Concerning neuromodulation as treatment of neurological and neuropsychiatric disorder: Insights gained from selective targeting of the subthalamic nucleus, para-subthalamic nucleus and zona incerta in rodents.

Neuromodulation such as deep brain stimulation (DBS) is advancing as a clinical intervention in several neurological and neuropsychiatric disorders, including Parkinson's disease, dystonia, tremor, and obsessive-compulsive disorder (OCD) for which DBS is already applied to alleviate severely afflicted individuals of symptoms. Tourette syndrome and drug addiction are two additional disorders for which DBS is in trial or proposed as treatment. However, some major remaining obstacles prevent this intervention from reaching its full therapeutic potential. Side-effects have been reported, and not all DBS-treated individuals are relieved of their symptoms. One major target area for DBS electrodes is the subthalamic nucleus (STN) which plays important roles in motor, affective and associative functions, with impact on for example movement, motivation, impulsivity, compulsivity, as well as both reward and aversion. The multifunctionality of the STN is complex. Decoding the anatomical-functional organization of the STN could enhance strategic targeting in human patients. The STN is located in close proximity to zona incerta (ZI) and the para-subthalamic nucleus (pSTN). Together, the STN, pSTN and ZI form a highly heterogeneous and clinically important brain area. Rodent-based experimental studies, including opto- and chemogenetics as well as viral-genetic tract tracings, provide unique insight into complex neuronal circuitries and their impact on behavior with high spatial and temporal precision. This research field has advanced tremendously over the past few years. Here, we provide an inclusive review of current literature in the pre-clinical research fields centered around STN, pSTN and ZI in laboratory mice and rats; the three highly heterogeneous and enigmatic structures brought together in the context of relevance for treatment strategies. Specific emphasis is placed on methods of manipulation and behavioral impact.

Most dynorphin neurons in the zona incerta-perifornical area are active in waking relative to non-rapid-eye movement and rapid-eye movement sleep.

Dynorphin is an endogenous opiate localized in many brain regions and spinal cord, but the activity of dynorphin neurons during sleep is unknown. Dynorphin is an inhibitory neuropeptide that is coreleased with orexin, an excitatory neuropeptide. We used microendoscopy to test the hypothesis that, like orexin, the dynorphin neurons are wake-active. Dynorphin-cre mice (n = 3) were administered rAAV8-Ef1a-Con/Foff 2.0-GCaMP6M into the zona incerta-perifornical area, implanted with a GRIN lens (gradient reflective index), and electrodes to the skull that recorded sleep. One month later, a miniscope imaged calcium fluorescence in dynorphin neurons during multiple bouts of wake, non-rapid-eye movement (NREM), and rapid-eye movement (REM) sleep. Unbiased data analysis identified changes in calcium fluorescence in 64 dynorphin neurons. Most of the dynorphin neurons (72%) had the highest fluorescence during bouts of active and quiet waking compared to NREM or REM sleep; a subset (20%) were REM-max. Our results are consistent with the emerging evidence that the activity of orexin neurons can be classified as wake-max or REM-max. Since the two neuropeptides are coexpressed and coreleased, we suggest that dynorphin-cre-driven calcium sensors could increase understanding of the role of this endogenous opiate in pain and sleep.

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.

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.

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.

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.

Probabilistic maps for deep brain stimulation - Impact of methodological differences.

BACKGROUND: Group analysis of patients with deep brain stimulation (DBS) has the potential to help understand and optimize the treatment of patients with movement disorders. Probabilistic stimulation maps (PSM) are commonly used to analyze the correlation between tissue stimulation and symptomatic effect but are applied with different methodological variations. OBJECTIVE: To compute a group-specific MRI template and PSMs for investigating the impact of PSM model parameters. METHODS: Improvement and occurrence of dizziness in 68 essential tremor patients implanted in caudal zona incerta were analyzed. The input data includes the best parameters for each electrode contact (screening), and the clinically used settings. Patient-specific electric field simulations (n = 488) were computed for all DBS settings. The electric fields were transformed to a group-specific MRI template for analysis and visualization. The different comparisons were based on PSMs representing occurrence (N-map), mean improvement (M-map), weighted mean improvement (wM-map), and voxel-wise t-statistics (p-map). These maps were used to investigate the impact from input data (clinical/screening settings), clustering methods, sampling resolution, and weighting function. RESULTS: Screening or clinical settings showed the largest impacts on the PSMs. The average differences of wM-maps were 12.4 and 18.2% points for the left and right sides respectively. Extracting clusters based on wM-map or p-map showed notable variation in volumes, while positioning was similar. The impact on the PSMs was small from weighting functions, except for a clear shift in the positioning of the wM-map clusters. CONCLUSION: The distribution of the input data and the clustering method are most important to consider when creating PSMs for studying the relationship between anatomy and DBS outcome.

Neural correlates of multidimensional motor outputs in an excitatory parafascicular-zona incerta circuit.

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.

Neural circuit control of innate behaviors.

All animals possess a plethora of innate behaviors that do not require extensive learning and are fundamental for their survival and propagation. With the advent of newly-developed techniques such as viral tracing and optogenetic and chemogenetic tools, recent studies are gradually unraveling neural circuits underlying different innate behaviors. Here, we summarize current development in our understanding of the neural circuits controlling predation, feeding, male-typical mating, and urination, highlighting the role of genetically defined neurons and their connections in sensory triggering, sensory to motor/motivation transformation, motor/motivation encoding during these different behaviors. Along the way, we discuss possible mechanisms underlying binge-eating disorder and the pro-social effects of the neuropeptide oxytocin, elucidating the clinical relevance of studying neural circuits underlying essential innate functions. Finally, we discuss some exciting brain structures recurrently appearing in the regulation of different behaviors, which suggests both divergence and convergence in the neural encoding of specific innate behaviors. Going forward, we emphasize the importance of multi-angle and cross-species dissections in delineating neural circuits that control innate behaviors.

Periaqueductal gray neurons encode the sequential motor program in hunting behavior of mice.

Sequential encoding of motor programs is essential for behavior generation. However, whether it is critical for instinctive behavior is still largely unknown. Mouse hunting behavior typically contains a sequential motor program, including the prey search, chase, attack, and consumption. Here, we reveal that the neuronal activity in the lateral periaqueductal gray (LPAG) follows a sequential pattern and is time-locked to different hunting actions. Optrode recordings and photoinhibition demonstrate that LPAGVgat neurons are required for the prey detection, chase and attack, while LPAGVglut2 neurons are selectively required for the attack. Ablation of inputs that could trigger hunting, including the central amygdala, the lateral hypothalamus, and the zona incerta, interrupts the activity sequence pattern and substantially impairs hunting actions. Therefore, our findings reveal that periaqueductal gray neuronal ensembles encode the sequential hunting motor program, which might provide a framework for decoding complex instinctive behaviors.

A cell type-specific cortico-subcortical brain circuit for investigatory and novelty-seeking behavior.

Exploring the physical and social environment is essential for understanding the surrounding world. We do not know how novelty-seeking motivation initiates the complex sequence of actions that make up investigatory behavior. We found in mice that inhibitory neurons in the medial zona incerta (ZIm), a subthalamic brain region, are essential for the decision to investigate an object or a conspecific. These neurons receive excitatory input from the prelimbic cortex to signal the initiation of exploration. This signal is modulated in the ZIm by the level of investigatory motivation. Increased activity in the ZIm instigates deep investigative action by inhibiting the periaqueductal gray region. A subpopulation of inhibitory ZIm neurons expressing tachykinin 1 (TAC1) modulates the investigatory behavior.

Incerta-thalamic Circuit Controls Nocifensive Behavior via Cannabinoid Type 1 Receptors.

Pain is a source of substantial discomfort. Abnormal activity in both the zona incerta (ZI) and posterior complex of the thalamus (Po) are implicated in neuropathic pain, but their exact roles remain unclear. In particular, the precise cell types and molecular mechanisms of the ZI-Po circuit that regulate nociception are largely uncharacterized. Here, we found that parvalbumin (PV)-positive neuronal projections from the ventral ZI (ZIv) to the Po (ZIv-Po) are critical for promoting nocifensive behaviors, whereas selectively inhibiting ZIv-Po activity reduces nocifensive withdrawal responses. Furthermore, cannabinoid type 1 receptors (CB1Rs) are expressed specifically at ZIv-Po axon terminals in this circuit, and cannabinoids attenuate nocifensive responses through presynaptic inhibition. Selective inhibition of the ZIv-Po circuit or administration of cannabinoids into the Po are sufficient to ameliorate pathological pain. These findings identify the critical role of the ZIv-Po circuit and its modulation by endocannabinoids in controlling nocifensive behaviors.

Zona incerta as a therapeutic target in Parkinson's disease.

The zona incerta has recently become an important target for deep-brain stimulation (DBS) in Parkinson's disease (PD). The present review summarizes clinical, animal and anatomical data which have indicated an important role of this structure in PD, and discusses potential mechanisms involved in therapeutic effects of DBS. Animal studies have suggested initially some role of neurons as well as GABAergic and glutamatergic receptors of the zona incerta in locomotion and generation of PD signs. Anatomical data have indicated that thanks to its multiple interconnections with the basal ganglia, thalamus, cerebral cortex, brainstem, spinal cord and cerebellum, the zona incerta is an important link in a neuronal chain transmitting impulses involved in PD pathology. Finally, clinical studies have shown that DBS of this structure alleviates parkinsonian bradykinesia, muscle rigidity and tremor. DBS of caudal zona incerta seemed to be the most effective therapeutic intervention, especially with regard to reduction of PD tremor as well as other forms of tremor.

Thalamic Input to Orbitofrontal Cortex Drives Brain-wide, Frequency-Dependent Inhibition Mediated by GABA and Zona Incerta.

Anatomical and behavioral data suggest that the ventrolateral orbitofrontal cortex (VLO), which exhibits extensive connectivity and supports diverse sensory and cognitive processes, may exert global influence over brain activity. However, this hypothesis has never been tested directly. We applied optogenetic fMRI to drive various elements of VLO circuitry while visualizing the whole-brain response. Surprisingly, driving excitatory thalamocortical projections to VLO at low frequencies (5-10 Hz) evoked widespread, bilateral decreases in brain activity spanning multiple cortical and subcortical structures. This pattern was unique to thalamocortical projections, with direct stimulations of neither VLO nor thalamus eliciting such a response. High-frequency stimulations (25-40 Hz) of thalamocortical projections evoked dramatically different-though still far-reaching-responses, in the form of widespread ipsilateral activation. Importantly, decreases in brain activity evoked by low-frequency thalamocortical input were mediated by GABA and activity in zona incerta. These findings identify specific circuit mechanisms underlying VLO control of brain-wide neural activities.

Zona incerta deep-brain stimulation in orthostatic tremor: efficacy and mechanism of improvement.

BACKGROUND: Orthostatic tremor is a rare hyperkinetic movement disorder that is characterized by a 13-18 Hz tremor in both legs while standing. Deep-brain stimulation of the caudal zona incerta has re-emerged as an alternate target for tremor control in various etiologies. OBJECT: Explore the clinical efficacy and mechanism of action of caudal zona incerta deep-brain stimulation in orthostatic tremor. METHODS: Four patients (63.1 ± 4.1 years, female = 50%) with orthostatic tremor were recruited for this open label study (63.1 ± 4.1 years, female = 50%). In two patients, the electrodes were externalized to determine the effectiveness of caudal zona incerta as a target. Surface EMG (leg muscles), EEG (leg motor cortex) and caudal zona incerta local field potential recordings were recorded. Data were recorded in sitting and standing positions with stimulation OFF and ON. RESULTS: EMG frequency analysis showed tremor frequency at 13-17 Hz. EMG-EEG coherence was found in the tremor frequency band and double tremor frequency band. EMG-caudal zona incerta coherence was higher in the tremor frequency band, while EEG coherence was higher in the double tremor frequency band. Upon stimulation, there was a selective reduction in tremor frequency band EEG-EMG coherence in all patients. All the patients had reduction in feeling of unsteadiness and increase in the stance duration. CONCLUSIONS: Bilateral caudal zona incerta deep-brain stimulation is effective in refractory orthostatic tremor. Two independent central oscillations were found at tremor and double tremor frequency. Zona incerta DBS produces improvement in OT patients possibly by modifying the abnormal oscillatory proprioceptive input from leg muscles. Frequent changes in deep-brain stimulation settings were required for maintaining the clinical benefit.

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.

A subcortical excitatory circuit for sensory-triggered predatory hunting in mice.

Predatory hunting plays a fundamental role in animal survival. Little is known about the neural circuits that convert sensory cues into neural signals to drive this behavior. Here we identified an excitatory subcortical neural circuit from the superior colliculus to the zona incerta that triggers predatory hunting. The superior colliculus neurons that form this pathway integrate motion-related visual and vibrissal somatosensory cues of prey. During hunting, these neurons send out neural signals that are temporally correlated with predatory attacks, but not with feeding after prey capture. Synaptic inactivation of this pathway selectively blocks hunting for prey without impairing other sensory-triggered behaviors. These data reveal a subcortical neural circuit that is specifically engaged in translating sensory cues into neural signals to provoke predatory hunting.

A cross-modality enhancement of defensive flight via parvalbumin neurons in zona incerta.

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.

Modulation of fear generalization by the zona incerta.

Fear expressed toward threat-associated stimuli is an adaptive behavioral response. In contrast, the generalization of fear responses toward nonthreatening cues is a maladaptive and debilitating dimension of trauma- and anxiety-related disorders. Expressing fear to appropriate stimuli and suppressing fear generalization require integration of relevant sensory information and motor output. While thalamic and subthalamic brain regions play important roles in sensorimotor integration, very little is known about the contribution of these regions to the phenomenon of fear generalization. In this study, we sought to determine whether fear generalization could be modulated by the zona incerta (ZI), a subthalamic brain region that influences sensory discrimination, defensive responses, and retrieval of fear memories. To do so, we combined differential intensity-based auditory fear conditioning protocols in mice with C-FOS immunohistochemistry and designer receptors exclusively activated by designer drugs (DREADDs)-based manipulation of neuronal activity in the ZI. C-FOS immunohistochemistry revealed an inverse relationship between ZI activation and fear generalization: The ZI was less active in animals that generalized fear. In agreement with this relationship, chemogenetic inhibition of the ZI resulted in fear generalization, while chemogenetic activation of the ZI suppressed fear generalization. Furthermore, targeted stimulation of GABAergic cells in the ZI reduced fear generalization. To conclude, our data suggest that stimulation of the ZI could be used to treat fear generalization in the context of trauma- and anxiety-related disorders.