Nigral inhibition of GABAergic neurons in mouse superior colliculus

Abstract

The current dominant concept for the control of saccadic eye movements by the basal ganglia is that release from tonic GABAergic inhibition by the substantia nigra pars reticulata (SNr) triggers burst firings of intermediate gray layer (SGI) neurons in the superior colliculus (SC) to allow saccade initiation. This hypothesis is based on the assumption that SNr cells inhibit excitatory projection neurons in the SGI. Here we show that nigrotectal fibers are connected to local GABAergic neurons in the SGI with a similar frequency to non-GABAergic neurons. This was accomplished by applying neuroanatomical tracing and slice electrophysiological experiments in GAD67-green fluorescent protein (GFP) knock-in mice, in which GABAergic neurons specifically express GFP. We also found that GABA(A), but not GABA(B), receptors subserve nigrotectal transmission. The present results revealed a novel aspect on the role of the basal ganglia in the control of saccades, e.g., the SNr not only regulates burst initiation but also modulates the spatiotemporal properties of premotor neurons via connections to local GABAergic neurons in the SC.

Figure 1.

Nigral fibers make synaptic contacts with SGI GABAergic neurons. A, A photomicrograph shows an example of the BDA injection site in the SNr in a GAD67–GFP knock-in mouse. B, Distribution of labeled SNr fibers in the SC ipsilateral to the BDA-injected SNr. The rectangle area in B is enlarged in C. C, A high-magnification photomicrograph shows BDA-labeled buttons encircling an SGI neuron. D–I, Double-fluorescence histochemistry for GFP and BDA in a different mouse. Low-magnification photographs (D–F) were taken with a fluorescence microscope, whereas high-magnification photographs (G–I) were taken with a confocal microscope. The rectangles in D–F are enlarged, respectively, in G–I. BDA was visualized with red fluorescence of Alexa 594. BDA-labeled axonal components are in close apposition to GFP-positive neuronal profiles (I, arrowheads). SO, Optic nerve layer.

Figure 2.

Nigrotectal tracts and the modified slice preparation method. A, Four coronal sections (50 μm thickness) are arranged from rostral on the left to caudal on the right. Section 1 represents the center of the BDA injection into the SNr, which approximately corresponds to bregma −3.0 mm section according to the mouse brain atlas by Paxinos and Franklin (2001). B, Four sagittal sections (100 μm thickness) are arranged from lateral on the left to medial on the right. Section 1 represents the center of BDA injection into the SNr, which approximately corresponds to lateral 1.65 mm based on the atlas. C, Schematic showing the methods to make modified slice preparations. a, First the brain was cut in an arc parallel to the rostrocaudal SNr–SC fibers with a bent blade. b, When the brain was placed on a stage with the cut surface facing downward, gravity pushed the originally curved rostrocaudal SNr–SC fibers into a plane. c, Thus, the SNr–SC fibers became nearly straight and were considerably preserved in the slices.

Figure 3.

Characteristics of nigral inhibition in SGI GABAergic neurons. A, Schematic of configuration for stimulating and recording electrodes. Five cathodal concentric bipolar electrodes with a tip distance of 300 μm were placed on the SNr. B, Representative responses evoked by nigral stimulation in SGI GABAergic neurons in the presence of CNQX (10 μm) and APV (50 μm). Membrane potential was held at 0 mV with a Cs-based intracellular pipette solution. In this neuron, only stimulation site 1 evoked IPSCs. Five traces are superimposed in each figure. C, Distributions of latency of the IPSCs evoked by SNr stimulation in GABAergic (filled rectangles) and non-GABAergic (open rectangles) SGI neurons. D, Evoked IPSCs were recorded at different holding potentials. E, Plots of IPSCs amplitudes versus holding potentials for seven neurons. F, Plots of means of normalized IPSCs amplitudes (amplitude of IPSCs at each holding potential was divided by that at 5 mV) versus holding potentials reveal that the IPSCs reversed at −51.8 ± 7.2 mV, which is close to the E_Cl⁻ (−51.4 mV) under the present experimental conditions. G, The SNr stimulation-evoked IPSCs were reversibly blocked by application of the GABA_A receptor antagonist gabazine (10 μm). H, Repetitive stimulation (20 pulses at 50 Hz)-evoked IPSCs were not affected by CGP52432 (3 μm) application (top traces). Addition of gabazine abolished the responses (bottom trace). In this experiment, membrane potential was held at −55 mV with K-based electrolyte in the presence of CNQX and APV. Stimulating electrodes were placed just ventral to the SGI.

Figure 4.

Location and morphology of SNr-recipient GABAergic neurons in the SGI. A, Left, Schematic of locations of recorded area in the SGI and stimulating electrode. Rectangle was enlarged in the right. Right, Each circle represents the location of IPSC-positive GABAergic neurons (n = 18). Numbers in the circles indicate the stimulation site within the SNr, the stimulation of which evoked IPSCs with a minimum current intensity. Morphologies of 7 of 18 GABAergic neurons (a–g) are drawn in B. B, Morphologies of biocytin-filled GABAergic neurons. Soma and dendrites are drawn in black, and axons are in red. High-magnification photomicrographs in a and f are obtained from the rectangle areas in the drawings, respectively. The labeled dendrites (arrowheads) and labeled terminals are located in the same focus level. SO, Optic nerve layer.

Figure 5.

Schematic of inhibitory circuit including the SC and SNr. GABAergic neurons in the SNr innervate both GABAergic and non-GABAergic neurons in the SGI. The SNr-recipient GABAergic neurons may mainly inhibit nearby non-GABAergic premotor neurons, which would project to the brainstem gaze centers.