BACKGROUND: Midline Tremor is defined as an isolated or combined tremor that affects the neck, trunk, jaw, tongue, and/or voice and could be part of Essential Tremor (ET), or dystonic tremor. The clinical efficacy of deep brain stimulation for Midline Tremor has been rarely reported. The Ventral Intermediate Nucleus and Globus Pallidus Internus are the preferred targets, but with variable outcomes. Thalamic Ventral-Oralis (VO) complex and Zona Incerta (ZI) are emerging targets for tremor control in various etiologies.
OBJECTIVE: To report on neuroradiological, neurophysiological targeting and long-term efficacy of thalamic Ventral-Oralis complex and Zona Incerta deep brain stimulation in Midline Tremor.
METHODS: Three patients (two males and one female) with Midline Tremor in dystonic syndromes were recruited for this open-label study. Clinical, surgical, neurophysiological intraoperative testing and long-term follow-up data are reported.
RESULTS: Intraoperative testing and reconstruction of volume of tissue activated confirmed the position of the electrodes in the area stimulated between the thalamic Ventral-Oralis complex and Zona Incerta in all patients. All three patients showed optimal control of both tremor and dystonic features at short-term (6 months) and long-term follow-up (up to 6 years). No adverse events occurred.
CONCLUSIONS: In the syndromes of Midline Tremor of various origins, the best target for DBS might be difficult to identify. Our results showed that thalamic Ventral-Oralis complex/Zona Incerta may be a viable and safe option even in specific forms of tremor with axial distribution.

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.

Understanding the neural basis of infant social behaviors is crucial for elucidating the mechanisms of early social and emotional development. In this work, we report a specific population of somatostatin-expressing neurons in the zona incerta (ZISST) of preweaning mice that responds dynamically to social interactions, particularly those with their mother. Bidirectional neural activity manipulations in pups revealed that widespread connectivity of preweaning ZISST neurons to sensory, emotional, and cognitive brain centers mediates two key adaptive functions associated with maternal presence: the reduction of behavior distress and the facilitation of learning. These findings reveal a population of neurons in the infant mouse brain that coordinate the positive effects of the relationship with the mother on an infant\'s behavior and physiology.

Motor learning (ML), which plays a fundamental role in growth and physical rehabilitation, involves different stages of learning and memory processes through different brain regions. However, the neural mechanisms that underlie ML are not sufficiently understood. Here, a previously unreported neuronal projection from the dorsal hippocampus (dHPC) to the zona incerta (ZI) involved in the regulation of ML behaviors is identified. Using recombinant adeno-associated virus, the projections to the ZI are surprisingly identified as originating from the dorsal dentate gyrus (DG) and CA1 subregions of the dHPC. Furthermore, projection-specific chemogenetic and optogenetic manipulation reveals that the projections from the dorsal CA1 to the ZI play key roles in the acquisition and consolidation of ML behaviors, whereas the projections from the dorsal DG to the ZI mediate the retrieval/retention of ML behaviors. The results reveal new projections from the dorsal DG and dorsal CA1 to the ZI involved in the regulation of ML and provide insight into the stages over which this regulation occurs.

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.

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.

Essential tremor (ET) is the most common movement disorder. Deep brain stimulation (DBS) targeting the ventral intermediate nucleus (VIM) is known to improve symptoms in patients with medication-resistant ET. However, the clinical effectiveness of VIM-DBS may vary, and other targets have been proposed. The authors aimed to investigate whether the same anatomical structure is responsible for tremor control both immediately after VIM-DBS and at later follow-up evaluations.
Of 68 electrodes from 41 patients with ET, the authors mapped the distances of the active contact from the VIM, the dentatorubrothalamic tract (DRTT), and the caudal zona incerta (cZI) and compared them using Friedman\'s ANOVA and the Wilcoxon signed-rank follow-up test. The same distances were also compared between the initially planned target and the final implantation site after intraoperative macrostimulation. Finally, the comparison among the three structures was repeated for 16 electrodes whose active contact was changed after a mean 37.5 months follow-up to improve tremor control.
After lead implantation, the VIM was statistically significantly closer to the active contact than both the DRTT (p = 0.008) and cZI (p < 0.001). This result did not change if the target was moved based on intraoperative macrostimulation. At the last follow-up, the active contact distance from the VIM was always significantly less than that of the cZI (p < 0.001), but the distance from the DRTT was reduced and even less than the distance from the VIM.
In patients receiving VIM-DBS, the VIM itself is the structure driving the anti-tremor effect and remains more effective than the cZI, even years after implantation. Nevertheless, the role of the DRTT may become more important over time and may help sustain the clinical efficacy when the habituation from the VIM stimulation ensues.

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.

The zona incerta (ZI) is a subthalamic structure that has been implicated in locomotion, fear, and anxiety. Recently interest has grown in its therapeutic efficacy in deep brain stimulation in movement disorders. This efficacy might be due to the ZI\'s functional projections to the other brain regions. Notwithstanding some evidence of anatomical connections between the ZI and the inferior olive (IO) and the pontine nuclei (PN), how the ZI modulates the neuronal activity in these regions remains to be determined. We first tested this by monitoring responses of single neurons in the PN and IO to optogenetic activation of channelrhodopsin-expressing ZI axons in wild-type mice, using an in vivo awake preparation. Stimulation of short, single pulses and trains of stimuli at 20 Hz elicited rapid responses in the majority of recorded cells in the PN and IO. Furthermore, the excitatory response of PN neurons scaled with the strength of ZI activation. Next, we used in vitro electrophysiology to study synaptic transmission at ZI-IO synapses. Optogenetic activation of ZI axons evoked a strong excitatory postsynaptic response in IO neurons, which remained robust with repeated stimulation at 20 Hz. Overall, our results demonstrate a functional connection within ZI-PN and ZI-IO pathways.

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.