDA receptor antagonist AP5 (90 µM) and the AMPA/kainate receptor antagonist CNQX (10 µM) reduced the effect of nicotine on the firing frequency. These results indicate that the nicotine-induced excitation of the dopamine neurones involves activation of ionotropic glutamate receptors.

To further analyse if this nicotine-mediated excitation involves activation of presynaptic receptors, spontaneous synaptic currents were studied using whole-cell patch clamp recordings (Grillner et al., 1999)). sEPSC were not frequent in these neurones; however, their occurrence increased by application of 4-AP (20-100 µM). They were recorded in the presence of PTX (100 µM), in order to abolish GABAA-mediated responses. Bath or local pressure application of nicotine did not affect neither the frequency nor the amplitude of the sEPSCs. The effect of nicotine was also studied on GABAergic mIPSCs. No effect of nicotine was, however, seen on the frequency or on the amplitude of the mIPSCs neither by local pressure application nor by bath application of nicotine.

Our results thus demonstrate that nicotine excites dopamine neurones through a specific activation of nicotinic receptors. The effect partly involves ionotropic glutamate receptor activation. We found, however, no evidence for activation of presynaptic nicotinic receptors on glutamate or GABA terminals onto the midbrain dopamine neurones. Our results thus confirm earlier studies (Calabresi et al. 1989, Pidiplichko et al. 1997, Picciotto et al. 1998) which suggest that nicotine acts primarily through nicotinic receptors located at the somato-dendritic membrane of the dopamine. The receptor subtype that mediates the nicotinic effect might be a4b2 or a7 since nicotine-induced currents mediated through both subtypes have been demonstrated in midbrain dopamine neurones (Pidiplichko et al. 1997, Picciotto et al. 1998). However, our results show that nicotine induced excitation of the dopamine neurones in vitro does not require activation of a7 receptors, since the relatively specific antagonist MLA did not prevent the increase in firing frequency. The reduced excitatory effect of nicotine in the presence of glutamate antagonists may be explained by a general reduction of the excitability of the dopamine neurones or by the possible release of glutamate from the dopamine neurones themselves (Sulzer et al. 1998).

DISCUSSION

In the present study the synaptic input to midbrain dopamine neurones and its modulation was studied in vitro. We have described and characterised an NMDA-mediated synaptic event that involves L-type Ca2+ channel activation, demonstrated that mGluRs and muscarinic receptors mediate presynaptic inhibition of glutamatergic and GABAergic synaptic input and provided additional support for the notion that nicotine activates these cells via postsynaptically located receptors. These findings thus allow for a more detailed analysis of the role of the glutamatergic and cholinergic afferent inputs in regulating the dopaminergic neuronal activity in the VTA and SNPC.

A. Regulation of neuronal activity in midbrain dopaminergic systems

1. Involvement of NMDA receptors and L-type Ca2+ channels

Glutamatergic afferent input to midbrain dopamine neurones seems to play a critical role for maintaining the spontaneous, burst firing activity of the dopamine neurones in vivo (Grenhoff et al. 1988, Svensson and Tung 1989, Charléty et al. 1991). In particular, NMDA receptors are involved in mediating this bursting activity (Chergui et al. 1993, 1994), which is functionally coupled to a markedly enhanced dopamine efflux in the terminal areas when compared to the efflux associated with regular firing (Gonon and Buda 1985, Gonon 1988, Bean and Roth 1991). The highly regular firing activity seen in vitro can be changed into burst activity if NMDA is bath applied together with apamine, a blocker of the KCa channels which reduces the postspike AHP (see Seutin et al. 1993). Moreover, repetitive stimulation of afferents in the slice preparation can induce burst firing of the dopamine neurones (Johnson 1998). Generally, activation of NMDA receptors seems to require a larger amount of glutamate release than AMPA/kainate receptors, since single stimulating pulses evoke synaptic potentials with only a small contribution of NMDA receptors (Mereu et al. 1991, Johnson and North 1992b), whereas high frequency stimulation is required to evoke synaptic potentials with a large contribution of NMDA receptors. The present study thus suggests that the burst activity seen in vivo results from a high level of activity of the glutamatergic afferents to the dopamine neurones. The requirement of higher amount of glutamate release for the activation of NMDA receptors is in one aspect surprising, because NMDA receptors have a higher affinity for glutamate than AMPA receptors (Hollmann and Heinemann 1994). Possible explanations might be: ⁽¹⁾ that the NMDA receptors are located more distantly from the release site than the AMPA receptors, ⁽²⁾ that the voltage dependence of the NMDA receptors requires a stronger depolarisation for its opening, and/or ⁽³⁾ that the AMPA receptors desensitise more rapidly than NMDA receptors (Trussel et al. 1993).

The involvement of L-type Ca2+ channels in mediating the slow NMDA EPSC is indeed interesting since these channels display a larger and longer activation than other Ca2+ channels. Therefore they may provide the additional Ca2+ influx into the glutamate terminal needed for the release of a sufficient amount of glutamate. Such a specific involvement in NMDA-mediated synaptic transmission may in turn allow the opportunity to selectively affect the NMDA-related activity, i.e. burst firing, of dopamine neurones by a specific pharmacological manipulation of L-type channels.

The glutamatergic afferents acting on NMDA receptors are most likely of critical importance for the normal functioning of dopaminergic systems. Accordingly, dysfunction in pathological states thought to be related to midbrain dopamine systems may thus depend, on altered level of glutamatergic afferent activity or dysfunctional L-type channels, and not only to intrinsic dysfunction of the dopamine neurones themselves.

2. Role of mGluRs

The role of mGluRs in VTA and SNPC seems quite complex because of the large variety of functions that have been described. Postsynaptic receptors have been demonstrated by the sensitivity of dopaminergic neurones to the mGluR agonist t-ACPD (Mercuri et al. 1993) and by the finding of slow mGluR-mediated postsynaptic events evoked by repetitive focal stimulation of the slice that can be either excitatory (Shen and Johnson 1997) or inhibitory (Fiorillo and Williams 1998). In addition, we here provide evidence that also presynaptic mGluRs are present on both glutamatergic and GABAergic terminals within the VTA and SNPC. These results were recently confirmed (Wigmore and Lacey 1998).

All postsynaptic effects of mGluR activation on midbrain dopamine neurones seem to be mediated by group I mGluRs (Shen and Johnson 1997, Fiorillo and Wiliams 1998). Interestingly, these phospholipase C-coupled receptors seem to be able to mediate two opposite actions on the dopamine neurones. The excitatory action, that was first described (Mercuri et al. 1993, Shen and Johnson 1997), is preceded by an inhibition if the agonist application or stimulating pulse is brief enough (Fiorillo and Williams 1998). Fiorillo and Williams (1998) have recently proposed that the pathway mediating this inhibition desensitises rapidly, while the excitatory effect persists. The desensitisation is most likely occurring downstream from the receptor since the excitation is not affected. A similar dual pathway activation by group I receptors has also been described at the presynaptic level (Herrero et al. 1998), where a rapidly desensitising enhancement of glutamate is followed by an inhibition of the release. The dual postsynaptic action by the group I mGluRs at the dopaminergic neurones is yet another example of the importance of the pattern of activity of the afferents for the final effect on the postsynaptic cells.

The presynaptic inhibition of both glutamatergic and GABAergic synaptic inputs demonstrated in the present study involves group II and III mGluRs but also, to a lesser degree, group I mGluRs. Presynaptic inhibition of glutamate and GABA release has previously been demonstrated in several regions of the brain such as cortex, hippocampus, striatum and thalamus (Baskys and Malenka 1991, Calabresi et al. 1993, Burke and Hablitz 1994, Salt and Eaton 1995). The presynaptic mGluRs located on the glutamatergic terminals projecting to the dopamine neurones might well function as autoreceptors as has already been demonstrated in the hippocampus (Scanziani et al. 1997). The autoreceptor function at glutamatergic afferents to dopamine neurones could be of importance in limiting glutamate-induced cytotoxicity resulting from excess glutamate release. The presynaptic inhibition of the GABA afferents to dopaminergic neurones would, in principle, have an opposite effect, i.e. enhancing excitation of the dopamine neurones. A functional role of this effect might be to augment the glutamatergic input when GABAergic input is activated at the same time. An overall function of the mGluRs as presynaptic inhibitory receptors on glutamatergic and GABAergic synaptic input may, consequently, be to allow for maintenance of homeostasis within the dopaminergic system.

Glutamatergic afferents to the midbrain dopamine neurones arise from several brain regions (prefrontal cortex, subthalamic nucleus, PPTg and LDTg), whereas the GABAergic input originates in the striatum and pallidum (Fonnum et al. 1978, Bolam and Smith 1990, Smith and Bolam 1990), and from intrinsic GABAergic interneurones of the midbrain (Di Chiara et al. 1979, Grofova et al. 1982, Stanford and Lacey 1996). Since it is not possible to determine the origin of the afferents that are activated by the stimulating electrodes in the slice preparation, it is likely that afferents from several areas are stimulated. The different afferents might contain different mGluR subtypes depending on their site of origin. Therefore it may, in principle, be possible to use specific pharmacological tools to selectively modulate some, but not other afferent inputs to the dopamine neurones.

3. Cholinergic control of midbrain dopamine neurones

It was previously known that both muscarinic and nicotinic receptors mediate a postsynaptic action on the dopamine neurones, but presynaptic modulation by cholinergic receptors of the synaptic input to the dopamine neurones had not previously been investigated. We show in this study that muscarinic, but not nicotinic receptors, presynaptically modulate the glutamatergic and GABAergic input. ACh acting on muscarinic or nicotinic postsynaptic receptors can thus not only exert a direct excitatory action of the dopamine neurones but may also via presynaptic muscarinic receptors modulate the synaptic input from other brain regions.

The muscarinic effect on dopamine neurones of the midbrain is, like that of mGluRs, dual with a postsynaptic excitation and a presynaptic inhibition. The postsynaptic action is sensitive to pirenzepine (Lacey et al. 1990) and is likely to be mediated through M1 receptors. However, studies using in situ hybridisation techniques have demonstrated that SNPC exclusively expresses mRNA for m5 receptors (Vilaro et al. 1990, Weiner et al. 1990). m5 receptors display an intermediate affinity for pirenzepine (Vilaro et al. 1990) and are, as the M1 receptors, coupled to the IP3 cascade. The M1-like postsynaptic action on the dopamine neurones might thus rather be mediated by activation of m5 receptors. Muscarinic agonists have previously been demonstrated to enhance NMDA-mediated synaptic transmission and NMDA-mediated postsynaptic inward currents in the auditory cortex (Aramakis et al. 1997, 1999). These authors demonstrated that the enhancement is mediated through M1-like receptors coupling to G-protein-mediated activation of the IP3 system. This mechanism might also be present in the midbrain dopamine neurones. Muscarine could, through activation of postsynaptic M1 or M5 receptors, thus not only increase the firing frequency of the dopamine neurones but also enhance ongoing NMDA receptor-mediated burst firing.

The action of muscarine on presynaptic receptors on glutamate and GABA afferents to the midbrain dopamine neurones described in the present work shows a pharmacological profile similar to M3 receptors. A functional role of the muscarinic presynaptic inhibition might be to prevent excess excitation when both glutamatergic and cholinergic afferents are active, whereas it could serve to prevent excessive inhibition of the synaptic input via inhibition of the GABAergic input.

The nicotinic effect on midbrain dopamine neurones in vitro can in all probability not be attributed to an enhanced glutamate release by presynaptic nicotine receptors, as was suggested by previous in vivo and in vitro experiments from our laboratory (Schilström et al. 1998a, Results, Paper V). Presynaptic facilitation of glutamate release was first described in the prefrontal cortex by Vidal and Changeux (1993), and was subsequently demonstrated in a number of other brain regions (McGehee et al. 1995, Gray et al. 1996). It was even suggested that presynaptic modulation of transmitter release may represent the predominant role of nicotinic receptors in the brain, especially considering the lack of evidence for fast synaptic transmission mediated through nicotinic receptors (McGehee et al. 1995). However, even if it now seems clear that nicotine can act on both pre- and postsynaptic receptors in the brain, its effect on VTA and SNPC neurones seems essentially to be postsynaptic.

Both nicotinic and muscarinic receptors in the VTA appears to contribute to rewarding brain stimulation of the lateral hypothalamus, since both nicotinic and muscarinic antagonists applied locally in the VTA reduce the rewarding effect of the brain stimulation (Yeomans and Batista 1997). However, the muscarinic receptors seem to contribute more than the nicotinic receptors. Yeomans and Batista (1997) proposed that rewarding stimulation of the lateral hypothalamus is mediated through an activation of the cholinergic neurones of the PPTg and the LDTg, that in turn activate the dopaminergic neurones of the midbrain. In contrast, nicotine self-administration does not seem to involve muscarinic receptors in the VTA or the cholinergic neurones in PPTg and LDTg (Corrigall et al. 1994).

B. Potential clinical implications

Several small clinical trials with Ca2+ antagonists, such as verapamil, diltiazem and dihydropyridines (all blockers of the L-type channels), in the treatment of psychiatric diseases have been published (see for review Hollister and Garza-Trevino 1996). The predominant diagnoses have included bipolar disorder, unipolar depression and schizophrenia. The effect on bipolar patients is relatively well documented and a therapeutic effect has frequently been observed in the manic phase, sometimes reversing it to a depression requiring treatment with antidepressants. Interestingly, in other studies the patients stabilised in their mood fluctuations between mania and depression. Unipolar depression has sometimes been found to be aggravated by Ca2+ antagonists, but in other cases to be improved. The effect of Ca2+ antagonists in schizophrenia appears equivocal, but some reports describing an effect similar to that of conventional antipsychotics have actually been published. Overall the potential usefulness of Ca2+ antagonists on psychiatric indications remains, as yet, to be conclusively established.

The putative role of the dopaminergic systems in the pathophysiology of e.g. bipolar disorder makes, in principle, the L-type-mediated NMDA transmission to the dopaminergic neurones described in the present study a tentative site of action for the Ca2+ antagonists, in particular as regards the mood stabilising action.

The clinical usefulness of mGluR agonists for the treatment of psychosis was recently proposed (see Moghaddam and Adams 1998). These authors showed that the phencyclidine (PCP)-induced glutamate release in the prefrontal cortex as well as the PCP-induced locomotor hyperactivity and stereotyped behaviour could be prevented by pre-treatment with an agonist to group II mGluRs. However, the PCP-induced increase in dopamine output in the nucleus accumbens and the prefrontal cortex was not affected. Needless to say, none of these effects of the mGluR agonist show a high predictive value for antipsychotic activity per se. The effect of the group II mGluR agonist might be mediated via presynaptic inhibition on the glutamatergic terminals in the prefrontal cortex, although the group II mGluR-mediated presynaptic inhibition of the glutamatergic input to the dopamine neurones demonstrated in Paper III did not seem to be reflected in any alteration of the basal extracellular concentrations of dopamine in terminal areas. Nevertheless, specific agonists or antagonists to mGluRs might prove useful in modulating the phasic responsivity of the dopamine neurones, since we here show that the presynatically located mGluRs are predominantly of group II or III (Paper III), whereas the postsynaptically located mGluRs have been shown to be of group I (Mercuri et al. 1993, Shen and Johnson 1997, Fiorillo and Williams 1998).

The psychotomimetic effect of antimuscarinics (see for review Yeomans 1995) as well as clinical results obtained already in the 1970’s (Davies et al. 1978), showing a reversal of the manic phase of bipolar disorder by the AChesterase inhibitor, physostigmine, suggests a possibility for muscarinic pharmacotherapy of psychiatric disorders involving potentially hyperactive or hyperreactive dopaminergic systems. For example, Yeomans (1995) suggested that blockade of muscarinic autoreceptors of the m2 subtype on the cholinergic neurones in the PPTg and LDTg leads to an increased activity of the cholinergic input to the midbrain dopamine neurones, thereby inducing psychosis. The demonstrated M3 receptor-mediated presynaptic inhibition of glutamatergic input to dopamine neurones could also help to explain an activation of the dopamine neurones by antimuscarinic agents. Indeed, M3 agonists are currently being evaluated as potential antipsychotic drugs (Shannon et al. 1998). One site of action might thus be the presynaptic receptors on glutamatergic terminals to the midbrain dopamine neurones described in this study.

Nicotine itself is currently being used clinically as a moderately effective treatment in smoking cessation, although more specific ligands to certain nicotinic receptor subtypes might provide interesting alternatives. The nicotinic receptor subtypes mediating nicotinic excitation of the dopamine neurones in VTA seems to be a4b2 and possibly a7 (Pidiplichko et al. 1997, Sorenson et al. 1998, Pichiotto et al. 1998, Schilström et al. 1998b) and these may allow for pharmaceutical development of novel therapies in this regard as well as in other dopamine-related neuropsychiatric disorders, such as Parkinson’s disease.

Generally the midbrain dopaminergic neurones, that have been implicated in reward-related behaviour, cognitive function and motor performances, thus have several afferent inputs, which control their activity in vivo. The incoming activity patterns of the different afferent inputs, which are critical for the postsynaptic response in the dopamine neurones, can thus be balanced against each other through presynaptic modulation via several G-protein-coupled receptors. This fine regulation of the afferent activity may in turn enable the dopamine neurones to adapt their activity to incoming signals from several brain regions (Hoffman et al. 1995). Thus, the dopaminergic neurones seem to play an integrative role in optimising the shaping of behaviour and motor acts in relation to the demands of the environment. By inference, distortions of this presynaptic neuromodulatory machinery might contribute to impaired capacity of the individual to adapt to the environment e.g. in psychiatric disorders or drug-dependent states, or to dysfunctional motor control and neurodegeneration in neurological disorders such as Parkinson’s disease.

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[ABSTRACT] [INTRODUCTION] [MATERIAL & METHODS] [RESULTS] [DISCUSSION] [REFERENCES] [Discussion Board]

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	[Cell Biology & Cytology]	[Neuroscience]

Nicola B. Mercuri, Pernilla Grillner
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