The Zona Incerta Regulates Communication between the Superior Colliculus and the Posteromedial Thalamus: Implications for Thalamic Interactions with the Dorsolateral Striatum

Abstract

There is uncertainty concerning the circuit connections by which the superior colliculus interacts with the basal ganglia. To address this issue, anterograde and retrograde tracers were placed, respectively, into the superior colliculus and globus pallidus of Sprague-Dawley rats. In this two-tracer experiment, the projections from the superior colliculus terminated densely in the ventral zona incerta (ZIv), but did not overlap the labeled neurons observed in the subthalamic nucleus. In cases in which anterograde and retrograde tracers were placed, respectively, in sensory-responsive sites in the superior colliculus and posteromedial (POm) thalamus, the labeled projections from superior colliculus innervated the ZIv regions that contained the labeled neurons that project to POm. We also confirmed this colliculo-incertal-POm pathway by depositing a mixture of retrograde and anterograde tracers at focal sites in ZIv to reveal retrogradely labeled neurons in superior colliculus and anterogradely labeled terminals in POm. When combined with retrograde tracer injections in POm, immunohistochemical processing proved that most ZIv projections to POm are GABAergic. Consistent with these findings, direct stimulation of superior colliculus evoked neuronal excitation in ZIv and caused inhibition of spontaneous activity in POm. Collectively, these results indicate that superior colliculus can activate the inhibitory projections from ZIv to the POm. This is significant because it suggests that the superior colliculus could suppress the interactions between POm and the dorsolateral striatum, presumably to halt ongoing behaviors so that more adaptive motor actions are selected in response to unexpected sensory events.

Significance statement: By demonstrating that the zona incerta regulates communication between the superior colliculus and the posteromedial thalamus, we have uncovered a circuit that partly explains the behavioral changes that occur in response to unexpected sensory stimuli. Furthermore, this circuit could explain why deep brain stimulation of the zona incerta is beneficial to patients who suffer from Parkinson's disease.

Keywords: neuronal tracing; sensorimotor; somatosensory; thalamostriatal; vision.

Figure 1.

Superior colliculus projects predominantly to ZIv, not to the STN. A, PSTH showing multiunit responses to whisker deflections recorded by a BDA-filled pipette in the lateral part of the superior colliculus. Binwidths, 10 ms. B, Deposit of BDA at the site of the neural responses shown in A. C, Section through the globus pallidus (GP) and striatum (St) processed for CO. Note the necrosis at the tracer injection site (arrow) in GP. C′, Adjacent section through GP shows the widest extent of the FG deposit in GP. D, CO-processed section through ZI and STN. D′, D″, Adjacent section viewed with conventional and fluorescent illumination indicates that BDA-labeled terminals and cell bodies in ZIv are separate from the FG-labeled cells in STN. Scale bars: B, C, C′, 500 μm; D, D′, D″, 250 μm.

Figure 2.

Light-responsive region in superior colliculus projects to ZIv region that innervates whisker-responsive region in POm. A, PSTH shows whisker-induced multiunit responses recorded in POm by an FG-filled pipette. This site did not respond to a 50 ms light flashed in both eyes. B, Section through POm indicates necrosis at the FG deposit marking the whisker-responsive site in A. B′, Same section under fluorescent illumination shows extent of the FG deposit. C, Multiunit responses to visual light flash recorded in the medial superior colliculus by a BDA-filled pipette. Neurons at this site responded weakly to whisker stimulation. D, CO-processed section of the recording site (arrow) in the medial superior colliculus. D′, Adjacent section shows the BDA deposit marking the light-responsive site in C. E, CO-processed section through ZI and STN. Inset indicates region displayed in F. F, Adjacent section shows BDA-filled cells and terminals in upper part of ZIv. Inset indicates the region displayed in G. G, Fluorescent illumination at high-magnification reveals BDA-labeled terminals intermingled with FG-labeled neurons in ZIv. Arrows indicate same blood vessels in F and G. Scale bars: B, B′, D, D′, E, 500 μm; F, 250 μm; G, 100 μm.

Figure 3.

Projections from the superior colliculus terminate in ZIv regions that project to POm. A, Whisker deflections at 2 Hz evoke POm multiunit responses recorded by a FG-filled pipette. B, Section processed for CO shows POm. B′, B″, Adjacent section viewed conventionally and with fluorescent microscopy depicts the FG deposit in POm. C, Multiunit response to whisker deflections recorded in superior colliculus of the same rat. D, D′, E, Adjacent sections processed (D) and unprocessed (D′, E) for CO depict the neuronal recording site and FR deposit in the intermediate layers of the superior colliculus. D′, Inset indicates the region shown in E. F, CO-processed section through ZI and STN. Inset indicates the region selected for confocal microscopy in subsequent panels. G, G′, G″, Confocal images illustrate overlap of FG-labeled neurons and FR-labeled terminals and neurons in ZIv. Blue asterisk indicates the same blood vessel in F. Scale bars: B, D, F, 500 μm; E, 250 μm; G, 50 μm. cp, Cerebral peduncle; fr, fasciculus retroflexus; ml, medial lemniscus; mt, mammilothalamic tract.

Figure 4.

Collicular projections overlap the ZI neurons that project to POm. A–C, Reconstructed plots of FG-filled neurons (gold) and FR-labeled terminal varicosities (red) in ZI. Numbers indicate distance from bregma in millimeters. A′–C′, Overlap analysis of FG-labeled neurons and FR-labeled terminals in ZI, using 50 μm² bins. Each panel corresponds to the plotted reconstruction shown directly above. For the overlap analysis, gold bins contain at least one FG-labeled neuron, red bins contain at least two FR-labeled terminals, and white bins contain at least one FG-labeled neuron and two FR-labeled terminals. Percentages indicate proportion of white bins. Scale bar, 1 mm. cp, Cerebral peduncle; ml, medial lemniscus; mt, mammilothalamic tract; ot, optic tract.

Figure 5.

Normalized distributions of labeling across the subthalamic subdivisions. A, Distribution of plotted terminals produced by anterograde tracer injections in whisker-responsive sites of the superior colliculus. B, Distribution of plotted cells produced by retrograde tracer injections in whisker responsive-sites in POm. C, Distribution of terminal-soma overlap based on the proportion of 50 μm² bins that contained at least one labeled cell and two labeled terminals.

Figure 6.

Afferent and efferent connectivity of a visually responsive region in ZIv. A, PSTH shows neural responses in lateral ZIv to a 50 ms visual light flash recorded by a pipette filled with both FG and BDA. B, C, BDA deposit at the light-sensitive site recorded in A. D, CO-processed section through the superior colliculus. E, F, Plotted reconstruction and photomicrograph of BDA-labeled neurons in superior colliculus; rectangle indicates region in F. G, CO-processed section through POm and VPM. H, I, Plotted reconstruction and photomicrograph of BDA-labeled terminal varicosities in POm; rectangle indicates the region in I. Scale bars: B, D, G, 500 μm; F, 250 μm; C, 100 μm; I, 50 μm. PAG, Periaqueductal gray; VPM, ventroposteromedial; VPL, ventroposterolateral.

Figure 7.

Afferent and efferent connections of a whisker-responsive region in ZIv. A, PSTH shows neural responses in ZIv recorded by a FG/BDA-filled pipette during whisker deflections administered at 2, 5, and 8 Hz. B, C, BDA deposit at the site of the whisker-responses recorded in A. Rectangles in B indicate regions in C and D. D, E, Low- and high-power views of BDA-labeled axonal terminals in POm. D, Inset indicates region in E. F, Thionin-stained section through the superior colliculus. F′, Adjacent section showing the laminar location and spatial extent of BDA-labeled neurons in the superior colliculus. Scale bars: B, 1 mm; D, F, 500 μm; C, 250 μm; E, 100 μm.

Figure 8.

Projections from ZIv to whisker-responsive sites in POm are GABAergic. A, Confocal image of retrogradely labeled neurons in ZIv after placing FG into a whisker-responsive site in POm. A′, Confocal image of the same stack processed for GABA using an antibody for GAD 65/67. A″, Overlap of FG and GAD fluorescence. White arrows indicate the strongest double-labeled neurons. Scale bar, 50 μm.

Figure 9.

Optical stimulation of superior colliculus evokes neuronal discharges in ZI. A, PSTHs display multiunit responses in the superior colliculus (top) recorded simultaneously with single neuron discharges in ZIv (bottom) during whisker deflections and optical stimulation (473 nm) of ChR2-expressing neurons in the colliculus. Mean waveform scale: 1 ms, 100 μV. B, B′, Adjacent sections illustrate the optrode location in the superior colliculus with respect to the laminar organization defined by CO processing and the expression of ChR2 produced by injections of AAV1.CamKIIa.hChR2(H134R)-eYFP.WPRE.hGH (Penn Vector Core). C, D, Low- and high-power views of the neuronal recording site in ZIv (arrow). Inset indicates the region depicted in D.

Figure 10.

Electrical stimulation in superior colliculus evokes inhibition of spontaneous activity in POm. A, PSTH from a representative POm recording shows the decrease in spontaneous discharges immediately after the superior colliculus is stimulated by a train of five 1 ms pulses administered at time 0 (arrow). Dashed lines indicate 99% confidence limits. Mean waveform scales: 1 ms, 100 μV. PSTH: 50 trials, 5 ms binwidths. B, C, Arrows indicate lesions that mark the recording and stimulation sites in POm and superior colliculus.

Figure 11.

Representative examples and mean responses of POm neurons to electrical stimulation of the superior colliculus. A, B, PSTHs indicate the changes in activity of two neurons in POm that displayed prolonged inhibition (A) or oscillations (B) in response to electrical stimulation of the superior colliculus (arrows). Dashed lines indicate 99% confidence limits. Mean waveform scales: 1 ms, 100 μV. C, Mean normalized response of 24 POm neurons. Individual neuronal activity was expressed as a proportion of the mean spontaneous rate before collicular activation, and then the sum of these normalized responses was averaged. Binwidths, 5 ms.

Figure 12.

Circuit connections by which the superior colliculus exerts opposing influences on Pf and POm. Superior colliculus activates Pf while simultaneously activating inhibitory projections from ZI to POm. Incertal-mediated inhibition of the POm projections to the dorsolateral striatum could disrupt well learned behavioral sequences that comprise sensorimotor habits.