Dopaminergic Presynaptic Modulation of Nigral Afferents: Its Role in the Generation of Recurrent Bursting in Substantia Nigra Pars Reticulata Neurons

Previous work has shown the functions associated with activation of dopamine presynaptic receptors in some substantia nigra pars reticulata (SNr) afferents: (i) striatonigral terminals (direct pathway) posses presynaptic dopamine D1-class receptors whose action is to enhance inhibitory postsynaptic currents (IPSCs) and GABA transmission. (ii) Subthalamonigral terminals posses D1- and D2-class receptors where D1-class receptor activation enhances and D2-class receptor activation decreases excitatory postsynaptic currents. Here we report that pallidonigral afferents posses D2-class receptors (D3 and D4 types) that decrease inhibitory synaptic transmission via presynaptic modulation. No action of D1-class agonists was found on pallidonigral synapses. In contrast, administration of D1-receptor antagonists greatly decreased striatonigral IPSCs in the same preparation, suggesting that tonic dopamine levels help in maintaining the function of the striatonigral (direct) pathway. When both D3 and D4 type receptors were blocked, pallidonigral IPSCs increased in amplitude while striatonigral connections had no significant change, suggesting that tonic dopamine levels are repressing a powerful inhibition conveyed by pallidonigral synapses (a branch of the indirect pathway). We then blocked both D1- and D2-class receptors to acutely decrease direct pathway (striatonigral) and enhance indirect pathways (subthalamonigral and pallidonigral) synaptic force. The result was that most SNr projection neurons entered a recurrent bursting firing mode similar to that observed during Parkinsonism in both patients and animal models. These results raise the question as to whether the lack of dopamine in basal ganglia output nuclei is enough to generate some pathological signs of Parkinsonism.


IntroductIon
The internal globus pallidus (GPi) and substantia nigra pars reticulata (SNr) are the basal ganglia (BG) output nuclei. Besides projecting to the thalamus to form the cortico-BG loops (Chevalier et al., 1985;Albin et al., 1989;Smith and Bolam, 1989;Alexander and Crutcher, 1990;DeLong, 1990;Smith et al., 1998;Haber, 2003), output nuclei also project to pons and brain stem to control descending pathways and central patterns generators (CPGs) that regulate muscular tone and automatic or rhythmic motor responses (Takakusaki et al., 2003(Takakusaki et al., , 2004Grillner et al., 2008). In birds, reptiles, and lower vertebrates in which the cortex is not well developed, the control of brain stem nuclei is a main function of the BG (Reiner et al., 1998;Grillner et al., 2005Grillner et al., , 2008Gale and Perkel, 2010). In the SNr, inhibitory postsynaptic currents (IPSCs) are in part provided by striatonigral direct pathway terminals (Grofová and Rinvik, 1970;Chevalier et al., 1985;Smith and Bolam, 1991;Deniau et al., 1996;Matuszewich and Yamamoto, 1999), which possess functional presynaptic dopamine D 1 -receptors whose activation increases direct pathway inhibition (Porceddu et al., 1986;Altar and Hauser, 1987;Floran et al., 1990;Radnikow and Misgeld, 1998;Chuhma et al., 2011). Enhancement of direct pathway inhibition facilitates movements while its reduction represses them (Albin Dopaminergic presynaptic modulation of nigral afferents: its role in the generation of recurrent bursting in substantia nigra pars reticulata neurons José de Jesús Aceves, Pavel E. Rueda-Orozco, Ricardo Hernández, Víctor Plata, Osvaldo Ibañez-Sandoval, Elvira Galarraga and José Bargas* División de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Distrito Federal México, México Previous work has shown the functions associated with activation of dopamine presynaptic receptors in some substantia nigra pars reticulata (SNr) afferents: (i) striatonigral terminals (direct pathway) posses presynaptic dopamine D 1 -class receptors whose action is to enhance inhibitory postsynaptic currents (IPSCs) and GABA transmission. (ii) Subthalamonigral terminals posses D 1 -and D 2 -class receptors where D 1 -class receptor activation enhances and D 2 -class receptor activation decreases excitatory postsynaptic currents. Here we report that pallidonigral afferents posses D 2 -class receptors (D 3 and D 4 types) that decrease inhibitory synaptic transmission via presynaptic modulation. No action of D 1 -class agonists was found on pallidonigral synapses. In contrast, administration of D 1 -receptor antagonists greatly decreased striatonigral IPSCs in the same preparation, suggesting that tonic dopamine levels help in maintaining the function of the striatonigral (direct) pathway. When both D 3 and D 4 type receptors were blocked, pallidonigral IPSCs increased in amplitude while striatonigral connections had no significant change, suggesting that tonic dopamine levels are repressing a powerful inhibition conveyed by pallidonigral synapses (a branch of the indirect pathway). We then blocked both D 1 -and D 2class receptors to acutely decrease direct pathway (striatonigral) and enhance indirect pathways (subthalamonigral and pallidonigral) synaptic force. The result was that most SNr projection neurons entered a recurrent bursting firing mode similar to that observed during Parkinsonism in both patients and animal models. These results raise the question as to whether the lack of dopamine in basal ganglia output nuclei is enough to generate some pathological signs of Parkinsonism.
transmitters (Ibáñez-Sandoval et al., 2006). Amplitude of first IPSC was used to build time courses of dopaminergic actions. Because striatonigral fibers pass through the GPe, D 2 -class selective agonists were tested in slices taken from animals with a stereotaxic lesion (ibotenic acid) of the GPe (1.4 mm AP, 3.4 L, and 4.7 mm V) and compared to recordings obtained without a lesion. The lesion further confirmed the differences of IPSCs from both sources. Ibotenic acid solution (dissolved in PBS adjusted to pH 7.4 with NaOH 3.0 μg/0.4 μl) was used to lesion the GPe. These values closely followed Paxinos and Watson (1982) coordinates system.
where A(I) = IPSC amplitude as a function of stimulus intensity, A max = maximal amplitude reached, k = slope factor, and I h = stimulus intensity necessary to reach IPSC amplitude equal to half maximal amplitude. All three parameters were significantly different when comparing IPSCs from striatonigral vs. pallidonigral afferents: A max : 430 ± 3 pA vs. 1512 ± 10 pA (n = 8; P < 0.0001); k = 3.6 ± 0.3 vs. 14 ± 5 (n = 8; P < 0.0001) and I h : 2.3 ± 0.2 vs. 1.1 ± 0.1 (n = 8; P < 0.005). These features coincide with a previous report (Connelly et al., 2010) and were verified qualitatively by evoking IPSCs from either the subthalamic nucleus (NST) or the GPe, however, in these where A(I) = IPSC amplitude as a function of stimulus intensity, A max = maximal amplitude reached, k = slope factor, and I h = stimulus intensity necessary to reach IPSC amplitude equal to half maximal amplitude. All parameters were significantly different. (g) Cluster plot showing that IPSCs from these sources can be separated. PPR = paired-pulse ratio. τ D = decay time constant of IPSCs. R S = rise time of IPSCs.

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www.frontiersin.org et al., 2011) with no action on pallidonigral terminals. On the other hand, physiological action of D 2 -receptor agonists on pallidonigral IPSCs is that of depression with no significant action on striatonigral terminals. Therefore we were forced to infer that reports about D 1 -mediated inhibition of striatonigral terminals (Miyazaki and Lacey, 1998) were either involving a non-specific action, a pallidal contamination, or both. To test this hypothesis we used larger micromolar concentrations of the D 1 -agonist while evoking IPSCs from both pathways. Figures 3A,B show that 5 μM SKF 81297 decreased IPSCs evoked from both set of terminals. Striatonigral IPSC decreased 82 ± 13% (n = 18; P < 0.001) and pallidonigral responses decreased by 35 ± 15% (n = 6; P < 0.005). These actions could not be blocked by micromolar concentrations of SCH 23390 (not shown), suggesting that they were not specific. In view of these results we built a concentration-response relationship (C-R plot) using a wide range of SKF 81297 concentrations while stimulating striatonigral afferents. This C-R plot can be seen in Figure 3C: it is biphasic. When the Hill equation was fitted to the ascending (specific part) EC 50 was 440 ± 60 nM and the Hill coefficient 1.6 ± 0.2, suggesting cooperativity and a specific action at submicromolar concentrations. Moreover, the fact that pallidonigral inputs are also affected when they do not respond when submicromolar concentrations of agonists are used confirmed non-specific actions.
The actions of selective dopamine receptor agonists for D 1 -and D 2 -receptor classes were tested. As it has been repeatedly demonstrated, the action of dopaminergic D 1 -class selective agonists at nanomolar concentrations was that of enhancing striatonigral IPSCs (Floran et al., 1990;Radnikow and Misgeld, 1998;Chuhma et al., 2011): striatonigral IPSC increased 153 ± 10% after 300 nM SKF 81297 (n = 15; P < 0.001) and the paired-pulse ratio (PPR = IPSC2/ IPSC1) decreased from 1.4 ± 0.13 in the control to 1.0 ± 0.12 during SKF 81297 (P < 0.001), confirming a presynaptic modulation. These actions were reversible and blocked by 100 nM of the D 1 -antagonist SCH 23390 (n = 5; not shown here but see below) indicating that at these concentrations the action is specific. In addition, here we show that the agonists have no significant action on pallidonigral IPSCs (cf. , Figures 2A,B).
Summarizing, physiological action of D 1 -receptor agonists on striatonigral terminals is that of IPSC enhancement as previously shown (Floran et al., 1990;Radnikow and Misgeld, 1998; Chuhma IPSCs. Note changes in PPR accompanying significant effects. Record 3 in each frame is the superimposition of records 1 and 2 after normalization of the first IPSC to better appreciate the PPR change when it is present.

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www.frontiersin.org D 1 -receptors in direct pathway striatonigral terminals are sensitive detectors of extracellular dopamine. Moreover, blockade of dopaminergic action can reduce striatonigral synaptic reliability to a minimum. In addition, Figures 4E,F illustrate the actions of selective antagonists for D 3 -and D 4 -type dopamine receptors: 500 nM U-9914A, a selective D 3 -type receptor antagonist enhanced pallidonigral IPSCs by 262 ± 16% after (n = 12; P < 0.001) while the PPR decreased from 0.87 ± 0.08 in the control to 0.51 ± 0.1 during the antagonist. On the other hand, 500 nM LY750, a selective D 4 -type receptor antagonist increased pallidonigral IPSCs by 170 ± 20% (n = 7; P < 0.001) while PPR decreased from 0.78 ± 0.07 in the control to 0.57 ± 0.09 during the blockade. Sulpiride a generic D 2 -class receptor antagonist had similar actions (not shown): pallidonigral IPSCs increased by 150 ± 14% (n = 3) while PPR decreased from 0.77 ± 0.03 in the control to 0.35 ± 0.01 during blockade. To summarize, D 3/4 -receptors in pallidonigral terminals (Murray et al., 1994;Bevan et al., 1996;Marshall et al., 2001;Rivera et al., 2003;Seeman et al., 2006;Acosta-García et al., 2009;Gasca-Martinez et al., 2010) are sensitive to extracellular dopamine, which has the role of tonically repressing the force Given the low concentrations of agonists needed to activate D 1 -and D 2 -class receptors in their respective terminals (striatonigral and pallidonigral) we inferred that, perhaps, endogenous extracellular dopamine exerts a tonic action on these receptors. Figure 4 shows that this hypothesis is correct. Figures 4A,B show that 50 nM of a D 1 -class receptor selective antagonist, SCH 23390, are enough to inhibit striatonigral IPSCs with no significative action on pallidonigral IPSC. Striatonigral IPSC decreased from 319 ± 75 in the control to 150 ± 11 pA after 50 nM SCH 23390 (n = 12; P < 0.02). Figures 4C,D confirm these findings and further show that potency and speediness of D 1 -action is concentration dependent. Striatonigral IPSC is greatly reduced -almost abolished -when a low micromolar antagonist concentration is maintained in the superfusion (Figure 4C). The effect is reversible (not shown). Traces chosen at different times during the time course, superimposed, and normalized to the amplitude of the first IPSC, show that the PPR is greatly increased from 1.9 ± 0.4 in the control to 2.4 ± 0.6 after SCH 23390 (n = 21; P < 0.001; when the IPSC is abolished PPR cannot be measured); confirming a presynaptic site of action. On the other hand, SCH 23390 did not produce any action on pallidonigral IPSC at any concentration ( Figure 4D). In summary, One of the most supported neuronal correlates of Parkinsonism: recurrent bursting in SNr neurons, was similar to that previously reported in vivo and in vitro (Hammond et al., 2007;Ibáñez-Sandoval et al., 2007;Walters et al., 2007;Walters and Bergstrom, 2009;Zold et al., 2009). Transitions from tonic to bursting firing mode can rarely be seen spontaneously in control preparations (Ibáñez-Sandoval et al., 2007).
To see whether excitatory subthalamonigral afferents, that is, the STN-GP circuit, was participating in this firing behavior, we added the NMDA-receptor antagonist APV (50 μM) to provoke a partial of these synapses. Some functional differences in the actions of these receptor types perhaps deserve further investigation (cf., Figures 4E,F).
Finally, because subthalamonigral terminals are also tonically controlled by presynaptic dopamine receptors (D 1 -and D 2 -class; Ibáñez-Sandoval et al., 2006) and because blockade of these receptors enhance subthalamonigral EPSCs (Ibáñez-Sandoval et al., 2006), we propose the following hypothesis based in the present and previous results (Radnikow and Misgeld, 1998;Acosta-García et al., 2009;Chuhma et al., 2011): that acute blockade of both D 1 -and D 2 -class (including D 3/4 -types) receptors (by 1 μM SCH 23390 plus 1 μM sulpiride) altogether may decrease direct pathway synapses (striatonigral) and, at the same time, enhance indirect pathway synapses (subthalamonigral and pallidonigral), both actions being required in physiopathological concentration has no action on pallidonigral IPSC. (e) 500 nM U-9914A, a selective D 3 -type receptor antagonist, significantly enhanced pallidonigral IPSC. Note a partial reversion. (F) 500 nM LY750, a selective D 4 -type receptor antagonist, significantly increased pallidonigral IPSC. Record 3 in each frame is the superimposition of representative records 1 and 2 after normalization of the first IPSC to better appreciate the PPR change when it is present.

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www.frontiersin.org D 1 -modulation. Notably, short-term facilitation works as a high pass filter of incoming inputs whereas short-term depression works as a low pass filter (Abbott and Regehr, 2004). In contrast, subthalamonigral excitatory synapses are controlled by both D 1 -and D 2 -class receptors (Ibáñez-Sandoval et al., 2006). Selective dopamine receptor agonists exert their specific actions at nanomolar or low micromolar concentrations but higher concentrations become non-specific, thus explaining previous contradictions (cf., Miyazaki and Lacey, 1998;Radnikow and Misgeld, 1998) and probably similar discrepancies within the striatal/accumbinal circuitry (Guzman et al., 2003;Taverna et al., 2005). To summarize: D 1 -receptor activation enhances GABA release in terminals from medium spiny neurons of the direct pathway whereas D 3/4 -receptor activation represses GABA release from pallidonigral terminals.

strIatonIgral and pallIdonIgral dopaMIne receptors sense extracellular aMbIent endogenous dopaMIne
Because nanomolar concentrations of selective receptor agonist are needed to modify transmission, we hypothesized that administrations of selective dopamine receptor antagonist would disclose the actions of endogenous dopamine present in the extracellular space. Therefore, to disclose the action of ambient endogenous dopamine we applied selective D 1 -and D 3/4 -receptor antagonists. Our findings were that according to the concentration, suppression of endogenous dopamine action greatly reduced striatonigral transmission while it enhanced pallidonigral transmission. These results indicated that extracellular dopamine concentrations are block of STN influence. The result was that the firing pattern became less bursty and more tonic (Ibáñez-Sandoval et al., 2007), however, firing was still abruptly interrupted by sudden hyperpolarizations, probably coming from enhanced pallidonigral inputs.

dIscussIon
The present work shows that extracellular dopamine concentrations are tonically being sensed by the synaptic terminals of inhibitory inputs to the SNr in opposite ways. Thus, blockade of D 1 -type receptors in striatonigral (direct pathway) afferents decreased striatonigral inhibition while blockade of D 3/4 -types receptors in pallidonigral terminals enhanced pallidonigral inhibition. Pallidonigral afferents are presynaptically controlled by D 2 -class but not D 1 -class receptors. Still other inputs to the SNr have to be studied to see whether they are presynaptically modulated.

strIatonIgral and pallIdonIgral Ipscs are dIfferent
Striatonigral IPSCs are smaller but last longer than pallidonigral IPSCs. In addition, they exhibit short-term facilitation and are positively regulated by D 1 -receptors (Floran et al., 1990;Radnikow and Misgeld, 1998;Connelly et al., 2010). Strong evidence for the last property has been obtained with optogenetic techniques (Chuhma et al., 2011), supporting the present and previous reports. In comparison, pallidonigral IPSCs are larger and exhibit different degrees of short-term depression (Connelly et al., 2010). The two classes of inhibition are so different that cannot be confused. Pallidonigral IPSCs are negatively regulated by D 2 -class receptors and have no . During acute blockade of dopamine D 1and D 2 -class receptors (1 μM SCH 23390 plus 1 μM sulpiride) SNr firing pattern shifted from tonic to a bursting pattern in n = 7 cells; two cells exhibited bursting before adding the blockers. To see whether besides striatonigral and pallidonigral afferents, subthalamonigral afferents were also contributing to this firing mode, the glutamate NMDA-receptor antagonist, 50 μM APV was added to the bath saline (bottom). The firing tended to return to a tonic firing pattern but it was frequently interrupted by sudden hyperpolarizations.

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www.frontiersin.org regulating synaptic release probability in both types of connections, increasing release probability in striatonigral synapses and decreasing release probability in pallidonigral synapses. Therefore, the following working hypothesis resulted from the present experiments: tonic levels of dopamine in SNr are necessary to maintain the normal function of direct pathway connections and maintain in check indirect pathway synapses from both pallidonigral terminals (present work) and subthalamonigral terminals (Ibáñez-Sandoval et al., 2007). The logical functional consequence of this hypothesis was tested: blockade of tonic dopamine action would enhance indirect pathway transmission and reduce direct pathway input thus yielding a previously observed neuronal correlate of Parkinsonism: SNr neurons shifted their tonic firing pattern to a bursting firing pattern typical of subjects with the disease (Takakusaki et al., 2004;Rivlin-Etzion et al., 2006;Hammond et al., 2007;Ibáñez-Sandoval et al., 2007;Walters et al., 2007;Zold et al., 2009).
Tonic spontaneous firing is preserved in the slice preparation in both GPe and STN (e.g., Beurrier et al., 1999;Chan et al., 2011). Calcium imaging techniques recording dozens of cells simultaneously within the striatal circuit show that there is always some spontaneous activity in striatal spiny neurons in control conditions (Carrillo-Reid et al., 2008). Moreover, there is a great amount of convergence between the striatum and the substantia nigra and inhibitory striatonigral events are of large amplitude (e.g., Chuhma et al., 2011). Thus, for a SNr neuron there always may be some striatal cell firing. Therefore, the lost balance between direct and indirect pathways by the acute blockade of dopamine receptors is the most probable cause of bursting in SNr neurons during the present experiments.
Postsynaptic dopamine receptors on SNr neurons cannot explain bursting because their function is to increase tonic firing frequency via a cation current; their blockade resulting in lower tonic frequency with irregularities, but not continuous bursting behavior (Lee and Tepper, 2007;Zhou et al., 2009).

functIonal consequences
Because the change in firing pattern of SNr neurons was achieved acutely by blocking dopamine receptors one can speculate what would happen during a chronic absence of dopamine in the output nuclei of the BG. The absence of dopamine (e.g., Parkinsonism) may reduce the function of direct pathway synapses in such a way that maintaining this state of affairs in the long-time would lead to loss of direct pathway synapses due to long-term synaptic plasticity. In contrast, maintaining a high function in pallidonigral and subthalamonigral synapses would produce long-term potentiation of these synapses. These events taken together may tend to change the circuitry permanently, making not only L-DOPA inefficient but, perhaps, making the chronic diseased circuitry radically different than the control or healthy circuit: that is, more dependent on the subthalamopallidal loop (Magill et al., 2001;Baufreton et al., 2009).
Bursting in SNr neurons leads to tremor and rigidity (Hemsley and Crocker, 1998;Takakusaki et al., 2004). Moreover, this configuration of synaptic weights would lead to the loss of high pass filtering of SNr inputs (direct pathway's short-term facilitation) and an increase in low pass filtering of SNr inputs (pallidonigral short-term depression; Abbott and Regehr, 2004) setting the stage to favor the entrance to an akinetic frequency lock (Hutchison et al., 2004;Avila et al., 2010). Consequently, therapeutic ways of activating the direct pathway in the chronic patient may be fundamental to avoid irreversible plastic changes in the network (Hammond et al., 2007;Walters et al., 2007;Walters and Bergstrom, 2009;Zold et al., 2009;Bateup et al., 2010;Kravitz et al., 2010).