Zona Incerta Neurons Research Articles

Dopaminergic Neurons in Zona Incerta Drives Appetitive Self-Grooming.

Dopaminergic (DA) neurons are known to play a key role in controlling behaviors. While DA neurons in other brain regions are extensively characterized, those in zona incerta (ZITH or A13) receive much less attention and their function remains to be defined. Here it is shown that optogenetic stimulation of these neurons elicited intensive self-grooming behaviors and promoted place preference, which can be enhanced by training but cannot be converted into contextual memory. Interestingly, the same stimulation increased DA release to periaqueductal grey (PAG) neurons and local PAG antagonism of DA action reduced the elicited self-grooming. In addition, A13 neurons increased their activity in response to various external stimuli and during natural self-grooming episodes. Finally, monosynaptic retrograde tracing showed that the paraventricular hypothalamus represents one of the major upstream brain regions to A13 neurons. Taken together, these results reveal that A13 neurons are one of the brain sites that promote appetitive self-grooming involving DA release to the PAG.

Zona incerta mediates early life isoflurane-induced fear memory deficits

The potential long-term effects of anesthesia on cognitive development, especially in neonates and infants, have raised concerns. However, our understanding of its underlying mechanisms and effective treatments is still limited. In this study, we found that early exposure to isoflurane (ISO) impaired fear memory retrieval, which was reversed by dexmedetomidine (DEX) pre-treatment. Measurement of c-fos expression revealed that ISO exposure significantly increased neuronal activation in the zona incerta (ZI). Fiber photometry recording showed that ZI neurons from ISO mice displayed enhanced calcium activity during retrieval of fear memory compared to the control group, while DEX treatment reduced this enhanced calcium activity. Chemogenetic inhibition of ZI neurons effectively rescued the impairments caused by ISO exposure. These findings suggest that the ZI may play a pivotal role in mediating the cognitive effects of anesthetics, offering a potential therapeutic target for preventing anesthesia-related cognitive impairments.

Up-regulating GABA transporter-3 in the zona incerta prevents surgery-induced memory impairment in mice

Clinical surgery can lead to severe neuroinflammation and cognitive dysfunctions. It has been reported that astrocytes mediate memory formation and postoperative cognitive dysfunction (POCD), however, the thalamic mechanism of astrocytes in mediating POCD remains unknown. Here, we report that reactive astrocytes in zona incerta (ZI) mediate surgery-induced recognition memory impairment in male mice. Immunostaining results showed that astrocytes are activated with GABA transporter-3 (GAT-3) being down-expressed, and neurons were suppressed in the ZI. Besides, our work revealed that reactive astrocytes caused increased tonic current in ZI neurons. Up-regulating the expression of GAT-3 in astrocytes ameliorates surgery-induced recognition memory impairment. Together, our work demonstrates that the reactive astrocytes in the ZI play a crucial role in surgery-induced memory impairment, which provides a new target for the treatment of surgery-induced neural dysfunctions.

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 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.

Unilateral optogenetic stimulation of Lhx6 neurons in the zona incerta increases REM sleep.

To determine how a waking brain falls asleep researchers have monitored and manipulated activity of neurons and glia in various brain regions. While imaging Gamma-Aminobutyric Acid (GABA) neurons in the zona incerta (ZI) we found a subgroup that anticipates onset of NREM sleep (Blanco-Centurion C, Luo S, Vidal-Ortiz A, Swank C, Shiromani PJ. Activity of a subset of vesicular GABA-transporter neurons in the ventral ZI anticipates sleep onset. Sleep. 2021;44(6):zsaa268. doi:10.1093/sleep/zsaa268.). To differentiate the GABA subtype we now image and optogenetically manipulate the ZI neurons containing the transcription factor, Lhx6. In the first study, Lhx6-cre mice (n = 5; female = 4) were given rAAV-DJ-EF1a-DIO-GCaMP6M into the ZI (isofluorane anesthesia), a GRIN lens implanted, and 21days later sleep and fluorescence in individual Lhx6 neurons were recorded for 4 hours. Calcium fluorescence was detected in 132 neurons. 45.5% of the Lhx6 neurons were REM-max; 30.3% were wake-max; 11.4% were wake + REM max; 9% were NREM-max; and 3.8% had no change. The NREM-max group of neurons fluoresced 30 seconds ahead of sleep onset. The second study tested the effects of unilateral optogenetic stimulation of the ZI Lhx6 neurons (n = 14 mice) (AAV5-Syn-FLEX-rc[ChrimsonR-tdTomato]. Stimulation at 1 and 5 Hz (1 minute on- 4 minutes off) significantly increased percent REM sleep during the 4 hours stimulation period (last half of day cycle). The typical experimental approach is to stimulate neurons in both hemispheres, but here we found that low-frequency stimulation of ZI Lhx6 neurons in one hemisphere is sufficient to shift states of consciousness. Detailed mapping combined with mechanistic testing is necessary to identify local nodes that can shift the brain between wake-sleep states.

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.

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.

An inhibitory circuit from central amygdala to zona incerta drives pain-related behaviors in 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.

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.

0126 Zona incerta Lhx6 neurons are most active during NREM and REM sleep and after prolonged waking

Abstract Introduction Emerging data shows that GABA neurons in the zona incerta (ZI) play a prominent role in regulating sleep. Transfer of the orexin gene into ZI neurons blocks cataplexy in narcoleptic orexin-knockout mice (Liu et al., JNeurosci, 2011) and vGAT GABA neurons in the ZI anticipate onset of NREM (Blanco-Centurion et al., SLEEP, 2021). To identify the subtype of GABA neurons regulating sleep, we examined the activity of Lhx6 neurons. This study tests the hypothesis that Lhx6 neurons show peak calcium fluorescence during sleep, and that the fluorescence is further increased after prolonged waking. Methods In Lhx6-cre mice (mice=4; all females; 4-5 mo), rAAV-DIO-GCaMP6s was delivered stereotaxically to the ZI (isofluorane anesthesia) and a GRIN lens, along with EEG and EMG electrodes were implanted. 21d later a miniscope (INSCOPIX) was attached, and after 3d of adaptation, sleep and fluorescence in individual Lhx6 neurons were recorded for 4h (baseline). On another day, the mice were kept awake for 6h (gentle handling; 9a-3p) and fluorescence in Lhx6 neurons was recorded for 2h during recovery sleep. The imaged data from the two recording periods (baseline and recovery sleep) was combined into a single data file and the change in fluorescence was determined against the mean image frame (F0). Previously, we and others found that the GCamP calcium fluorescence is a direct measure of action potentials and serves as a marker of activity. Results 97 neurons were automatically extracted (PCA-ICA analysis; blinded without knowledge of sleep state). In 66 neurons (68%) the average fluorescence was significantly higher during REM, NREM or both, compared to waking (Mixed Model ANOVA; SPSS25; P<0.01). In this population the fluorescence was significantly higher during recovery sleep compared to baseline (P<0.001) indicating increased activity of the sleep-active neurons during recovery sleep. With ensuing sleep, the increase in fluorescence gradually returned to baseline levels, attesting to the fluorescence as a marker of homeostatic sleep pressure. In 14 neurons (14%), fluorescence was highest in waking as compared to the other states during baseline, and in these neurons, fluorescence did not increase after sleep loss. Interestingly, six neurons (6%) were most active in waking and NREM but silent in REM (REM-off). Conclusion This is the first study to measure fluorescence in individual neurons after sleep loss. We find that the fluorescence in two-thirds of ZI Lhx6 neurons is tightly linked to sleep, and that the average fluorescence is further increased after prolonged waking. Microendoscopy is superior to indirect measures such as c-FOS or photometry in gauging sleep pressure in individual neurons. Support (If Any) VA BX000798, 1K6BX004216.

Zona Incerta Modulates Pain-Associated Behaviors in Mice

Pain is an unpleasant experience with both sensory and affective components. However, the neural circuits underlying modulation of pain-related behaviors remains unclear. In this study, we identified an inhibitory neural circuit that originates in central amygdala protein kinase C-delta (CeA-PKCδ) neurons and terminates in the ventral sector of the zona incerta (ZI), a subthalamic structure previously linked to pain processing. Behavioral experiments show that chemogenetic inhibition of GABAergic ZI neurons induces bilateral hypersensitivity in uninjured mice as well as contralateral hypersensitivity after nerve injury. In contrast, chemogenetic activation of VGAT cells in the ZI reverses nerve injury-induced hypersensitivity. Furthermore, we found that chemogenetic activation of ZI-VGAT neurons reduced pain-related aversion during formalin conditioned place aversion (FCPA) without affecting spontaneous nociceptive responses induced by formalin. Taken together, our results suggest an indispensable role of ZI in regulating somatosensory and affective components of pain. Grant support from NCCIH.

Parvalbumin Neurons in Zona Incerta Regulate Itch in Mice.

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.

A primate temporal cortex-zona incerta pathway for novelty seeking.

Primates interact with the world by exploring visual objects; they seek opportunities to view novel objects even when these have no extrinsic reward value. How the brain controls this novelty seeking is unknown. Here we show that novelty seeking in monkeys is regulated by the zona incerta (ZI). As monkeys made eye movements to familiar objects to trigger an opportunity to view novel objects, many ZI neurons were preferentially activated by predictions of novel objects before the gaze shift. Low-intensity ZI stimulation facilitated gaze shifts, whereas ZI inactivation reduced novelty seeking. ZI-dependent novelty seeking was not regulated by neurons in the lateral habenula or by many dopamine neurons in the substantia nigra, traditionally associated with reward seeking. But the anterior ventral medial temporal cortex, an area important for object vision and memory, was a prominent source of novelty predictions. These data uncover a functional pathway in the primate brain that regulates novelty seeking.

Zona incerta neurons projecting to the ventral tegmental area promote action initiation towards feeding.

Key points 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. Both the zona incerta (ZI) and the ventral tegmental area (VTA) have been implicated in feeding behaviour. The ZI provides prominent input to the VTA, although it has not yet been investigated whether this projection regulates feeding. Therefore, we investigated the role of ZI to VTA projection neurons in the regulation of several aspects of feeding behaviour. We determined the effects of (in)activation of ZI to VTA projection neurons on feeding microstructure, food‐motivated behaviour under a progressive ratio schedule of reinforcement, locomotor activity and core body temperature. To activate or inactivate ZI neurons projecting to the VTA, we used a combination of canine adenovirus‐2 in the VTA, as well as Cre‐dependent designer receptors exclusively activated by designer drugs (DREADD) or tetanus toxin (TetTox) light chain in the ZI. TetTox‐mediated inactivation of ZI to VTA projection neurons reduced food‐motivated behaviour and feeding by reducing meal frequency. Conversely, DREADD‐mediated chemogenetic activation of ZI to VTA projection neurons promoted food‐motivated behaviour and feeding. (In)activation of ZI to VTA projection neurons did not affect locomotor activity or directly regulate core body temperature. Taken together, ZI neurons projecting to the VTA exert bidirectional control overfeeding behaviour. More specifically, activity of ZI to VTA projection neurons facilitate action initiation towards feeding, as reflected in both food‐motivated behaviour and meal initiation, without affecting general activity.

LIM homeobox 6 (Lhx6)+ neurons in the ventral zona incerta project to the core portion of the lateral supramammillary nucleus in the rat

There was a recent report suggesting that LIM homeobox 6 (Lhx6)+ GABA-releasing neurons of the ventral zona incerta (ZI) promote sleep. We demonstrated in the previous study that Lhx6+ ZI neurons are activated during paradoxical sleep (PS) hypersomnia which was induced by 48-hour PS deprivation, implying their roles in the control of PS like melanin-concentrating hormone (MCH) cells. Since the core portion of the lateral supramammillary nucleus (SUMl) is the major hypothalamic area activating the dentate gyrus as well as other limbic cortices during PS, we examined in the present study whether Lhx6+ ZI cells provide efferent projections to the SUMl, using the retrograde-tracing method. The majority of Lhx6+ neurons projecting to the SUMl occupied the ventral border (or ventral one-third) of the ventral ZI. Based on the quantitative analysis, the mean number of retrogradely-labeled Lhx6+ neurons was comparable to that of retrogradely-labeled MCH cells in the ZI. However, the total (i.e., single- plus double-labeled) number of Lhx6+ cells was approximately three times larger than that of MCH cells in the ZI. Thus, the proportion (about 7.8%) of retrogradely-labeled Lhx6+ neurons over the total Lhx6+ cells was approximately one-third of the percentage (about 20.9%) of retrogradely-labeled MCH neurons over the total MCH cells. On the other hand, a combination of retrogradely-labeled, Lhx6 and MCH cells occupied approximately 43.7% of the total retrogradely-labeled neurons in the ventral ZI. The present observations suggested that Lhx6+ neurons in the ventral ZI might play an important role in the regulation of PS, partly via the neural network involving the SUMl.

Distributions of hypothalamic neuron populations coexpressing tyrosine hydroxylase and the vesicular GABA transporter in the mouse.

The hypothalamus contains catecholaminergic neurons marked by the expression of tyrosine hydroxylase (TH). As multiple chemical messengers coexist in each neuron, we determined if hypothalamic TH-immunoreactive (ir) neurons express vesicular glutamate or GABA transporters. We used Cre/loxP recombination to express enhanced GFP (EGFP) in neurons expressing the vesicular glutamate (vGLUT2) or GABA transporter (vGAT), then determined whether TH-ir neurons colocalized with native EGFPVglut2 - or EGFPVgat -fluorescence, respectively. EGFPVglut2 neurons were not TH-ir. However, discrete TH-ir signals colocalized with EGFPVgat neurons, which we validated by in situ hybridization for Vgat mRNA. To contextualize the observed pattern of colocalization between TH-ir and EGFPVgat , we first performed Nissl-based parcellation and plane-of-section analysis, and then mapped the distribution of TH-ir EGFPVgat neurons onto atlas templates from the Allen Reference Atlas (ARA) for the mouse brain. TH-ir EGFPVgat neurons were distributed throughout the rostrocaudal extent of the hypothalamus. Within the ARA ontology of gray matter regions, TH-ir neurons localized primarily to the periventricular hypothalamic zone, periventricular hypothalamic region, and lateral hypothalamic zone. There was a strong presence of EGFPVgat fluorescence in TH-ir neurons across all brain regions, but the most striking colocalization was found in a circumscribed portion of the zona incerta (ZI)-a region assigned to the hypothalamus in the ARA-where every TH-ir neuron expressed EGFPVgat . Neurochemical characterization of these ZI neurons revealed that they display immunoreactivity for dopamine but not dopamine β-hydroxylase. Collectively, these findings indicate the existence of a novel mouse hypothalamic population that may signal through the release of GABA and/or dopamine.

Targeted recombination in active populations as a new mouse genetic model to study sleep-active neuronal populations: Demonstration that Lhx6+ neurons in the ventral zona incerta are activated during paradoxical sleep hypersomnia.

The cFos immunostaining allowed the identification of multiple populations of neurons involved in the generation of paradoxical sleep. We adopted the transgenic (targeted recombination in active populations) mouse model, which following injection of tamoxifen, allows expression of Cre-dependent reporter constructs (i.e., mCherry) in neurons expressing cFos during waking or paradoxical sleep hypersomnia following automatic paradoxical sleep deprivation. Three groups of mice were subjected to two periods of waking, one period of waking and one of paradoxical sleep hypersomnia, or two periods of paradoxical sleep hypersomnia. A high percentage of double-labelled neurons was observed in the lateral hypothalamic area and zona incerta of two periods of waking and two periods of paradoxical sleep hypersomnia in mice, but not in those of one period of waking and one of paradoxical sleep hypersomnia in animals. Melanin-concentrating hormone neurons in the lateral hypothalamic area and Lhx6+ cells in the zona incerta constituted 5.7±1.5% and 8.8±2.3% of all mCherry+ cells and 20.6±4.8% and 24.6±5.9% of all cFos+ neurons in two periods of paradoxical sleep hypersomnia in animals. In addition, melanin-concentrating hormone cells as well as Lhx6+ neurons rarely expressed mCherry (or cFos) in the waking condition, in contrast to orexin neurons, which constituted approximately 30% of mCherry+ and cFos+ neurons. Our results validate the TRAP methodology and open the way to use it for identifying the neurons activated during waking and paradoxical sleep hypersomnia. Furthermore, they indicate for the first time that Lhx6+ neurons in the zona incerta, like melanin-concentrating hormone cells in the lateral hypothalamic area, are activated during paradoxical sleep hypersomnia but not during waking. These results indicate that Lhx6+ neurons might play a role in the control of paradoxical sleep, like the melanin-concentrating hormone cells.

Zona incerta GABAergic neurons integrate prey-related sensory signals and induce an appetitive drive to promote hunting.

The neural substrates for predatory hunting, an evolutionarily conserved appetitive behavior, remain largely undefined. Photoactivation of zona incerta (ZI) GABAergic neurons strongly promotes hunting of both live and artificial prey. Conversely, photoinhibition of these neurons or deletion of their GABA function severely impairs hunting. Here electrophysiological recordings reveal that ZI neurons integrate prey-related multisensory signals and discriminate prey from non-prey targets. Visual or whisker sensory deprivation reduces calcium responses induced by prey introduction and attack and impair hunting. ZI photoactivation largely corrects the hunting impairment caused by sensory deprivations. Motivational and reinforcing assays reveal that ZI photoactivation is associated with a strong appetitive drive, causing repetitive self-stimulatory behaviors. These ZI neurons project to the periaqueductal gray matter to induce hunting and motivation. Thus, we have delineated the function of ZI GABAergic neurons in hunting, which integrates prey-related sensory signals into prey detection and attack and induces a strong appetitive motivational drive.