Proliferation of external globus pallidus-subthalamic nucleus synapses following degeneration of midbrain dopamine neurons

Abstract

The symptoms of Parkinson's disease (PD) are related to changes in the frequency and pattern of activity in the reciprocally connected GABAergic external globus pallidus (GPe) and glutamatergic subthalamic nucleus (STN). In idiopathic and experimental PD, the GPe and STN exhibit hypoactivity and hyperactivity, respectively, and abnormal synchronous rhythmic burst firing. Following lesion of midbrain dopamine neurons, abnormal STN activity emerges slowly and intensifies gradually until it stabilizes after 2-3 weeks. Alterations in cellular/network properties may therefore underlie the expression of abnormal firing. Because the GPe powerfully regulates the frequency, pattern, and synchronization of STN activity, electrophysiological, molecular, and anatomical measures of GPe-STN transmission were compared in the STN of control and 6-hydroxydopamine-lesioned rats and mice. Following dopamine depletion: (1) the frequency (but not the amplitude) of mIPSCs increased by ∼70%; (2) the amplitude of evoked IPSCs and isoguvacine-evoked current increased by ∼60% and ∼70%, respectively; (3) mRNA encoding α1, β2, and γ2 GABA(A) receptor subunits increased by 15-30%; (4) the density of postsynaptic gephyrin and γ2 subunit coimmunoreactive structures increased by ∼40%, whereas the density of vesicular GABA transporter and bassoon coimmunoreactive axon terminals was unchanged; and (5) the number of ultrastructurally defined synapses per GPe-STN axon terminal doubled with no alteration in terminal/synapse size or target preference. Thus, loss of dopamine leads, through an increase in the number of synaptic connections per GPe-STN axon terminal, to substantial strengthening of the GPe-STN pathway. This adaptation may oppose hyperactivity but could also contribute to abnormal firing patterns in the parkinsonian STN.

Figure 1.

Chronic dopamine depletion increased the frequency and prolonged the decay kinetics of mIPSCs. A, Representative recordings of mIPSCs in STN neurons derived from control and 6-OHDA-treated animals. The frequency but not the amplitude of mIPSCs was greater following dopamine depletion. B, Cumulative probability (cum. prob.) plots of the sample populations confirmed that dopamine depletion significantly reduced the intervals between mIPSCs but had no effect on the conductance underlying mIPSCs. C, The mean frequency of mIPSCs was significantly enhanced by dopamine depletion, whereas the mean conductance underlying mIPSCs was unaltered. D, The decay of mIPSCs was prolonged by chronic dopamine depletion, as evidenced by representative mean peak-scaled mIPSCs and box plots. *p < 0.05.

Figure 2.

Chronic dopamine depletion increased the magnitude of evoked GABA_A receptor-mediated synaptic transmission but had no effect on short-term synaptic plasticity. A–H, Following chronic dopamine depletion, the magnitude of synaptic transmission evoked at 10 Hz (A–D) and 50 Hz (E–H) was enhanced with no alteration in short-term synaptic plasticity. A, E, Representative recordings. B, F, Peak and normalized conductances plotted against IPSC number. C, D, G, H, Chronic dopamine depletion significantly increased the conductances underlying IPSC1 and IPSC25 (C, G) but had no effect on the ratio of conductances underlying IPSC2 to IPSC1 or IPSC25 to IPSC1 (D, H). *p < 0.05.

Figure 3.

Chronic dopamine depletion increased the conductance evoked by the GABA_A receptor agonist isoguvacine. A, Representative traces of isoguvacine-evoked currents in STN neurons derived from control and 6-OHDA-treated animals. B, The conductance evoked by isoguvacine was significantly increased by chronic dopamine depletion. *p < 0.05.

Figure 4.

Chronic dopamine depletion increased the transcription of genes encoding commonly expressed GABA_A receptor subunits. Quantitative PCR of the STN derived from control and 6-OHDA-lesioned animals revealed that the abundance of mRNA encoding GABA_A α1, β2, and γ2 receptor subunits was significantly increased by chronic dopamine depletion. *p < 0.05.

Figure 5.

Chronic dopamine depletion increased the densities of postsynaptic gephyrin-immunoreactive and γ2 GABA_A receptor subunit-immunoreactive structures. A1–B3, Representative confocal micrographs of gephyrin (A1, B1: green), γ2 GABA_A receptor subunit (A2, B2: red), and gephyrin plus γ2 GABA_A receptor subunit immunoreactivity (A3, B3: yellow) in the STN of a control (A) and 6-OHDA-treated (B) animal. The densities of immunoreactive structures in the STN were increased by chronic dopamine depletion compared with control both in representative micrographs (A, B) and across the sample population (C). The scale bar in A1 applies to each micrograph. *p < 0.05.

Figure 6.

Chronic dopamine depletion did not alter the densities of presynaptic vGAT- and bassoon-immunoreactive structures. A1–B3, Representative confocal micrographs of bassoon (A1, B1: green), vGAT (A2, B2: red), and bassoon plus vGAT immunoreactivity (A3, B3: yellow) in the STN of a control (A) and 6-OHDA-depleted (B) animal. The densities of immunoreactive structures in the STN were not altered by chronic dopamine depletion compared with control in the representative micrographs (A, B) or across the sample population (C). The scale bar in A1 applies to each micrograph. *p < 0.05.

Figure 7.

Chronic dopamine depletion increased the number of bassoon-immunoreactive structures associated with large vGAT-immunoreactive axon terminals. A–F, Representative through-focus confocal micrographs of bassoon (green) and vGAT (red) coimmunoreactive (yellow) axon terminals in the STN of control (A–C) and 6-OHDA-lesioned (D–F) animals. Three examples (A–C and D–F) per condition are illustrated. G, The number of bassoon-immunoreactive structures per vGAT-immunoreactive axon terminal (arrows) was significantly increased by chronic dopamine depletion compared with control both in the representative examples and across the sample population. The scale bar in A applies to each micrograph. *p < 0.05.

Figure 8.

Chronic dopamine depletion increased the number synapses per vGAT-immunoreactive axon terminal. A–F, Representative electron micrographs of vGAT-immunoreactive axon terminals in the STN of control (A–C) and 6-OHDA-lesioned (D–F) animals. vGAT-immunoreactive axon terminals were characterized by the presence of flocculent, electron-dense DAB reaction product, which adhered to intracellular organelles, particularly vesicles. Three examples per condition are illustrated (A–C and D–F). vGAT-immunoreactive structures formed synapses (black arrows) with the dendrites (d) and somata (s) of STN neurons. Vesicles associated with each synapse are also indicated (white arrows). In some cases, vGAT-immunoreactive structures did not form a synaptic connection in the ultrathin section that was examined (A). Although the dimensions of vGAT-immunoreactive structures, synaptic targets, and synapses were similar in control and dopamine-depleted animals, individual vGAT-immunoreactive axon terminals formed significantly more synapses in dopamine-depleted animals (A–G). Scale bar in A applies to each micrograph. *p < 0.05.