Despite recent clinical observations linking the zona incerta (ZI) to anxiety, little is known about whether and how the ZI processes anxiety. Here, we subject mice to anxious experiences and observe an increase in ZI c-fos–labeled neurons and single-cell calcium activity as well as an efficient effect of ZI infusion of diazepam, a classical anxiolytic drug. We further identify that somatostatin (SOM)–, calretinin (CR)–, and vesicular glutamate transporter-2 (Vglut2)–expressing cells display unique electrophysiological profiles; however, they similarly respond to anxiety-provoking stimuli and to diazepam. Optogenetic manipulations reveal that each of these ZI neuronal populations triggers specific anxiety-related behavioral phenotypes. Activation of SOM-expressing neurons induced anxiety, while photoactivation of CR-positive cells and photoinhibition of Vglut2-expressing neurons produce anxiolysis. Furthermore, activation of CR- and Vglut2-positive cells provokes rearing and jumps, respectively. Our findings provide the first experimental evidence that ZI subpopulations encode and modulate different components of anxiety.

Stimulation of zona incerta in rodent models has been shown to modulate behavioral reactions to noxious stimuli. Sensory changes observed in Parkinsonian patients with subthalamic deep brain stimulation suggest that this effect is translatable to humans. Here, we utilized the serendipitous placement of subthalamic deep brain stimulation leads in 6 + 5 Parkinsonian patients to directly investigate the effects of zona incerta stimulation on human pain perception. We found that stimulation at 20 Hz, the physiological firing frequency of zona incerta, reduces experimental heat pain by a modest but significant amount, achieving a 30% reduction in one fifth of implants. Stimulation at higher frequencies did not modulate heat pain. Modulation was selective for heat pain and was not observed for warmth perception or pressure pain. These findings provide a mechanistic explanation of sensory changes seen in subthalamic deep brain stimulation patients and identify zona incerta as a potential target for neuromodulation of pain.

Zona incerta (ZI) is a functionally mysterious subthalamic nucleus containing mostly inhibitory neurons. Here, we discover that GABAergic neurons in the rostral sector of ZI (ZIr) directly innervate excitatory but not inhibitory neurons in the dorsolateral and ventrolateral compartments of periaqueductal gray (PAG), which can drive flight and freezing behaviors respectively. Optogenetic activation of ZIr neurons or their projections to PAG reduces both sound-induced innate flight response and conditioned freezing response, while optogenetic suppression of these neurons enhances these defensive behaviors, likely through a mechanism of gain modulation. ZIr activity progressively increases during extinction of conditioned freezing response, and suppressing ZIr activity impairs the expression of fear extinction. Furthermore, ZIr is innervated by the medial prefrontal cortex (mPFC), and silencing mPFC prevents the increase of ZIr activity during extinction and the expression of fear extinction. Together, our results suggest that ZIr is engaged in modulating defense behaviors.

Pain and itch are intricately entangled at both circuitry and behavioral levels. Emerging evidence indicates that parvalbumin (PV)-expressing neurons in zona incerta (ZI) are critical for promoting nocifensive behaviors. However, the role of these neurons in itch modulation remains elusive. Herein, by combining FOS immunostaining, fiber photometry, and chemogenetic manipulation, we reveal that ZI PV neurons act as an endogenous negative diencephalic modulator for itch processing. Morphological data showed that both histamine and chloroquine stimuli induced FOS expression in ZI PV neurons. The activation of these neurons was further supported by the increased calcium signal upon scratching behavior evoked by acute itch. Behavioral data further indicated that chemogenetic activation of these neurons reduced scratching behaviors related to histaminergic and non-histaminergic acute itch. Similar neural activity and modulatory role of ZI PV neurons were seen in mice with chronic itch induced by atopic dermatitis. Together, our study provides direct evidence for the role of ZI PV neurons in regulating itch, and identifies a potential target for the remedy of chronic itch.

The zona incerta is a subthalamic nucleus made up mostly of GABAergic neurons. It has wide-ranging inputs and outputs and is believed to have many integrative functions that link sensory stimuli with motor responses to guide behavior. However, its role is not well established perhaps because few studies have measured the activity of zona incerta neurons in behaving animals under different conditions. To record the activity of zona incerta neurons during exploratory and cue-driven goal-directed behaviors, we used electrophysiology in head-fixed mice moving on a spherical treadmill and fiber photometry in freely moving mice. We found two groups of neurons based on their sensitivity to movement, with a minority of neurons responding to whisker stimuli. Furthermore, zona incerta GABAergic neurons robustly code the occurrence of exploratory and goal-directed movements, but not their direction. To understand the function of these activations, we performed genetically targeted lesions and optogenetic manipulations of zona incerta GABAergic neurons during exploratory and goal-directed behaviors. The results showed that the zona incerta has a role in modulating the movement associated with these behaviors, but this has little impact on overall performance. Zona incerta neurons distribute a broad corollary signal of movement occurrence to their diverse projection sites, which regulates behavior.

Energy deprivation triggers food seeking to ensure homeostatic consumption, but the neural coding of motivational vigor in food seeking during physical hunger remains unknown. Here, we report that ablation of dopamine (DA) neurons in zona incerta (ZI) but not ventral tegmental area potently impaired food seeking after fasting. ZI DA neurons and their projections to paraventricular thalamus (PVT) were quickly activated for food approach but inhibited during food consumption. Chemogenetic manipulation of ZI DA neurons bidirectionally regulated feeding motivation to control meal frequency but not meal size for food intake. Activation of ZI DA neurons promoted, but silencing of these neurons blocked, contextual memory associate with food reward. In addition, selective activation of ZI DA projections to PVT promoted food seeking for food consumption and transited positive-valence signals. Together, these findings reveal that ZI DA neurons encode motivational vigor in food seeking for food consumption through their projections to PVT.

Top-down projections convey a family of signals encoding previous experiences and current aims to the sensory neocortex, where they converge with external bottom-up information to enable perception and memory. Whereas top-down control has been attributed to excitatory pathways, the existence, connectivity, and information content of inhibitory top-down projections remain elusive. Here, we combine synaptic two-photon calcium imaging, circuit mapping, cortex-dependent learning, and chemogenetics in mice to identify GABAergic afferents from the subthalamic zona incerta as a major source of top-down input to the neocortex. Incertocortical transmission undergoes robust plasticity during learning that improves information transfer and mediates behavioral memory. Unlike excitatory pathways, incertocortical afferents form a disinhibitory circuit that encodes learned top-down relevance in a bidirectional manner where the rapid appearance of negative responses serves as the main driver of changes in stimulus representation. Our results therefore reveal the distinctive contribution of long-range (dis)inhibitory afferents to the computational flexibility of neocortical circuits.

The zona incerta (ZI) is involved in various functions and may serve as an integrative node of the circuits for global behavioral modulation. However, the long-range connectivity of different sectors in the mouse ZI has not been comprehensively mapped. Here, we obtained whole-brain images of the input and output connections via fluorescence micro-optical sectioning tomography and viral tracing. The principal regions in the input-output circuits of ZI GABAergic neurons were topologically organized. The 3D distribution of cortical inputs showed rostro-caudal correspondence with different ZI sectors, while the projection fibers from ZI sectors were longitudinally organized in the superior colliculus. Clustering results show that the medial and lateral ZI are two different major functional compartments, and they can be further divided into more subdomains based on projection and input connectivity. This study provides a comprehensive anatomical foundation for understanding how the ZI is involved in integrating different information, conveying motivational states, and modulating global behaviors.

The zona incerta (ZI) is a small gray matter region of the deep brain first identified in the 19th century, yet direct in vivo visualization and characterization has remained elusive. Noninvasive detection of the ZI and surrounding region could be critical to further our understanding of this widely connected but poorly understood deep brain region and could contribute to the development and optimization of neuromodulatory therapies. We demonstrate that high resolution (submillimetric) longitudinal (T1) relaxometry measurements at high magnetic field strength (7 T) can be used to delineate the ZI from surrounding white matter structures, specifically the fasciculus cerebellothalamicus, fields of Forel (fasciculus lenticularis, fasciculus thalamicus, and field H), and medial lemniscus. Using this approach, we successfully derived in vivo estimates of the size, shape, location, and tissue characteristics of substructures in the ZI region, confirming observations only previously possible through histological evaluation that this region is not just a space between structures but contains distinct morphological entities that should be considered separately. Our findings pave the way for increasingly detailed in vivo study and provide a structural foundation for precise functional and neuromodulatory investigation.

The zona incerta is a subthalamic nucleus proposed to link sensory stimuli with motor responses to guide behavior, but its functional role is not well established. Using mice of either sex, we studied the effect of manipulating zona incerta GABAergic cells on the expression of a signaled locomotor action, known as signaled active avoidance. We found that modulation of GABAergic zona incerta cells, but not of cells in the adjacent thalamic reticular nucleus (NRT), fully controls the expression of signaled active avoidance responses. Inhibition of zona incerta GABAergic cells drives active avoidance responses, while excitation of these cells blocks signaled active avoidance mainly by inhibiting cells in the midbrain pedunculopontine tegmental nucleus (PPT). The zona incerta regulates signaled locomotion in the midbrain.

The parafascicular nucleus (Pf) in medial thalamus is interconnected with prefrontal cortex and basal ganglia. Though much research has determined its importance in cognitive regulation of behaviour, its projections to regions in subthalamus remain less known. Such connections include those to zona incerta (ZI), located immediately dorsal to subthalamic nuclei (STN) regulating motor output, and whose role in a motor context is only beginning to be investigated. We thus examined circuits from parafascicular (Pf) thalamus to ZI, and its activity during locomotion and spontaneous behaviours in mice. We found that a distinct group of CaMKIIα-positive excitatory parafascicular neurons, separated from VGLUT2-positive excitatory neurons, project widely into ZI, more than adjacent STN. Our results from fibre photometry and decoding with general linear model (GLM) indicate that PF-ZI pathways do not specifically correlate with amount of locomotion or movement velocity, but instead show more specified activity during relative directional changes of movements observed in turning, sniffing behaviours. These results hint at the PF-ZI pathway having a distinct role in directing action specificity and have implications for subcortical bases in dimensional control of behaviours.

The ability to adjust defensive behavior is critical for animal survival in dynamic environments. However, neural circuits underlying the modulation of innate defensive behavior remain not well-understood. In particular, environmental threats are commonly associated with cues of multiple sensory modalities. It remains to be investigated how these modalities interact to shape defensive behavior. In this study, we report that auditory-induced defensive flight behavior can be facilitated by somatosensory input in mice. This cross-modality modulation of defensive behavior is mediated by the projection from the primary somatosensory cortex (SSp) to the ventral sector of zona incerta (ZIv). Parvalbumin (PV)-positive neurons in ZIv, receiving direct input from SSp, mediate the enhancement of the flight behavior via their projections to the medial posterior complex of thalamus (POm). Thus, defensive flight can be enhanced in a somatosensory context-dependent manner via recruiting PV neurons in ZIv, which may be important for increasing survival of prey animals.

The cuneiform nucleus (CnF) regulates locomotor activity, which is canonically viewed as being primarily involved in initiating locomotion and regulating speed. Recent research shows greater context dependency in the locomotor functions of this nucleus. Glutamatergic neurons, which contain vesicular glutamate transporter 2 (vGLUT2), regulate context-dependent locomotor speed in the CnF and play a role in defensive behavior. Here, we identify projections from the medial zona incerta (mZI) to CnF vGLUT2 neurons that promote exploratory behavior. Using fiber photometry recordings in male mice, we find that mZI gamma-aminobutyric acid (GABA) neurons increase activity during periods of exploration. Activation of mZI GABAergic neurons is associated with reduced spiking of CnF neurons. Additionally, activating both retrogradely labeled mZI-CnF GABAergic projection neurons and their terminals in the CnF increase exploratory behavior. Inhibiting CnF vGLUT2 neuronal activity also increases exploratory behavior. These findings provide evidence for the context-dependent dynamic regulation of CnF vGLUT2 neurons, with the mZI-CnF circuit shaping exploratory behavior.

The zona incerta (ZI) and ventral tegmental area (VTA) are brain areas that are both implicated in feeding behaviour. The ZI projects to the VTA, although it has not yet been investigated whether this projection regulates feeding. We experimentally (in)activated the ZI to VTA projection by using dual viral vector technology, and studied the effects on feeding microstructure, the willingness to work for food, general activity and body temperature. Activity of the ZI to VTA projection promotes feeding by facilitating action initiation towards food, as reflected in meal frequency and the willingness to work for food reward, without affecting general activity or directly modulating body temperature. We show for the first time that activity of the ZI to VTA projection promotes feeding, which improves the understanding of the neurobiology of feeding behaviour and body weight regulation.

The subthalamic nucleus (STN) and the zona incerta (ZI) are two major structures of the subthalamus. The STN has strong connections between the basal ganglia and related nuclei. The ZI has strong connections between brainstem reticular nuclei, sensory nuclei, and nonspecific thalamic nuclei. Both the STN and ZI receive heavy projections from a subgroup of layer V neurons in the cerebral cortex. The major goal of this study was to investigate the following two questions about the cortico-subthalamic projections using the lentivirus anterograde tracing method in the rat: 1) whether cortical projections to the STN and ZI have independent functional organizations or a global organization encompassing the entire subthalamus as a whole; and 2) how the cortical functional zones are represented in the subthalamus. This study revealed that the subthalamus receives heavy projections from the motor and sensory cortices, that the cortico-subthalamic projections have a large-scale functional organization that encompasses both the STN and two subdivisions of the ZI, and that the group of cortical axons that originate from a particular area of the cortex sequentially innervate and form separate terminal fields in the STN and ZI. The terminal zones formed by different cortical functional areas have highly overlapped and fuzzy borders, as do the somatotopic representations of the sensorimotor cortex in the subthalamus. The present study suggests that the layer V neurons in the wide areas of the sensorimotor cortex simultaneously control STN and ZI neurons. Together with other known afferent and efferent connections, possible new functionality of the STN and ZI is discussed.

No abstract available

Mutations in the aristaless-related homeobox (ARX) gene result in a spectrum of structural and functional nervous system disorders including lissencephaly, movement disorders, intellectual disabilities, and epilepsy. Some patients also have symptoms indicating hypothalamic dysfunction, but little is known about the role of ARX in diencephalic development. To begin evaluating diencephalic defects, we examined the expression of a panel of known genes and gene products that label specific diencephalic nuclei in 2 different Arx mutant mouse lines at E18.5. Male mice engineered to have a polyalanine expansion mutation (Arx) revealed no expression differences in any diencephalic nucleus when compared with wild-type littermates. In contrast, mice null for Arx (Arx) lost expression of specific markers of the thalamic reticular nucleus and zona incerta (ZI) while retaining expression in other thalamic nuclei and in the hypothalamus. Tyrosine hydroxylase, a marker of the dopaminergic A13 subnucleus of ZI, was among those lost, suggesting a requirement for Arx in normal thalamic reticular nucleus and ZI development and, specifically, for A13 dopaminergic fate. Because the ZI and A13 regions make connections to several hypothalamic nuclei, such misspecification may contribute to the "hypothalamic dysfunction" observed in some patients.

Parkinson's disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantia nigra and leads to progressive motor dysfunction. While studies have focused on the basal ganglia network, recent evidence suggests neuronal systems outside the basal ganglia are also related to PD pathogenesis. The zona incerta (ZI) is a predominantly inhibitory subthalamic region for global behavioral modulation. This study investigates the role of GABAergic neurons in the ZI in a mouse model of 6-hydroxydopamine (6-OHDA)-induced PD. First, we found a decrease in GABA-positive neurons in the ZI, and then the mice used chemogenetic/optogenetic stimulation to activate or inhibit GABAergic neurons. The motor performance of PD mice was significantly improved by chemogenetic/optogenetic activation of GABAergic neurons, and repeated chemogenetic activation of ZI GABAergic neurons increased the dopamine content in the striatum. Our work identifies the role of ZI GABAergic neurons in regulating motor behaviors in 6-OHDA-lesioned PD model mice.

Central amygdala neurons expressing protein kinase C-delta (CeA-PKCδ) are sensitized following nerve injury and promote pain-related responses in mice. The neural circuits underlying modulation of pain-related behaviors by CeA-PKCδ neurons, however, remain unknown. In this study, we identified a neural circuit that originates in CeA-PKCδ neurons and terminates in the ventral region of the zona incerta (ZI), a subthalamic structure previously linked to pain processing. Behavioral experiments show that chemogenetic inhibition of GABAergic ZI neurons induced bilateral hypersensitivity in uninjured mice and contralateral hypersensitivity after nerve injury. In contrast, chemogenetic activation of GABAergic ZI neurons reversed nerve injury-induced hypersensitivity. Optogenetic manipulations of CeA-PKCδ axonal terminals in the ZI further showed that inhibition of this pathway reduces nerve injury-induced hypersensitivity whereas activation of the pathway produces hypersensitivity in the uninjured paws. Altogether, our results identify a novel nociceptive inhibitory efferent pathway from CeA-PKCδ neurons to the ZI that bidirectionally modulates pain-related behaviors in mice.

Defensive behaviors induced by innate fear or Pavlovian fear conditioning are crucial for animals to avoid threats and ensure survival. The zona incerta (ZI) has been demonstrated to play important roles in fear learning and fear memory, as well as modulating auditory-induced innate defensive behavior. However, whether the neuronal subtypes in the ZI and specific circuits can mediate the innate fear response is largely unknown. Here, we found that somatostatin (SST)-positive neurons in the rostral ZI of mice were activated by a visual innate fear stimulus. Optogenetic inhibition of SST-positive neurons in the rostral ZI resulted in reduced flight responses to an overhead looming stimulus. Optogenetic activation of SST-positive neurons in the rostral ZI induced fear-like defensive behavior including increased immobility and bradycardia. In addition, we demonstrated that manipulation of the GABAergic projections from SST-positive neurons in the rostral ZI to the downstream nucleus reuniens (Re) mediated fear-like defensive behavior. Retrograde trans-synaptic tracing also revealed looming stimulus-activated neurons in the superior colliculus (SC) that projected to the Re-projecting SST-positive neurons in the rostral ZI (SC-ZIrSST-Re pathway). Together, our study elucidates the function of SST-positive neurons in the rostral ZI and the SC-ZIrSST-Re tri-synaptic circuit in mediating the innate fear response.