Ole Kiehn

Summary: Tissue-clearing and light-sheet microscopy have transformed volumetric imaging of intact organs, yet limited mechanistic understanding of dehydration-based clearing continues to constrain rational protocol design and broader applicability. Here, we define the cardinal chemical and physical principles underlying dehydration-based tissue-clearing and establish a new pipeline for large-volume imaging. To maximize imaging performance, we developed the “mesoSPIM-ultra”, an upgraded mesoSPIM platform with a temperature-controlled sample chamber, a large field-of-view (FoV) camera and specialized optics to achieve long-working-distance, high-resolution imaging of cleared samples. We applied this approach to investigate the projectome of Chx10 ⁺ neurons, a cell population with complex axonal morphologies along the entire mouse spinal-cord and brain, and implicated in ipsilateral orienting behaviors. By combining behavioral analysis with post-hoc single-neuron reconstructions, we revealed previously inaccessible branching architectures and long-range projections extending from the brainstem to the spinal cord. Together, our work establishes a mechanistic foundation for tissue-clearing and scalable imaging.

Animals continuously adapt their behavior to balance survival and fulfilling essential needs. This balancing act involves prioritization of safety over the pursuit of other needs. However, the specific deep brain circuits that regulate safety-seeking behaviors in conjunction with motor circuits remain poorly understood. Here, we identify a class of glutamatergic neurons in the mouse lateral hypothalamic area (LHA) that target the midbrain locomotor-promoting pedunculopontine nucleus (PPN). Following activation, this LHA–PPN pathway orchestrates context-dependent locomotion, prioritizing safety-directed movement over other essential needs such as foraging or social contact. Remarkably, the neuronal activity of this circuit correlates directly with safety-seeking behavior. The circuit may respond to both intrinsic and extrinsic cues, having a pivotal role in ensuring survival. Our findings uncover a circuit motif within the lateral hypothalamus that, when recruited, prioritizes critical needs through the recruitment of an appropriate motor action.

Animals continously adapt their behavior to balance survival and fulfilling essential needs. This balancing act involves prioritization of safety over the pursuit of other needs. However, the specific deep brain circuits that regulate safety-seeking behaviors in conjuction with motor circuits remain poorly understood. Here we identify a class of glutamatergic neurons in the lateral hypothalamic area (LHA) that target the midbrain locomotor-promoting pedunculopontine nucleus (PPN). Upon activation, this LHA-PPN pathway orchestrates context-dependent locomotion, prioritizing safety-directed movement over other essential needs such as foraging or mating. Remarkably, the neuronal activity of these circuits correlates directly with safety-seeking behavior. These circuits may respond to both intrinsic and external cues, playing a pivotal role in ensuring survival. Our findings uncover a circuit motif within the lateral hypothalamus that when recruited, prioritizes critical needs through the recruitment of an appropriate motor action.

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of somatic motor neurons. A major focus has been directed to motor neuron intrinsic properties as a cause for degeneration, while less attention has been given to the contribution of spinal interneurons. In the present work, we applied multiplexing detection of transcripts and machine learning–based image analysis to investigate the fate of multiple spinal interneuron populations during ALS progression in the SOD1 ^G93A mouse model. The analysis showed that spinal inhibitory interneurons are affected early in the disease, before motor neuron death, and are characterized by a slow progressive degeneration, while excitatory interneurons are affected later with a steep progression. Moreover, we report differential vulnerability within inhibitory and excitatory subpopulations. Our study reveals a strong interneuron involvement in ALS development with interneuron specific degeneration. These observations point to differential involvement of diverse spinal neuronal circuits that eventually may be determining motor neuron degeneration.

Locomotion allows us to move and interact with our surroundings. Spinal networks that control locomotion produce rhythm, left-right and flexor-extensor coordination. Several glutamatergic populations, Shox2 non-V2a, Hb9-derived interneurons and recently, spinocerebellar neurons have been proposed to be involved in the mouse rhythm generating networks. However, these cells make up only a smaller fraction of the excitatory cells in the ventral spinal cord. Here we set out to identify additional populations of excitatory spinal neurons that may be involved in rhythm generation or other functions in the locomotor network. For this, we use RNA-sequencing from glutamatergic, non-glutamatergic, and Shox2 cells in the neonatal mice from both sexes followed by differential gene expression analyses. These analyses identified transcription factors that are highly expressed by glutamatergic spinal neurons and differentially expressed between Shox2 neurons and glutamatergic neurons. From this latter category, we identified the Lhx9-derived neurons that have a restricted spinal expression pattern and do not overlap with Shox2 neurons. They are purely glutamatergic and ipsilaterally projecting. Ablation of the glutamatergic transmission or acute inactivation of the neuronal activity of Lhx9-derived neurons lead to a decrease in the frequency of locomotor-like activity with no change in coordination pattern. Optogenetic activation of Lhx9-derived neurons promotes locomotor-like activity and modulates the frequency of the ongoing drug-induced locomotor activity. Calcium activities of Lhx9-derived neurons in the upper lumbar cord show strong left-right out of phase rhythmicity during locomotor-like activity. Our study identifies a distinct population of spinal excitatory neurons that regulates the frequency of locomotor output with a suggested role in rhythm-generation in the mouse alongside other spinal populations.Significance StatementIpsilaterally-projecting excitatory interneurons play a crucial role in the generation of locomotor rhythms in the vertebrate spinal cord. While the Shox2, Hb9, and spinocerebellar tract neurons are known components involved in this behavior they represent only a fraction of the entire excitatory population in the spinal cord. In this study, we identify additional excitatory spinal populations that could be essential contributors to the locomotor circuitry and shed light on a new population of ipsilaterally-projecting excitatory neurons expressing the transcription factor Lhx9. Our findings show that this newly identified population has the capacity to modulate the locomotor frequency, suggesting a potential involvement in the rhythm generating network along other spinal neuronal populations.

The basal ganglia are essential for executing motor actions. How the basal ganglia engage spinal motor networks has remained elusive. Medullary Chx10 gigantocellular (Gi) neurons are required for turning gait programs, suggesting that turning gaits organized by the basal ganglia are executed via this descending pathway. Performing deep brainstem recordings of Chx10 Gi Ca ²⁺ activity in adult mice, we show that striatal projection neurons initiate turning gaits via a dominant crossed pathway to Chx10 Gi neurons on the contralateral side. Using intersectional viral tracing and cell-type-specific modulation, we uncover the principal basal ganglia–spinal cord pathway for locomotor asymmetries in mice: basal ganglia → pontine reticular nucleus, oral part (PnO) → Chx10 Gi → spinal cord. Modulating the restricted PnO → Chx10 Gi pathway restores turning competence upon striatal damage, suggesting that dysfunction of this pathway may contribute to debilitating turning deficits observed in Parkinson’s disease. Our results reveal the stratified circuit architecture underlying a critical motor program.

Astrocytes, the most abundant glial cells in the central nervous system, respond to a wide variety of neurotransmitters binding to metabotropic receptors. Here, we investigated the intracellular calcium responses of spinal cord astrocytes to dopamine and noradrenaline, two catecholamines released by specific descending pathways. In a slice preparation from the spinal cord of neonatal mice, puff application of dopamine resulted in intracellular calcium responses that remained in the endfeet. Noradrenaline induced stronger responses that also started in the endfeet but spread to neighbouring compartments. The intracellular calcium responses were unaffected by blocking neuronal activity or inhibiting various neurotransmitter receptors, suggesting a direct effect of dopamine and noradrenaline on astrocytes. The intracellular calcium responses induced by noradrenaline and dopamine were inhibited by the D1 receptor antagonist SCH 23390. We assessed the functional consequences of these astrocytic responses by examining changes in arteriole diameter. Puff application of dopamine or noradrenaline resulted in vasoconstriction of spinal arterioles. However, blocking D1 receptors or manipulating astrocytic intracellular calcium levels did not abolish the vasoconstrictions, indicating that the observed intracellular calcium responses in astrocyte endfeet were not responsible for the vascular changes. Our findings demonstrate a compartmentalized response of spinal cord astrocytes to catecholamines and expand our understanding of astrocyte-neurotransmitter interactions and their potential roles in the physiology of the central nervous system. Spinal cord astrocytes, responding to dopamine and noradrenaline, exhibit compartmentalized calcium responses that start in endfeet. Dopamine induces localized responses, while noradrenaline triggers stronger, spreading responses. The two type of responses depend on D1 receptors.image

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of somatic motor neurons. Major focus has been directed to motor neuron intrinsic properties as a cause for degeneration while less attention has been given to the contribution of spinal interneurons. In the present work, we applied multiplexing detection of transcripts and machine learning-based image analysis to investigate the fate of multiple spinal interneuron populations during ALS progression in the SOD1 ^G93A mouse model. The analysis showed that spinal inhibitory interneurons are affected early in disease, before motor neurons death, and are characterized by a slow progressive degeneration, while excitatory interneurons are affected later with a steep progression. Moreover, we report differential vulnerability within inhibitory and excitatory subpopulations, with interneurons directly projecting onto motor neurons being preferentially affected. Our study reveals a strong interneuron involvement in ALS development with interneuron specific degeneration. This points to differential involvement of diverse spinal neuronal circuits that eventually may be determining for motor neuron degeneration. Teaser: A new approach to study the motor neuron disorder Amyotrophic Lateral Sclerosis shows the early and differential dysregulation of spinal interneurons.

Arrest of ongoing movements is an integral part of executing motor programs. Behavioral arrest may happen upon termination of a variety of goal-directed movements or as a global motor arrest either in the context of fear or in response to salient environmental cues. The neuronal circuits that bridge with the executive motor circuits to implement a global motor arrest are poorly understood. We report the discovery that the activation of glutamatergic Chx10-derived neurons in the pedunculopontine nucleus (PPN) in mice arrests all ongoing movements while simultaneously causing apnea and bradycardia. This global motor arrest has a pause-and-play pattern with an instantaneous interruption of movement followed by a short-latency continuation from where it was paused. Mice naturally perform arrest bouts with the same combination of motor and autonomic features. The Chx10-PPN-evoked arrest is different to ventrolateral periaqueductal gray-induced freezing. Our study defines a motor command that induces a global motor arrest, which may be recruited in response to salient environmental cues to allow for a preparatory or arousal state, and identifies a locomotor-opposing role for rostrally biased glutamatergic neurons in the PPN.