Coupled Activation of the Hyperdirect and Cerebellothalamic Pathways with Zona Incerta Deep Brain Stimulation.

BACKGROUND: Deep brain stimulation (DBS) of the subthalamic nucleus (STN) or ventral intermediate nucleus (VIM) are established targets for the treatment of Parkinson's disease (PD) or essential tremor (ET), respectively. However, DBS of the zona incerta (ZI) can be effective for both disorders. VIM DBS is assumed to achieve its therapeutic effect via activation of the cerebellothalamic (CBT) pathway, whereas the activation of the hyperdirect (HD) pathway likely plays a role in the mechanisms of STN DBS. Interestingly, HD pathway axons also emit collaterals to the ZI and red nucleus (RN) and the CBT pathway courses nearby to the ZI. OBJECTIVE: The aim was to examine the ability of ZI DBS to mutually activate the HD and CBT pathways in a detailed computational model of human DBS. METHODS: We extended a previous model of the human HD pathway to incorporate axon collaterals to the ZI and RN. The anatomical framework of the model system also included representations of the CBT pathway and internal capsule (IC) fibers of passage. We then performed detailed biophysical simulations to quantify DBS activation of the HD, CBT, and IC pathways with electrodes located in either the STN or ZI. RESULTS: STN DBS and ZI DBS both robustly activated the HD pathway. However, STN DBS was limited by IC activation at higher stimulus amplitudes. Alternatively, ZI DBS avoided IC activation while simultaneously activating the HD and CBT pathways. CONCLUSIONS: From both neuroanatomical and biophysical perspectives, ZI DBS represents an advantageous target for coupled activation of the HD and CBT pathways. © 2024 International Parkinson and Movement Disorder Society.

Inhibitory medial zona incerta pathway drives exploratory behavior by inhibiting glutamatergic cuneiform neurons.

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.

Low-frequency stimulation in the zona incerta attenuates seizure via driving GABAergic neuronal activity.

BACKGROUND: Managing refractory epilepsy presents a significant a substantial clinical challenge. Deep brain stimulation (DBS) has emerged as a promising avenue for addressing refractory epilepsy. However, the optimal stimulation targets and effective parameters of DBS to reduce seizures remian unidentified. OBJECTIVES: This study endeavors to scrutinize the therapeutic potential of DBS within the zona incerta (ZI) across diverse seizure models and elucidate the associated underlying mechanisms. METHODS: We evaluated the therapeutic potential of DBS with different frequencies in the ZI on kainic acid (KA)-induced TLE model or M1-cortical seizures model, pilocarpine-induced M1-cortical seizure models, and KA-induced epilepsy model. Further, employing calcium fiber photometry combined with cell-specific ablation, we sought to clarified the causal role of ZI GABAergic neurons in mediating the therapeutic effects of DBS. RESULTS: Our findings reveal that DBS in the ZI alleviated the severity of seizure activities in the KA-induced TLE model. Meanwhile, DBS attenuated seizure activities in KA- or pilocarpine-induced M1-cortical seizure model. In addition, DBS exerts a mitigating influence on KA induced epilepsy model. DBS in the ZI showed anti-seizure effects at low frequency spectrum, with 5 Hz exhibiting optimal efficacy. The low-frequency DBS significantly increased the calcium activities of ZI GABAergic neurons. Furthermore, selective ablation of ZI GABAergic neurons with taCasp3 blocked the anti-seizure effect of low-frequency DBS, indicating the anti-seizure effect of DBS is mediated by the activation of ZI GABAergic neurons. CONCLUSION: Our results demonstrate that low-frequency DBS in the ZI attenuates seizure via driving GABAergic neuronal activity. This suggests that the ZI represents a potential DBS target for treating both hippocampal and cortical seizure through the activation of GABAergic neurons, thereby holding therapeutic significance for seizure treatment.

Glutamatergic Projection from the Ventral Tegmental Area to the Zona Incerta Regulates Fear Response.

Innate defensive behavior is important for animal survival. The Vglut2+ neurons in the ventral tegmental area (VTA) have been demonstrated to play important roles in innate defensive behaviors, but the neural circuit mechanism is still unclear. Here, we find that VTA - zona incerta (ZI) glutamatergic projection is involved in regulating innate fear responses. Combining calcium signal recording and chemogentics, we find that VTA-Vglut2+ neurons respond to foot shock stimulus. Inhibition of VTA-Vglut2+ neurons reduces foot shock-evoked freezing, while chemogentic activation of these neurons results in an enhanced fear response. Using viral tracing and immunofluorescence, we show that VTA - Vglut2+ neurons send direct excitatory outputs to the ZI. Moreover, we find that the activity of VTAVglut2 - ZI projection is pivotal in modulating fear response. Together, our study reveals a new VTA - ZI glutamatergic circuit in mediating innate fear response and provides a potential target for treating post-traumatic stress disorder.

Disentangling the identity of the zona incerta: a review of the known connections and latest implications.

The zona incerta (ZI) is a subthalamic region composed by loosely packed neurochemically mixed neurons, juxtaposed to the main ascending and descending bundles. The extreme neurochemical diversity that characterizes this area, together with the diffuseness of its connections with the entire neuraxis and its hard-to-reach positioning in the brain caused the ZI to keep its halo of mystery for over a century. However, in the last decades, a rich albeit fragmentary body of knowledge regarding both the incertal anatomical connections and functional implications has been built mostly based on rodent studies and its lack of cohesion makes difficult to depict an integrated, exhaustive picture regarding the ZI and its roles. This review aims to provide a unified resource that summarizes the current knowledge regarding the anatomical profile of interactions of the ZI in rodents and non-human primates and the functional significance of its connections, highlighting the aspects still unbeknown to research.

Zona incerta distributes a broad movement signal that modulates behavior.

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.

Zona incerta dopamine neurons encode motivational vigor in food seeking.

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.

Long-Term Follow-Up of Unilateral Deep Brain Stimulation Targeting the Caudal Zona Incerta in 13 Patients with Parkinsonian Tremor.

INTRODUCTION: Deep brain stimulation (DBS) is an established treatment for Parkinson's disease (PD) and other movement disorders. The ventral intermediate nucleus of the thalamus is considered as the target of choice for tremor disorders, including tremor-dominant PD not suitable for DBS in the subthalamic nucleus (STN). In the last decade, several studies have shown promising results on tremor from DBS in the posterior subthalamic area (PSA), including the caudal zona incerta (cZi) located posteromedial to the STN. The aim of this study was to evaluate the long-term effect of unilateral cZi/PSA-DBS in patients with tremor-dominant PD. METHODS: Thirteen patients with PD with medically refractory tremor were included. The patients were evaluated using the motor part of the Unified Parkinson Disease Rating Scale (UPDRS) off/on medication before surgery and off/on medication and stimulation 1-2 years (short-term) after surgery and at a minimum of 3 years after surgery (long-term). RESULTS: At short-term follow-up, DBS improved contralateral tremor by 88% in the off-medication state. This improvement persisted after a mean of 62 months. Contralateral bradykinesia was improved by 40% at short-term and 20% at long-term follow-up, and the total UPDRS-III by 33% at short-term and by 22% at long-term follow-up with stimulation alone. CONCLUSIONS: Unilateral cZi/PSA-DBS seems to remain an effective treatment for patients with severe Parkinsonian tremor several years after surgery. There was also a modest improvement on bradykinesia.

Distinct circuits in anterior cingulate cortex encode safety assessment and mediate flexibility of fear reactions.

Safety assessment and threat evaluation are crucial for animals to live and survive in the wilderness. However, neural circuits underlying safety assessment and their transformation to mediate flexibility of fear-induced defensive behaviors remain largely unknown. Here, we report that distinct neuronal populations in mouse anterior cingulate cortex (ACC) encode safety status by selectively responding under different contexts of auditory threats, with one preferably activated when an animal staysing in a self-deemed safe zone and another specifically activated in more dangerous environmental settings that led to escape behavior. The safety-responding neurons preferentially target the zona incerta (ZI), which suppresses the superior colliculus (SC) via its GABAergic projection, while the danger-responding neurons preferentially target and excite SC. These distinct corticofugal pathways antagonistically modulate SC responses to threat, resulting in context-dependent expression of fear reactions. Thus, ACC serves as a critical node to encode safety/danger assessment and mediate behavioral flexibility through differential top-down circuits.

[Activation of GABAergic neurons in the zona incerta accelerates anesthesia induction with sevoflurane and propofol without affecting anesthesia maintenance or awakening in mice].

OBJECTIVE: To explore the regulatory effects of GABAergic neurons in the zona incerta (ZI) on sevoflurane and propofol anesthesia. METHODS: Forty-eight male C57BL/6J mice divided into 8 groups (n=6) were used in this study. In the study of sevoflurane anesthesia, chemogenetic experiment was performed in 2 groups of mice with injection of either adeno-associated virus carrying hM3Dq (hM3Dq group) or a virus carrying only mCherry (mCherry group). The optogenetic experiment was performed in another two groups of mice injected with an adeno-associated virus carrying ChR2 (ChR2 group) or GFP only (GFP group). The same experiments were also performed in mice for studying propofol anesthesia. Chemogenetics or optogenetics were used to induce the activation of GABAergic neurons in the ZI, and their regulatory effects on anesthesia induction and arousal with sevoflurane and propofol were observed; EEG monitoring was used to observe the changes in sevoflurane anesthesia maintenance after activation of the GABAergic neurons. RESULTS: In sevoflurane anesthesia, the induction time of anesthesia was significantly shorter in hM3Dq group than in mCherry group (P < 0.05), and also shorter in ChR2 group than in GFP group (P < 0.01), but no significant difference was found in the awakening time between the two groups in either chemogenetic or optogenetic tests. Similar results were observed in chemogenetic and optogenetic experiments with propofol (P < 0.05 or 0.01). Photogenetic activation of the GABAergic neurons in the ZI did not cause significant changes in EEG spectrum during sevoflurane anesthesia maintenance. CONCLUSION: Activation of the GABAergic neurons in the ZI promotes anesthesia induction of sevoflurane and propofol but does not affect anesthesia maintenance or awakening.

Glutamatergic Neurons in the Zona Incerta Modulate Pain and Itch Behaviors in Mice.

Emerging evidence suggest that parvalbumin neurons in zona incerta (ZI) modulate pain and itch behavior in opposite manners. However, the role of ZI glutamatergic neurons, a unique incertal neuronal subpopulation residing in the caudal division, in pain and itch modulation remains unknown. In the present study, by combining chemogenetic manipulation, fiber photometry, and behavioral tests, we proved that incertal glutamatergic neurons served as an endogenous negative diencephalic modulator for both pain and itch processing. We demonstrated that ZI vesicular glutamate transporter 2 (VGluT2) neurons exhibited increased calcium signal upon hindpaw withdrawal in response to experimental mechanical and thermal stimuli. Behavioral tests further showed that pharmacogenetic activation of this specific type of neurons reduced nocifensive withdrawal responses in both naïve and inflammatory pain mice. Similar neural activity and modulatory role of ZI VGluT2 neurons were also observed upon histaminergic and non-histaminergic acute itch stimuli. Together, our study would expedite our understandings of brain mechanisms underlying somatosensory processing and modulation, and supply a novel therapeutic target for the management of chronic pain and itch disorders.

The Rostral Zona Incerta: A Subcortical Integrative Hub and Potential Deep Brain Stimulation Target for Obsessive-Compulsive Disorder.

BACKGROUND: The zona incerta (ZI) is involved in mediating survival behaviors and is connected to a wide range of cortical and subcortical structures, including key basal ganglia nuclei. Based on these connections and their links to behavioral modulation, we propose that the ZI is a connectional hub for mediating between top-down and bottom-up control and a possible target for deep brain stimulation for obsessive-compulsive disorder. METHODS: We analyzed the trajectory of cortical fibers to the ZI in nonhuman and human primates based on tracer injections in monkeys and high-resolution diffusion magnetic resonance imaging in humans. The organization of cortical and subcortical connections within the ZI were identified in the nonhuman primate studies. RESULTS: Monkey anatomical data and human diffusion magnetic resonance imaging data showed a similar trajectory of fibers/streamlines to the ZI. Prefrontal cortex/anterior cingulate cortex terminals all converged within the rostral ZI, with dorsal and lateral areas being most prominent. Motor areas terminated caudally. Dense subcortical reciprocal connections included the thalamus, medial hypothalamus, substantia nigra/ventral tegmental area, reticular formation, and pedunculopontine nucleus and a dense nonreciprocal projection to the lateral habenula. Additional connections included the amygdala, dorsal raphe nucleus, and periaqueductal gray. CONCLUSIONS: Dense connections with dorsal and lateral prefrontal cortex/anterior cingulate cortex cognitive control areas and the lateral habenula and the substantia nigra/ventral tegmental area, coupled with inputs from the amygdala, hypothalamus, and brainstem, suggest that the rostral ZI is a subcortical hub positioned to modulate between top-down and bottom-up control. A deep brain stimulation electrode placed in the rostral ZI would not only involve connections common to other deep brain stimulation sites but also capture several critically distinctive connections.

Inhibitory top-down projections from zona incerta mediate neocortical memory.

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.

Somatostatin-Positive Neurons in the Rostral Zona Incerta Modulate Innate Fear-Induced Defensive Response 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.

Projections from LIM homeobox 6 (Lhx6) + zona incerta neurons to the cholinergic or monoaminergic nuclei of the rat.

A recent report suggested that LIM homeobox 6 (Lhx6) + GABA-releasing neurons of the ventral zona incerta (VZI) promote sleep, particularly paradoxical sleep (PS). While their potential involvement in sleep still needs to be firmly confirmed, little is known about their specific input/output connections with widespread brain regions, including those involved in sleep. Thus, the present study was designed to examine whether Lhx6-expressing neurons (in parallel to intermingled MCH-expressing ones) may send efferent projections to cholinergic and/or monoaminergic nuclei from basal forebrain (BF) to brainstem (BS). Based on the present observations, the proportions of Lhx6+ neuronal projection to the BF and BS cholinergic nuclei over the total number of Lhx6+ VZI cells were approximately 5.9% and 6.9%, respectively. Likewise, the proportions of Lhx6+ neuronal projection to the dorsal raphe and locus coeruleus over the total number of Lhx6+ VZI cells were about 4.3% and 3.9%, respectively. In addition, Lhx6+ cells projecting to the cholinergic or monoaminergic nuclei were scattered along the entire dorsal-to-ventral extent of the VZI. Based on the present as well as our previous observations, it is suggested that Lhx6+ VZI neurons might play an important role in the regulation of PS, partly via the neural network involving the cholinergic as well as monoaminergic nuclei of the rat.

Activity of GABA neurons in the zona incerta and ventral lateral periaqueductal grey is biased towards sleep.

STUDY OBJECTIVES: As in various brain regions the activity of gamma-aminobutyric acid (GABA) neurons is largely unknown, we measured in vivo changes in calcium fluorescence in GABA neurons in the zona incerta (ZI) and the ventral lateral periaqueductal grey (vlPAG), two areas that have been implicated in regulating sleep. METHODS: vGAT-Cre mice were implanted with sleep electrodes, microinjected with rAAV-DIO-GCaMP6 into the ZI (n = 6) or vlPAG (n = 5) (isoflurane anesthesia) and a GRIN (Gradient-Index) lens inserted atop the injection site. Twenty-one days later, fluorescence in individual vGAT neurons was recorded over multiple REM cycles. Regions of interest corresponding to individual vGAT somata were automatically extracted with PCA-ICA analysis. RESULTS: In the ZI, 372 neurons were identified. Previously, we had recorded the activity of 310 vGAT neurons in the ZI and we combined the published dataset with the new dataset to create a comprehensive dataset of ZI vGAT neurons (total neurons = 682; mice = 11). In the vlPAG, 169 neurons (mice = 5) were identified. In both regions, most neurons were maximally active in REM sleep (R-Max; ZI = 51.0%, vlPAG = 60.9%). The second most abundant group was W-Max (ZI = 23.9%, vlPAG = 25.4%). In the ZI, but not in vlPAG, there were neurons that were NREMS-Max (11.7%). vlPAG had REMS-Off neurons (8.3%). In both areas, there were two minor classes: wake/REMS-Max and state indifferent. In the ZI, the NREMS-Max neurons fluoresced 30 s ahead of sleep onset. CONCLUSIONS: These descriptive data show that the activity of GABA neurons is biased in favor of sleep in two brain regions implicated in sleep.

Activation of GABAergic neurons in the zona incerta accelerates anesthesia induction with sevoflurane and propofol without affecting anesthesia maintenance or awakening in mice / 南方医科大学学报

OBJECTIVE@#To explore the regulatory effects of GABAergic neurons in the zona incerta (ZI) on sevoflurane and propofol anesthesia.@*METHODS@#Forty-eight male C57BL/6J mice divided into 8 groups (n=6) were used in this study. In the study of sevoflurane anesthesia, chemogenetic experiment was performed in 2 groups of mice with injection of either adeno-associated virus carrying hM3Dq (hM3Dq group) or a virus carrying only mCherry (mCherry group). The optogenetic experiment was performed in another two groups of mice injected with an adeno-associated virus carrying ChR2 (ChR2 group) or GFP only (GFP group). The same experiments were also performed in mice for studying propofol anesthesia. Chemogenetics or optogenetics were used to induce the activation of GABAergic neurons in the ZI, and their regulatory effects on anesthesia induction and arousal with sevoflurane and propofol were observed; EEG monitoring was used to observe the changes in sevoflurane anesthesia maintenance after activation of the GABAergic neurons.@*RESULTS@#In sevoflurane anesthesia, the induction time of anesthesia was significantly shorter in hM3Dq group than in mCherry group (P < 0.05), and also shorter in ChR2 group than in GFP group (P < 0.01), but no significant difference was found in the awakening time between the two groups in either chemogenetic or optogenetic tests. Similar results were observed in chemogenetic and optogenetic experiments with propofol (P < 0.05 or 0.01). Photogenetic activation of the GABAergic neurons in the ZI did not cause significant changes in EEG spectrum during sevoflurane anesthesia maintenance.@*CONCLUSION@#Activation of the GABAergic neurons in the ZI promotes anesthesia induction of sevoflurane and propofol but does not affect anesthesia maintenance or awakening.

Somatostatin-Positive Neurons in the Rostral Zona Incerta Modulate Innate Fear-Induced Defensive Response in Mice / 神经科学通报·英文版

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.