Michael G. Lacey

A Postsynaptic Interaction between Dopamine D1 and NMDA Receptors Promotes Presynaptic Inhibition in the Rat Nucleus Accumbens via Adenosine Release

The mechanism underlying dopamine D1receptor-mediated attenuation of glutamatergic synaptic input to nucleus accumbens (NAcc) neurons was investigated in slices of rat forebrain, using whole-cell patch-clamp recording. The depression by dopamine of EPSCs evoked by single-shock cortical stimulation was stimulus-dependent. Synaptic activation of NMDA-type glutamate receptors was critical for this effect, because dopamine-induced EPSC depressions were blocked by the competitive NMDA receptor antagonistd/l-2-amino-5-phosphonopentanoate (AP5). Application of NMDA also depressed the EPSC, and both this effect and the dopamine depressions were blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), implicating adenosine release in the EPSC depression. A1 receptor agonists also depressed EPSCs by a presynaptic action, causing increased paired-pulse facilitation, but this was insensitive to AP5. Activation of D1 receptors enhanced both postsynaptic inward currents evoked by NMDA application and the isolated NMDA receptor-mediated component of synaptic transmission. The biochemical processes underlying the dopamine-induced EPSC depression did not involve either protein kinase A or the production of cAMP and its metabolites, because this effect was resistant to the protein kinase inhibitors H89 and H7 and the cAMP-specific phosphodiesterase inhibitor rolipram. We conclude that activation of postsynaptic D1receptors enhances the synaptic activation of NMDA receptors in nucleus accumbens neurons, thereby promoting a transsynaptic feedback inhibition of glutamatergic synaptic transmission via release of adenosine. Unusually for D1 receptors, this phenomenon occurs independently of adenylyl cyclase stimulation. This process may contribute to the locomotor stimulant action of dopaminergic agents in the NAcc.

Excitation of rat substantia nigra pars reticulata neurons by 5-hydroxytryptaminein vitro: Evidence for a direct action mediated by 5-hydroxytryptamine2C receptors

Single-unit extracellular and whole-cell patch clamp recording were used to study the actions of exogenously applied 5-hydroxytryptamine on substantia nigra pars reticulata neurons in parasaggital slices of rat midbrain. Seventy-six per cent of substantia nigra pars reticulata cells (254/334) recorded extracellularly were excited by 5-hydroxytryptamine (EC50 = 9.56 microM); in the remainder, inhibitions (13.5%), biphasic responses (4.2%) or lack of response (6.3%) were observed. Using whole-cell patch recording, 5-hydroxytryptamine (10 microM) caused either an inward current (9/9 cells) or a depolarization (3/3 cells) at membrane potentials in the range -50 to -90 mV, which was resistant to tetrodotoxin (4/4 cells), indicating that the predominant, excitatory action of 5-hydroxytryptamine was due to a direct action on substantia nigra pars reticulata neurons. The 5-hydroxytryptamine excitation (recorded extracellularly) was reduced to 24 +/- 6% of control values by methysergide (0.1 microM) and to 17 +/- 5% of control by ketanserin (10 microM), but was unaffected by the 5-hydroxytryptamine antagonists spiperone (0.1 microM), yohimbine (0.1 microM), pindolol (1 microM), GR113808A (1 microM) or ICS 205930 (10 microM). In addition, the 5-hydroxytryptamine excitation was mimicked by the 5-hydroxytryptamine2C receptor--preferring agonist alpha-methyl 5-hydroxytryptamine (10 microM), but the agonists CP93, 129 (0.1-1 microM) and (+/-)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (0.1-1 microM) were without effect. Taken together, this pharmacology indicated involvement of the 5-hydroxytryptamine2C receptor in the 5-hydroxytryptamine excitation, while other candidate receptors known to be present in rat substantia nigra pars reticulata (5-hydroxytryptamine1B, 5-hydroxytryptamine2A and 5-hydroxytryptamine4) could be excluded from consideration. While in accord with current information on the location of 5-hydroxytryptamine receptor subtypes in substantia nigra pars reticulata, and the consequence of activation of neuronal 5-hydroxytryptamine2C receptors, these results contrast with data from in vivo experiments which suggest that the net effect of 5-hydroxytryptamine is to inhibit substantia nigra pars reticulata neurons. The reason for this apparent discrepancy may lie in detailed consideration of the microcircuitry of the substantia nigra pars reticulata. This may lead to a re-evaluation of the influence of 5-hydroxytryptamine on this basal ganglia output relay nucleus, and its role in motor control and the gating of generalized seizure activity.

Metabotropic glutamate receptors depress glutamate-mediated synaptic input to rat midbrain dopamine neurones in vitro

Glutamate (AMPA receptor‐mediated) excitatory postsynaptic potentials (e.p.s.ps.), evoked by electrical stimulation rostral to the recording site, were examined by intracellular microelectrode recording from dopamine neurones in parasagittal slices of rat ventral midbrain. The e.p.s.p. was depressed by the group III metabotropic glutamate (mGlu) receptor agonist L‐2‐amino‐4‐phosphonobutyric acid (L‐AP4; 0.01–30 μM) by up to 60% with an EC50 of 0.82 μM. The depression induced by L‐AP4 (3 μM) was reversed by the group III preferring mGlu receptor antagonist, α‐methyl‐4‐phosphonophenylglycine (MPPG; 250 μM). The group I and II mGlu agonist, 1S,3R‐aminocyclopentanedicarboxylic acid (ACPD; 3–30 μM) also depressed the e.p.s.p. in a concentration‐dependent manner. The effect of ACPD (10 μM) was reversed by (+)‐α‐methyl‐4‐carboxyphenylglycine (MCPG; 1 mM; 4 cells). This effect of ACPD was also partially antagonized (by 50.3±15.7%, 4 cells) by MPPG (250 μM). The selective agonist at group I mGlu receptors, dihydroxyphenylglycine (DHPG; 100 μM), decreased e.p.s.p. amplitude by 27.1±8.2% (7 cells), as did the group II mGlu receptor‐selective agonist (1S,1′R,2′R,3′R)‐2‐(2,3‐dicarboxycyclopropyl)glycine (DCG‐IV; 1 μM) by 26.7±4.3% (5 cells). DHPG (10–100 μM) caused a depolarization of the recorded cell, as did ACPD (3–30 μM), whereas no such postsynaptic effect of either L‐AP4 or DCG‐IV was observed. These results provide evidence for the presence of presynaptic inhibitory metabotropic glutamate autoreceptors from the mGlu receptor groups II and III on descending glutamatergic inputs to midbrain dopamine neurones. Group I mGlu receptors mediate a postsynaptic depolarization, and can also depress glutamatergic transmission, but may not necessarily be localized presynaptically. These sites represent novel drug targets for treatment of schizophrenia and movement disorders of basal ganglia origin.

Modulation by dopamine D1-like receptors of synaptic transmission and NMDA receptors in rat nucleus accumbens is attenuated by the protein kinase C inhibitor Ro 32-0432

Dopamine, acting at a D1-like receptor, depresses the release of glutamate in the nucleus accumbens (NAcc) in brain slices, thereby reducing the amplitude of the excitatory postsynaptic current (EPSC). This effect depends upon an inhibitory feedback action of adenosine, liberated following facilitation of postsynaptic NMDA receptors by D1 receptor activation, an action independent of adenylyl cyclase stimulation or cyclic AMP-dependent protein kinase (PKA; Harvey, J., Lacey, M.G., 1997. J. Neurosci. 17, 5271). Using whole-cell recording from NAcc neurones, the dopamine depression of the EPSC was blocked by pre-treatment of brain slices with the selective protein kinase C (PKC) inhibitor Ro 32-0432, but only minimally attenuated by intracellular dialysis of single cells with Ro 32-0432 in the recording pipette. With synaptic transmission blocked by tetrodotoxin, inward currents caused by application of NMDA were enhanced by the D1 receptor agonist SKF 81297A in half the cells tested. In a separate population of cells dialysed intracellularly with Ro 32-0432, SKF 81297A was without effect on NMDA current amplitude. These findings indicate a functional role for phospholipase C-coupled D1-like receptors in both modulating synaptic transmission in NAcc and potentiating NMDA receptors on a subset of NAcc neurones, via PKC activation.

Rat substantia nigra pars reticulata neurones are tonically inhibited via GABAA, but not GABAB, receptors in vitro

Extracellular single unit recordings were made from substantia nigra pars reticulata (SNr) neurones in slices of rat brain. Cells fired spontaneous action potentials at 11.4 +/- 0.8 Hz. The GABAA receptor agonist isoguvacine (1-10 microM) reduced firing rate in a concentration-dependent manner [50% of maximal inhibition (IC50) with 3.2 microM], as did the GABAB agonist baclofen (0.3-10 microM; IC50 1.4 microM). The GABAA antagonist bicuculline (30 microM) not only blocked the action of isoguvacine, but also increased the basal firing rate to 187.5 +/- 12.6% of control. The GABAB antagonist CGP 55845A (0.1 microM), while blocking the inhibitory action of baclofen, was without effect on spontaneous firing rate, as was strychnine (10 microM), the antagonist of glycine and taurine, and also Met-enkephalin (10 microM). Tiagabine (50 microM), the blocker of GABA uptake, caused an inhibition of firing which could be reversed with bicuculline (30 microM) but not CGP 55845A (1 microM). We conclude that the firing rate of SNr neurones is under tonic inhibition by GABA in vitro, which can be relieved by antagonists of GABAA, but not GABAB receptors, and enhanced by blockade of GABA reuptake. The source of this GABA tone is likely to be from recurrent axon collaterals of SNr neurones themselves.

Overwhelmingly asynchronous firing of rat subthalamic nucleus neurones in brain slices provides little evidence for intrinsic interconnectivity

In Parkinsons disease the neurones of the subthalamic nucleus show increased synchrony and oscillatory burst discharge, thought to reflect a breakdown of parallel processing in basal ganglia circuitry. To understand better the mechanisms underlying this transition, we sought to mimic this change in firing pattern within sagittal slices of rat midbrain. The firing patterns of up to four simultaneously extracellularly recorded subthalamic nucleus (STN) neurones were analysed using burst and oscillation detection programs, and correlated activity between pairs of neurones assessed. In control conditions all but 11 of 488 (2%) neurones fired in a predominantly tonic pattern (with mean oscillation frequency >3 Hz), with no significantly cross-correlated activity in any of 393 pairs of neurones. The glutamate antagonists DL-2-amino-phosphonopentanoic acid (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6-methyl-2-(phenylethynyl)pyridine (MPEP) did not change the firing rate or pattern of these cells, providing no evidence for a role of glutamatergic collaterals within the STN under these conditions. The GABA(A) receptor antagonist bicuculline and GABA(B) receptor antagonist (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl]phenylmethyl phosphinic acid (CGP 55845) were also without effect on firing rate or pattern in these cells, suggesting that there was no active input from other GABAergic basal ganglia nuclei in this slice. The dopamine receptor antagonist haloperidol caused no significant change to firing rate or pattern of firing in these cells, suggesting that there was no active dopaminergic input in this slice. Excitations of STN neurones by muscarine, (+)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD), N-methyl-D-aspartic acid (NMDA) or dopamine were all unaccompanied by a change in firing pattern or any significant correlated activity between STN neurone pairs. Burst firing could be induced in STN neurones with either the potassium channel blocker tetraethylammonium (TEA; 10 mM; in 100/138 [72%] of cells) or with a combination of NMDA and the calcium-activated potassium channel blocker apamin (in 101/216 [47%] of cells). Burst firing in TEA was unchanged by CNOX and APV, MPEP, CGP55845, haloperidol, dopamine, and ACPD, although muscarine produced a significant increase in oscillation frequency. Burst firing in NMDA and apamin was unchanged by CNQX and APV, dopamine, muscarine and ACPD, although bicuculline caused a significant increase in oscillation frequency. Such burst firing was not accompanied by synchrony in any condition, either alone, or during application of excitatory agents or glutamate or GABA antagonists. As the bursting seen here was unaccompanied by the synchronous activity that has often been observed (pathologically) in vivo, it probably reflects solely intrinsic STN neuronal properties, rather than network activity. No functional role was found for glutamatergic collaterals within the STN, either when cells are firing tonically or burst firing. The circuitry needed to produce synchrony in the STN is most likely not intrinsic to the STN itself, but requires connections with other basal ganglia nuclei, and/or the cortex, which are not present in this preparation.

Presynaptic inhibition by dopamine of a discrete component of GABA release in rat substantia nigra pars reticulata

1 Whole‐cell patch clamp recordings were made from substantia nigra pars reticulata (SNr) neurones in rat midbrain slices. Monosynaptic IPSCs were evoked by electrical stimulation of the cerebral peduncle in the presence of the glutamate receptor antagonists CNQX (6‐cyano‐7‐nitroquinoxaline‐2,3‐dione) and AP5 (2‐amino‐5‐phosphonopentanoic acid). 2 IPSCs were predominantly outward at −70 mV (in 124/135 cells), with a reversal potential of −83 mV, a time to peak of 2.6 ms and a decay time constant of 6.5 ms. Faster inward IPSCs were also observed in thirty‐five cells, with a time to peak of 1.0 ms, a decay time constant of 2.3 ms, and a reversal potential of −61 mV. Both IPSCs were sensitive to the GABAA receptor antagonists picrotoxin or bicuculline. 3 In cells recorded with Cs+‐filled pipettes, the outward IPSC reversal potential was shifted to −76 mV, closer to the estimated Cl− equilibrium potential of −56 mV, while that of the inward IPSC was unchanged at −64 mV. 4 The outward IPSC was reversibly depressed by up to 100 % by dopamine in a concentration‐dependent manner with an IC50 of 10.5 μm, while the inward IPSC was relatively insensitive. 5 Dopamine was without effect on cell holding current, or on outward IPSC reversal potential, but it increased paired‐pulse IPSC facilitation, consistent with a presynaptic site of action. 6 The D1‐like dopamine receptor agonist SKF 38393 (10 μm) depressed the outward IPSC by 43 %, while the D2‐like dopamine receptor agonist quinpirole (10 μm) was without effect. 7 It is concluded that GABA‐ergic synaptic input onto distal rather than proximal regions of SNr neurones is susceptible to presynaptic inhibition via a D1‐like receptor. These inputs are probably from striato‐nigral fibres, and their inhibition by dopamine is likely to influence the patterning of basal ganglia output.

Electrophysiological investigation of adenosine trisphosphate-sensitive potassium channels in the rat substantia nigra pars reticulata

Adenosine trisphosphate-sensitive potassium (K-ATP) channels in the substantia nigra pars reticulata were studied in rat brain slices using whole-cell patch clamp recording. Substantia nigra pars reticula neurons were identified as such by their spontaneous action potential firing at mean rate of 15.3 Hz1 virtual absence of hyperpolarization-activated inward current Ih1 and unresponsiveness to dopamine (30 microM), quinirole (10 microM) and (Met)enkephalin (10 microM). Intracellular dialysis with Mg(2+0-ATP-free pipette solutions caused a slowly developing membrane hyperpolarization (13 +/- 4 mV), accompanied by a cessation of action potential firing, or an outward current (79 +/- 30 pA at around -60 mV), which were reversed b the sulphonylurea K-ATO channel blockers tolbutamide (100 microM) and glibenclamide (3 microM). When Mg(2+0-ATP (2 mM) was included in the recording pipette no membrane hyperpolarization or outward current was observed. Neither the sulphonylureas nor the potassium channel activator lemakalim (200 MicroM) altered membrane potential, firing rate or holding current under these recording conditions. The outward current induced by dialysis with Mg(2+)-ATP-free solutions reversed polarity negative to -94 +/- 9 mV (9 cells), close to the estimated K+ equilibrium potential (-105 mV) for the conditions used, and was associated with a conductance increase that was blocked by Ba2+ (100 microM). The current blocked by the sulphonylureas had a similar reversal potential (-97 +/- 7 MV; 13 cells), and both currents were voltage independent over the range -50 to -100 mV with slope conductance of approximately 2.0 nS. Outward synaptic current were evoked by single shock electrical simulation, in the presence of glutamate receptor antagonists, at a holding potential of -50 mV. These synaptic currents were blocked by bicuculline (10 microM) and reversed polarity at around -65 mV, close to the Cl- equilibrium potential, and were thus mediated by GABAA receptors. They were reversibly depressed by 37 +/- 14% in lemakalim (200 microM) in 6/12 cells tested, an effect that was partially reversed by tolbutamide (200 microM). It is concluded that functional K-ATP channels are present both presynaptically and postsynaptically in the substantia nigra pars reticulata. Postsynaptic K-ATP channels may control excitability in conditions where intracellular ATP is reduced, whereas presynaptic K-ATP channels, sensitive to the potassium channel activator lemakalim, can modulate the release of GABA, which probably arises from fibres of extranigral origin. Pharmacological differences between these two sites could be exploited to treat epilepsies, dyskinesias and akinesia.

A Kv3-like persistent, outwardly rectifying, Cs+-permeable, K+ current in rat subthalamic nucleus neurones.

1 A persistent outward K+ current (IPO), activated by depolarization from resting potential, has been identified and characterized in rat subthalamic nucleus (SThN) neurones using whole‐cell voltage‐clamp recording in brain slices. 2 I PO both rapidly activated (τ= 8 ms at +5 mV) and deactivated (τ= 2 ms at −68 mV), while showing little inactivation. Tail current reversal potentials varied with extracellular K+ concentration in a Nernstian manner. 3 Intracellular Cs+ did not alter either IPO amplitude or the voltage dependence of activation, but blocked transient (A‐like) outward currents activated by depolarization. When extracellular K+ was replaced with Cs+, IPO tail current reversal potentials were dependent upon the extracellular Cs+ concentration, indicating an ability to conduct Cs+, as well as K+. 4 I PO was blocked by Ba2+ (1 mm), 4‐aminopyridine (1 mm) and tetraethylammonium (TEA; 20 mm), with an IC50 for TEA of 0.39 mm. 5 The IPO conductance appeared maximal (38 nS) at around +27 mV, half‐maximal at −13 mV, with the threshold for activation at around −38 mV. 6 TEA (1 mm) blocked the action potential after‐hyperpolarization and permitted accommodation of action potential firing at frequencies greater than around 200 Hz. 7 We conclude that IPO, which shares many characteristics of currents attributable to Kv3.1 K+ channels, enables high‐frequency spike trains in SThN neurones.

Functional interconnectivity between the globus pallidus and the subthalamic nucleus in the mouse brain slice

In accordance with its central role in basal ganglia circuitry, changes in the rate of action potential firing and pattern of activity in the globus pallidus (GP)–subthalamic nucleus (STN) network are apparent in movement disorders. In this study we have developed a mouse brain slice preparation that maintains the functional connectivity between the GP and STN in order to assess its role in shaping and modulating bursting activity promoted by pharmacological manipulations. Fibre‐tract tracing studies indicated that a parasagittal slice cut 20 deg to the midline best preserved connectivity between the GP and the STN. IPSCs and EPSCs elicited by electrical stimulation confirmed connectivity from GP to STN in 44/59 slices and from STN to GP in 22/33 slices, respectively. In control slices, 74/76 (97%) of STN cells fired tonically at a rate of 10.3 ± 1.3 Hz. This rate and pattern of single spiking activity was unaffected by bath application of the GABAA antagonist picrotoxin (50 μm, n= 9) or the glutamate receptor antagonist (6‐cyano‐7‐nitroquinoxaline‐2, 3‐dione (CNQX) 10 μm, n= 8). Bursting activity in STN neurones could be induced pharmacologically by application of NMDA alone (20 μm, 3/18 cells, 17%) but was more robust if NMDA was applied in conjunction with apamin (20–100 nm, 34/77 cells, 44%). Once again, neither picrotoxin (50 μm, n= 5) nor CNQX (10 μm, n= 5) had any effect on the frequency or pattern of the STN neurone activity while paired STN and GP recordings of tonic and bursting activity show no evidence of coherent activity. Thus, in a mouse brain slice preparation where functional GP–STN connectivity is preserved, no regenerative synaptically mediated activity indicative of a dynamic network is evident, either in the resting state or when neuronal bursting in both the GP and STN is generated by application of NMDA/apamin. This difference from the brain in Parkinsons disease may be attributed either to insufficient preservation of cortico‐striato‐pallidal or cortico‐subthalamic circuitry, and/or an essential requirement for adaptive changes resulting from dopamine depletion for the expression of network activity within this tissue complex.