Dopamine differentially modulates the excitability of striatal neurons of the direct and indirect pathways in lamprey

Abstract

The functions of the basal ganglia are critically dependent on dopamine. In mammals, dopamine differentially modulates the excitability of the direct and indirect striatal projection neurons, and these populations selectively express dopamine D1 and D2 receptors, respectively. Although the detailed organization of the basal ganglia is conserved throughout the vertebrate phylum, it was unknown whether the differential dopamine modulation of the direct and indirect pathways is present in non-mammalian species. We aim here to determine whether the receptor expression and opposing dopaminergic modulation of the direct and indirect pathways is present in one of the phylogenetically oldest vertebrates, the river lamprey. Using in situ hybridization and patch-clamp recordings, we show that D1 receptors are almost exclusively expressed in the striatal neurons projecting directly to the homolog of the substantia nigra pars reticulata. In addition, the majority of striatal neurons projecting to the homolog of the globus pallidus interna/globus pallidus externa express D1 or D2 receptors. As in mammals, application of dopamine receptor agonists differentially modulates the excitability of these neurons, increasing the excitability of the D1-expressing neurons and decreasing the excitability of D2-expressing neurons. Our results suggest that the segregated expression of the D1 and D2 receptors in the direct and indirect striatal projection neurons has been conserved across the vertebrate phylum. Because dopamine receptor agonists differentially modulate these pathways, increasing the excitability of the direct pathway and decreasing the excitability of the indirect pathway, this organization may be conserved as a mechanism that biases the networks toward action selection.

Figure 1.

Striatonigral neurons express functional D₁ receptors that excite neurons. A, Striatal neurons (Str) retrogradely labeled after injections of Neurobiotin into the SNr. B, DIG-labeled D₁ receptor riboprobe expressed in a subpopulation of striatal neurons. C, Merged image showing the overlap between retrogradely labeled cells and D₁ receptor mRNA. Inset shows at higher magnification an example of two cells that are retrogradely labeled and express the D₁ receptor. Yellow arrows indicate neurons that are retrogradely labeled but do not express D₁ receptor mRNA. Green arrows indicate neurons that are retrogradely labeled and express D₁ receptor mRNA. D, Quantification showing the percentage of retrogradely labeled neurons that express D₁ receptor mRNA. E, Striatal neurons retrogradely labeled after injections of Neurobiotin into the SNr. F, DIG-labeled D₂ receptor riboprobe expressed in a subpopulation of striatal neurons. G, Merged image showing the lack of overlap between retrogradely labeled cells and D₂ receptor mRNA. Inset shows at higher magnification an example of one cell that is retrogradely labeled and is devoid of D₂ receptor expression. Yellow arrows indicate neurons that are retrogradely labeled but do not express D₂ receptor mRNA. H, Quantification showing the percentage of retrogradely labeled neurons that express D₂ receptor mRNA. I, Evoked APs in a retrogradely labeled striatonigral neuron during subsequent application of 10 μm SKF 81297 (red, 2nd trace and bar below) and 100 μm TNPA (blue, last trace and bar below) shown in a corresponding plot of time and the number of spikes evoked by the same near-rheobase current step. This neuron only responded to application of SKF 81297, which increased spiking seen in the plot, whereas TNPA had no effect on the number of evoked spikes. Ctrl, Control. J, Application of SKF 81297 enhanced evoked spiking in striatonigral neurons, and all but one of the tested neurons were unresponsive to sequential application of TNPA (K), *p < 0.05. Scale bars, 200 μm.

Figure 2.

One subgroup of striatopallidal neurons expresses functional D₂ receptors that reduce excitability. A, Striatal neurons retrogradely labeled after injections of Neurobiotin into the dorsal pallidum (DP). B, DIG-labeled D₂ receptor riboprobe expressed in a subpopulation of striatal neurons. C, Merged image showing the overlap between retrogradely labeled cells and D₂ receptor mRNA. Inset shows at higher magnification an example of two cells that are retrogradely labeled and that express the D₂ receptor. Yellow arrows indicate neurons that are retrogradely labeled express D₂ receptor mRNA. Green arrows indicate neurons that are retrogradely labeled and express D₂ receptor mRNA. D, Quantification showing the percentage of retrogradely labeled neurons that express D₂ receptor mRNA. E, Evoked APs in a retrogradely labeled striatopallidal neuron during sequential application of 10 μm SKF 81297 (red trace and bar) and 100 μm TNPA (blue trace and bar). This neuron does not respond to application of SKF 81297, whereas application of TNPA potently reduces the number of evoked spikes. Ctrl, Control. F, Application of SKF 81297 did not significantly affected excitability, although a few of the neurons slightly increased or decreased their spiking. G, In these seven cells (n = 7 of 16), application of TNPA had a pronounced effect on evoked spikes that were reduced by the D₂ activation, ***p < 0.001. Scale bars, 200 μm.

Figure 3.

One subgroup of striatopallidal neurons expresses functional D₁ receptors that enhance excitability. A, Striatal neurons retrogradely labeled after injections of Neurobiotin into the dorsal pallidum (DP). B, DIG-labeled D₁ receptor riboprobe expressed in a subpopulation of striatal neurons. C, Merged image showing the overlap between retrogradely labeled cells and D₁ receptor mRNA. Inset shows at higher magnification an example of one cell that is retrogradely labeled and express the D₁ receptor. Yellow arrows indicate neurons that are retrogradely labeled but do not express D₁ receptor mRNA. Green arrows indicate neurons that are retrogradely labeled and express D₁ receptor mRNA. D, Quantification showing the percentage of retrograde labeled neurons that express D₁ receptor mRNA. E, Evoked APs in a retrogradely labeled striatopallidal neuron during sequential application of 10 μm SKF 81297 (red trace and bar) and 100 μm TNPA (blue trace and bar). This neuron responds to application of SKF 81297 by increasing spike discharge, whereas application of TNPA does not affect the number of evoked spikes. Ctrl, Control. F, Enhanced spiking by SKF 81297 was seen in 6 of 16 cells, and five of these six cells were unresponsive to TNPA (G), **p < 0.01. Scale bars, 200 μm.

Figure 4.

D₁ receptor activation by the agonist SKF 81297 excites depolarized neurons. A, Evoked response patterns of striatal neurons held at depolarized membrane potentials (−55 to −65 mV). Application of SKF 81297 (10 μm) enhances the firing. A₁, Voltage responses of a neuron (striatonigral) to hyperpolarizing and depolarizing 1 s current steps of 5 pA per step, elicited from a depolarized potential at −55 mV in control aCSF (left, control) or during bath application SKF 81297 (right). SKF 81297 increases the number of evoked spikes (arrow) and PIR spikes (arrow). The hyperpolarizing voltage responses are similar in control and SKF 81297, indicating that there is no change in input resistance. A₂, Current–frequency diagram of the same neuron showing the increased number of evoked spikes during SKF 81297 (gray circles) compared with control (black squares). A₃, Application of SKF 81297 increases the number of evoked spikes in D₁ receptor-stimulated neurons, measured near rheobase, ***p < 0.001. A₄, SKF 81297 increased PIR spikes in four neurons capable of producing such hyperpolarization-activated APs, quantified by the total number of PIR spikes in response to 8–10 consecutive hyperpolarizations from −100 mV to baseline as in A₁, **p < 0.01. B, Same protocols as in A but at hyperpolarized potentials at approximately −80 mV. B₁, B₂, SKF 81297 (B₁, right traces) has no effect on evoked potentials when they are elicited from −80 mV compared with control (B₁, left traces), and the neuron (same as in A) does not fire PIR spikes from this negative potential. B₃, This was consistent for almost all cells tested. B₄, Only one of the four neurons with PIR spikes at depolarized potentials were capable of producing such APs from hyperpolarized levels.

Figure 5.

D₂ receptor activation by the agonist TNPA inhibits neurons. A, Evoked response patterns of striatal neurons held at depolarized levels before and during application of TNPA (100 μm) that reduce excitability. A₁, Voltage responses of a neuron to hyperpolarizing and depolarizing 1 s current steps of 3 pA per step, elicited at membrane potentials at approximately −55 mV in control aCSF (left, control) or during bath application TNPA (right). TNPA reduces the number of evoked spikes (arrow) and PIR spikes (arrow). The hyperpolarizing voltage responses are similar in control and TNPA, indicating that there is no change in input resistance. A₂, Current–frequency diagram of the same neuron showing the decreased number of evoked spikes during TNPA (gray circles) compared with control (black squares). A₃, Application of TNPA potently reduces the number of evoked spikes in D₂-stimulated neurons, measured at near-rheobase positive current injection, ***p < 0.001. A₄, TNPA strongly reduced PIR spikes in all neurons capable of producing such hyperpolarization-activated APs, quantified by the total number of PIR spikes in response to 8–10 consecutive hyperpolarizations from −100 mV to baseline as in A₁, ***p < 0.001. B, Same protocols as in A but at hyperpolarized potentials at approximately −80 mV. B_1, B₂, TNPA (B₁, right traces) reduces evoked potentials also when they are elicited from a hyperpolarized baseline approximately −80 mV compared with control (B₁, left traces). The neuron (same as in A) does not fire PIR spikes from this negative potential. B₃, The reduced spiking was consistent for all cells tested, **p < 0.01. B₄, Only one of all neurons with PIR spikes at depolarized potentials were capable of producing such APs from hyperpolarized levels.

Figure 6.

Summary of the organization of the direct and indirect pathways in lamprey. A, Schematic drawing showing the evolutionarily conserved architecture of the basal ganglia. Blue, pink, and red arrows indicate GABAergic, dopaminergic, and glutamatergic projections, respectively. B, Schematic sagittal section through the lamprey brain showing the location of the known basal ganglia nuclei and the connectivity of the direct and indirect pathways from the striatum to the output nuclei. Enk, Enkephalin; ntp, nucleus tuberculi posterior; OT, optic tectum; SP, substance P; PPN, pedunculopontine nucleus; Th, thalamus.