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, 39 (23), 4448-4460

Glutamatergic Innervation Onto Striatal Neurons Potentiates GABAergic Synaptic Output

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Glutamatergic Innervation Onto Striatal Neurons Potentiates GABAergic Synaptic Output

Foteini Paraskevopoulou et al. J Neurosci.

Abstract

Striatal output pathways are known to play a crucial role in the control of movement. One possible component for shaping the synaptic output of striatal neuron is the glutamatergic input that originates from cortex and thalamus. Although reports focusing on quantifying glutamatergic-induced morphological changes in striatum exist, the role of glutamatergic input in regulating striatal function remains poorly understood. Using primary neurons from newborn mice of either sex in a reduced two-neuron microcircuit culture system, we examined whether glutamatergic input modulates the output of striatal neurons. We found that glutamatergic input enhanced striatal inhibition in vitro With a glutamatergic partner from either cortex or thalamus, we attributed this potentiation to an increase in the size of quantal IPSC, suggesting a strengthening of the postsynaptic response to GABAergic signaling. Additionally, a differential effect of cortical and thalamic innervation onto striatal GABAergic neurons output was revealed. We observed that cortical, but not thalamic input, enhanced the number of releasable GABAergic synaptic vesicles and morphological synapses. Importantly, these alterations were reverted by blockade of neuronal activity and glutamate receptors, as well as disruption of BDNF-TrkB signaling. Together, our data indicate, for first time, that GABAergic synapse formation in corticostriatal pairs depends on two parallel, but potentially intersecting, signaling pathways that involve glutamate receptor activation in striatal neurons, as well as BDNF signaling. Understanding how cortical and thalamic inputs refine striatal output will pave the way toward dissecting basal ganglia activity in both physiological and pathological conditions.SIGNIFICANCE STATEMENT Striatal GABAergic microcircuits are critical for motor function. However, the mechanisms controlling striatal output, particularly at the level of synaptic strength, are unclear. Using two-neuron culture system, we quantified the synaptic output of individual striatal GABAergic neurons paired with a glutamatergic partner and studied the influence of the excitatory connections that are known to be interregionally formed in vivo We found that glutamatergic input potentiated striatal inhibitory output, potentially involving an increased feedback and/or feedforward inhibition. Moreover, distinct components of glutamatergic innervation, such as firing activity or release of neurotrophic factors were shown to be required for the glutamatergic-induced phenotype. Investigation, therefore, of two-neuron in vitro microcircuits could be a powerful tool to explore synaptic mechanisms or disease pathophysiology.

Keywords: BDNF; GABAergic neuron; cell culture; excitatory input; paired recordings; striatum.

Figures

Figure 1.
Figure 1.
Striatal GABAergic output is modulated by glutamatergic input. A–C, Schematic diagram illustrating autaptic and heterosynaptic connections in striatal (dark blue represents GABAergic only), CS (pink represents glu-GABA), and TS (green represents glu-GABA) pairs. D–Q, Functional analysis of striatal autapses (light blue traces and dots), striatal pairs (blue traces and dark blue dots), CS pairs (pink traces and dots), and TS pairs (green traces and dots). D–F, Representative traces of GABAergic response to paired pulse stimulation with 50 ms interstimulus interval and to a 5 s pulse of 500 mm hypertonic sucrose solution (dark represents autaptic; light represents heterosynaptic). G–J, Scatter plots showing total evoked IPSC amplitudes (G), RRP size (H), Pvr% (I), and PPR (J). K–M, Representative traces showing miniature postsynaptic current activity (dark represents autaptic; light represents heterosynaptic). N–Q, Scatter plots showing mean mIPSC amplitudes (N), charge (O), frequency (P), and RRP vesicle number (Q). R, Representative traces of glutamatergic response to paired pulse stimulation with 25 ms interstimulus interval (dark represents autaptic; light represents heterosynaptic; pink represents CS pairs; green represents TS pairs). S, T, Scatter plots showing total evoked EPSC amplitudes (S) and PPR (T). U, V, Bars graphs showing the mean PSC amplitude of autaptic and heterosynaptic responses of glutamatergic (U) and GABAergic neurons in homotypic or heterotypic pairs (V). Data are mean ± SEM. ns refers to not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. See also Figure 1-1.
Figure 2.
Figure 2.
Cortical input increases the number of GABAergic synapses in striatal neurons. A, B, Morphological analysis of striatal autapses (light blue dots), striatal pairs (dark blue dots), CS pairs (pink dots), and TS pairs (green dots). A, Representative images of neuronal morphology showing immunoreactivity for MAP2, VGAT, and VGLUT1 (cortical synapses) or VGLUT2 (thalamic synapses). B, C, Scatter plots showing the number of VGAT synapses per neuron (B), the number of VGLUT1 or VGLUT2 synapses per neuron (C), and mean membrane capacitance measurements as obtained from the membrane test (D). Data are mean ± SEM. ns refers to not significant, **p ≤ 0.01, ****p ≤ 0.0001. See also Figure 2-1).
Figure 3.
Figure 3.
Activity modulates GABAergic synapse output in CS pairs. A–F, Functional analysis of striatal pairs (blue color scale dots), CS pairs (red color scale dots). Scatter plots showing total evoked IPSC amplitudes (A), RRP size (B), Pvr% (C), mIPSC amplitudes (D), RRP vesicle number (E), and mean membrane capacitance measurements as obtained from the membrane test (F). Data are mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. See also Figure 3-1).
Figure 4.
Figure 4.
Exogenous BDNF promotes growth and synapse formation in striatal autaptic neurons. A–D, Morphological analysis of striatal autapses. Light blue represents control. Yellow represents BDNF-treated. Purple represents Trk-antagonist-treated. Green represents BDNF and Trk-antagonist-treated. A, Representative images of neuronal morphology showing immunoreactivity for MAP2 and VGAT. B–D, Scatter plots showing neuronal soma area (B), number of VGAT synapses per neuron (C), and mean total dendritic length (D). E–L, Bar graphs showing evoked IPSC amplitudes (E), RRP size (F), PPR (G), mean mIPSC amplitudes (H), frequency (J), RRP vesicles number (I), normalized response amplitude to 5 μm GABA (K), and membrane capacitance (L). Data are mean ± SEM. *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001. See also Figure 4-1.
Figure 5.
Figure 5.
BDNF release modulates GABAergic synapse output in CS pairs. A, Bar graph showing real-time RT-PCR analysis for mRNA expression of Bdnf gene in striatal and cortical neuronal mass cultures. B–H, Functional analysis of striatal pairs (dark blue represents untreated; purple represents Trk-antagonist-treated), CS pairs (pink represents untreated; brown represents Trk-antagonist-treated). Scatter plots showing mean evoked IPSC amplitudes (B), RRP size (C), PPR (D), mIPSC amplitudes (E), mIPSC frequency (F), RRP vesicle number (G), and mean membrane capacitance measurements as obtained from the membrane test (H). J, K, Representative images of neuronal morphology showing immunoreactivity for MAP2, VGAT, and VGLUT1. Scatter plots showing the number of VGAT synapses per neuron (I), the number of VGLUT1 synapses per neuron (K). Data are mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. See also Figure 5-1.

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