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.

High-throughput gait acquisition system for freely moving mice.

Normal and pathological locomotion can be discriminated by analyzing an animal's gait on a linear walkway. This step is labor intensive and introduces experimental bias due to the handling involved while placing and removing the animal between trials. We designed a system consisting of a runway embedded within a larger arena, which can be traversed ad libitum by unsupervised, freely moving mice, triggering the recording of short clips of locomotor activity. Multiple body parts were tracked using DeepLabCut and fed to an analysis pipeline (GaitGrapher) to extract gait metrics. We compared the results from unsupervised against the standard experimenter-supervised approach and found that gait parameters analyzed via the new approach were similar to a previously validated approach (Visual Gait Lab). These data show the utility of incorporating an unsupervised, automated, approach for collecting kinematic data for gait analysis.NEW & NOTEWORTHY The acquisition and analysis of walkway data is a time-consuming task. Here, we provide an unmonitored approach for collecting gait metrics that reduces the handling and stress of mice and saves time. A detailed pipeline is outlined that provides for the collection and analysis of data using an integrated suite of tools.

The Mesencephalic Locomotor Region: Beyond Locomotor Control.

The mesencephalic locomotor region (MLR) was discovered several decades ago in the cat. It was functionally defined based on the ability of low threshold electrical stimuli within a region comprising the cuneiform and pedunculopontine nucleus to evoke locomotion. Since then, similar regions have been found in diverse vertebrate species, including the lamprey, skate, rodent, pig, monkey, and human. The MLR, while often viewed under the lens of locomotion, is involved in diverse processes involving the autonomic nervous system, respiratory system, and the state-dependent activation of motor systems. This review will discuss the pedunculopontine nucleus and cuneiform nucleus that comprises the MLR and examine their respective connectomes from both an anatomical and functional angle. From a functional perspective, the MLR primes the cardiovascular and respiratory systems before the locomotor activity occurs. Inputs from a variety of higher structures, and direct outputs to the monoaminergic nuclei, allow the MLR to be able to respond appropriately to state-dependent locomotion. These state-dependent effects are roughly divided into escape and exploratory behavior, and the MLR also can reinforce the selection of these locomotor behaviors through projections to adjacent structures such as the periaqueductal gray or to limbic and cortical regions. Findings from the rat, mouse, pig, and cat will be discussed to highlight similarities and differences among diverse species.

Hop Mice Display Synchronous Hindlimb Locomotion and a Ventrally Fused Lumbar Spinal Cord Caused by a Point Mutation in Ttc26.

Identifying the spinal circuits controlling locomotion is critical for unravelling the mechanisms controlling the production of gaits. Development of the circuits governing left-right coordination relies on axon guidance molecules such as ephrins and netrins. To date, no other class of proteins have been shown to play a role during this process. Here, we have analyzed hop mice, which walk with a characteristic hopping gait using their hindlimbs in synchrony. Fictive locomotion experiments suggest that a local defect in the ventral spinal cord contributes to the aberrant locomotor phenotype. Hop mutant spinal cords had severe morphologic defects, including the absence of the ventral midline and a poorly defined border between white and gray matter. The hop mice represent the first model where, exclusively found in the lumbar domain, the left and right components of the central pattern generators (CPGs) are fused with a synchronous hindlimb gait as a functional consequence. These defects were associated with abnormal developmental processes, including a misplaced notochord and reduced induction of ventral progenitor domains. Whereas the underlying mutation in hop mice has been suggested to lie within the Ttc26 gene, other genes in close vicinity have been associated with gait defects. Mouse embryos carrying a CRISPR replicated point mutation within Ttc26 displayed an identical morphologic phenotype. Thus, our data suggest that the assembly of the lumbar CPG network is dependent on fully functional TTC26 protein.

Contributions of h- and Na+/K+ Pump Currents to the Generation of Episodic and Continuous Rhythmic Activities.

Developing spinal motor networks produce a diverse array of outputs, including episodic and continuous patterns of rhythmic activity. Variation in excitability state and neuromodulatory tone can facilitate transitions between episodic and continuous rhythms; however, the intrinsic mechanisms that govern these rhythms and their transitions are poorly understood. Here, we tested the capacity of a single central pattern generator (CPG) circuit with tunable properties to generate multiple outputs. To address this, we deployed a computational model composed of an inhibitory half-center oscillator (HCO). Following predictions of our computational model, we tested the contributions of key properties to the generation of an episodic rhythm produced by isolated spinal cords of the newborn mouse. The model recapitulates the diverse state-dependent rhythms evoked by dopamine. In the model, episodic bursting depended predominantly on the endogenous oscillatory properties of neurons, with Na+/K+ ATPase pump (I Pump) and hyperpolarization-activated currents (I h ) playing key roles. Modulation of either I Pump or I h produced transitions between episodic and continuous rhythms and silence. As maximal activity of I Pump decreased, the interepisode interval and period increased along with a reduction in episode duration. Decreasing maximal conductance of I h decreased episode duration and increased interepisode interval. Pharmacological manipulations of I h with ivabradine, and I Pump with ouabain or monensin in isolated spinal cords produced findings consistent with the model. Our modeling and experimental results highlight key roles of I h and I Pump in producing episodic rhythms and provide insight into mechanisms that permit a single CPG to produce multiple patterns of rhythmicity.

Fiber photometry for monitoring cerebral oxygen saturation in freely-moving rodents.

Hemodynamic parameters, such as tissue oxygen saturation and blood volume fraction, are important markers of brain physiology. They are also widely used surrogate markers of electrophysiological activity. Here, we present a single fiber spectroscopic (SFS) system for monitoring cerebral oxygen saturation in localized, non-line-of-sight brain regions in freely-moving rodents. We adapted the implantation ferrule and patch cable design from commercialized optogenetics and fiber photometry systems, enabling stereotaxic fiber implantation, longitudinal tissue access and measurement from freely-moving animals. The optical system delivers and collects light from the brain through a 200 µm-core-diameter, 0.39NA multimode fiber. We robustly measured oxygen saturation from phantoms with different optical properties mimicking brain tissue. In mice, we demonstrated, for the first time, measurements of oxygen saturation from a highly-localized, targeted brain region over 31 days and continuous measurements from a freely-moving animal for over an hour. These results suggest that single fiber spectroscopy has enormous potential for functional brain monitoring and investigating neurovascular coupling in freely-moving animals. In addition, this technique can potentially be combined with fiber photometry systems to correct for hemodynamic artifacts in the fluorescence detection.

A dynamic role for dopamine receptors in the control of mammalian spinal networks.

Dopamine is well known to regulate movement through the differential control of direct and indirect pathways in the striatum that express D1 and D2 receptors respectively. The spinal cord also expresses all dopamine receptors; however, how the specific receptors regulate spinal network output in mammals is poorly understood. We explore the receptor-specific mechanisms that underlie dopaminergic control of spinal network output of neonatal mice during changes in spinal network excitability. During spontaneous activity, which is a characteristic of developing spinal networks operating in a low excitability state, we found that dopamine is primarily inhibitory. We uncover an excitatory D1-mediated effect of dopamine on motoneurons and network output that also involves co-activation with D2 receptors. Critically, these excitatory actions require higher concentrations of dopamine; however, analysis of dopamine concentrations of neonates indicates that endogenous levels of spinal dopamine are low. Because endogenous levels of spinal dopamine are low, this excitatory dopaminergic pathway is likely physiologically-silent at this stage in development. In contrast, the inhibitory effect of dopamine, at low physiological concentrations is mediated by parallel activation of D2, D3, D4 and α2 receptors which is reproduced when endogenous dopamine levels are increased by blocking dopamine reuptake and metabolism. We provide evidence in support of dedicated spinal network components that are controlled by excitatory D1 and inhibitory D2 receptors that is reminiscent of the classic dopaminergic indirect and direct pathway within the striatum. These results indicate that network state is an important factor that dictates receptor-specific and therefore dose-dependent control of neuromodulators on spinal network output and advances our understanding of how neuromodulators regulate neural networks under dynamically changing excitability.

The Effects of a Ketogenic Diet on Sensorimotor Function in a Thoracolumbar Mouse Spinal Cord Injury Model.

Spinal cord injury and peripheral nerve injuries are traumatic events that greatly impact quality of life. One factor that is being explored throughout patient care is the idea of diet and the role it has on patient outcomes. But the effects of diet following neurotrauma need to be carefully explored in animal models to ensure that they have beneficial effects. The ketogenic diet provides sufficient daily caloric requirements while being potentially neuroprotective and analgesic. In this study, animals were fed a high-fat, low-carbohydrate diet that led to a high concentration of blood ketone that was sustained for as long as the animals were on the diet. Mice fed a ketogenic diet had significantly lower levels of tyrosine and tryptophan, but the levels of other monoamines within the spinal cord remained similar to those of control mice. Mice were fed a standard or ketogenic diet for 7 d before and 28 d following the injury. Our results show that mice hemisected over the T10-T11 vertebrae showed no beneficial effects of being on a ketogenic diet over a 28 d recovery period. Similarly, ligation of the common peroneal and tibial nerve showed no differences between mice fed normal or ketogenic diets. Tests included von Frey, open field, and ladder-rung crossing. We add to existing literature showing protective effects of the ketogenic diet in forelimb injuries by focusing on neurotrauma in the hindlimbs. The results suggest that ketogenic diets need to be assessed based on the type and location of neurotrauma.

Visual Gait Lab: A user-friendly approach to gait analysis.

BACKGROUND: Gait analysis forms a critical part of many lab workflows, ranging from those interested in preclinical neurological models to others who use locomotion as part of a standard battery of tests. Unfortunately, while paw detection can be semi-automated, it becomes generally a time-consuming process with error corrections. Improvement in paw tracking would aid in better gait analysis performance and experience. NEW METHOD: Here we show the use of Visual Gait Lab (VGL), a high-level software with an intuitive, easy to use interface, that is built on DeepLabCut™. VGL is optimized to generate gait metrics and allows for quick manual error corrections. VGL comes with a single executable, streamlining setup on Windows systems. We demonstrate the use of VGL to analyze gait. RESULTS: Training and evaluation of VGL were conducted using 200 frames (80/20 train-test split) of video from mice walking on a treadmill. The trained network was then used to visually track paw placements to compute gait metrics. These are processed and presented on the screen where the user can rapidly identify and correct errors. COMPARISON WITH EXISTING METHODS: Gait analysis remains cumbersome, even with commercial software due to paw detection errors. DeepLabCut™ is an alternative that can improve visual tracking but is not optimized for gait analysis functionality. CONCLUSIONS: VGL allows for gait analysis to be performed in a rapid, unbiased manner, with a set-up that can be easily implemented and executed by those without a background in computer programming.

An economical solution to record and control wheel-running for group-housed mice.

BACKGROUND: The effects of exercise on brain function are widely known; however, there is a need for inexpensive, practical solutions for monitoring and metering the activity of multiple mice. NEW METHOD: A contoured running wheel that has a built-in radio-frequency identification (RFID) receiver to monitor the activity of several mice in a single cage is presented. This system is scalable , the interface is easy to use, and the wheel can be dynamically locked so that each group-housed mouse receives a set exercise regimen. RESULTS: We were able to reliably monitor three mice that were group-housed. We were able to reliably meter the amount of exercise performed by the mice using the servo-controlled lock. COMPARISON WITH EXISTING METHODS: Current methods allow a wheel to be locked when a set distance is reached. However, an issue with this method is that the set distance includes the cumulative activity of all mice in the cage so one mouse could contribute a disproportionate amount to the total distance. Our solution ensures that the wheel is locked when an individual mouse reaches the target distance, but remains unlocked for individuals that have not reached the programmed distance. CONCLUSIONS: The dynamic locking wheel (DynaLok) is designed to allow a researcher to provide individually designed exercise plans for multi-housed mice; therefore, users are able to house mice conventionally rather than in individual cages. DynaLok reduces animal housing costs, allows for new experimental exercise regimens to be developed, and is scalable and cost-effective.

Orexinergic Modulation of Spinal Motor Activity in the Neonatal Mouse Spinal Cord.

The role of orexin during development, and especially in terms of spinal cord function, is not well understood. It is for this reason that we focused on the network actions of orexin during the first week of development. We found that orexinergic fibers were present in the lumbar spinal cord of postnatal day 0 (P0) to P3 mice. The fibers were expressed mainly in the dorsal horn, but occasional fibers were observed in the ventral horn. Both orexin (OX) A and OXB increased the motoneurons (MNs) tonic neurogram discharge. However, only OXA was found to significantly increase spontaneous bursting activity and the frequency of fictive locomotor bursts. We show that OXA is able to act directly on MNs. To test the contribution of the recurrent MN collaterals, we blocked the nicotinic cholinergic drive and observed that OXA retained its ability to increase fictive locomotor activity. Additionally, we recorded neurograms from ventral lateral funiculi, where OXA had no effect on population discharge. These effects were also confirmed by recording from descending commissural interneurons via patch recordings. The loci of the effects of OXA were further investigated in a dorsal horn-removed preparation where OXA also shows an increase in the discharge from ventral root neurograms but no increase in the frequency of spontaneous or fictive locomotion burst activity. In summary, multiple lines of evidence from our work demonstrate the robust effects of orexins on spinal cord networks and MNs at the time of birth.

Optogenetic Activation of A11 Region Increases Motor Activity.

Limbic brain regions drive goal-directed behaviors. These behaviors often require dynamic motor responses, but the functional connectome of limbic structures in the diencephalon that control locomotion is not well known. The A11 region, within the posterior diencephalon has been postulated to contribute to motor function and control of pain. Here we show that the A11 region initiates movement. Photostimulation of channelrhodopsin 2 (ChR2) transfected neurons in A11 slice preparations showed that neurons could follow stimulation at frequencies of 20 Hz. Our data show that photostimulation of ChR2 transfected neurons in the A11 region enhances motor activity often leading to locomotion. Using vGluT2-reporter and vGAT-reporter mice we show that the A11 tyrosine hydroxylase positive (TH) dopaminergic neurons are vGluT2 and vGAT negative. We find that in addition to dopaminergic neurons within the A11 region, there is another neuronal subtype which expresses the monoenzymatic aromatic L-amino acid decarboxylase (AADC), but not TH, a key enzyme involved in the synthesis of catecholamines including dopamine. This monoaminergic-based motor circuit may be involved in the control of motor behavior as part of a broader diencephalic motor region.

Parallel descending dopaminergic connectivity of A13 cells to the brainstem locomotor centers.

The mesencephalic locomotor region (MLR) is an important integrative area for the initiation and modulation of locomotion. Recently it has been realized that dopamine (DA) projections from the substantia nigra pars compacta project to the MLR. Here we explore DA projections from an area of the medial zona incerta (ZI) known for its role in motor control onto the MLR. We provide evidence that dopaminergic (DAergic) A13 neurons have connectivity to the cuneiform nucleus (CnF) and pedunculopontine tegmental nucleus (PPTg) of the MLR. No ascending connectivity to the dorsolateral striatum was observed. On the other hand, DAergic A13 projections to the medullary reticular formation (MRF) and the lumbar spinal cord were sparse. A small number of non-DAergic neurons within the medial ZI projected to the lumbar spinal cord. We then characterized the DA A13 cells and report that these cells differ from canonical DA neurons since they lack the Dopamine Transporter (DAT). The lack of DAT expression, and possibly the lack of a dopamine reuptake mechanism, points to a longer time of action compared to typical dopamine neurons. Collectively our data suggest a parallel descending DAergic pathway from the A13 neurons of the medial ZI to the MLR, which we expect is important for modulating movement.

Single-Cell Transcriptomics and Fate Mapping of Ependymal Cells Reveals an Absence of Neural Stem Cell Function.

Ependymal cells are multi-ciliated cells that form the brain's ventricular epithelium and a niche for neural stem cells (NSCs) in the ventricular-subventricular zone (V-SVZ). In addition, ependymal cells are suggested to be latent NSCs with a capacity to acquire neurogenic function. This remains highly controversial due to a lack of prospective in vivo labeling techniques that can effectively distinguish ependymal cells from neighboring V-SVZ NSCs. We describe a transgenic system that allows for targeted labeling of ependymal cells within the V-SVZ. Single-cell RNA-seq revealed that ependymal cells are enriched for cilia-related genes and share several stem-cell-associated genes with neural stem or progenitors. Under in vivo and in vitro neural-stem- or progenitor-stimulating environments, ependymal cells failed to demonstrate any suggestion of latent neural-stem-cell function. These findings suggest remarkable stability of ependymal cell function and provide fundamental insights into the molecular signature of the V-SVZ niche.

Marking the differences in motoneurons.

A subgroup of the neurons that control muscles becomes less excitable shortly before the symptoms of ALS develop.

Integration of Descending Command Systems for the Generation of Context-Specific Locomotor Behaviors.

Over the past decade there has been a renaissance in our understanding of spinal cord circuits; new technologies are beginning to provide key insights into descending circuits which project onto spinal cord central pattern generators. By integrating work from both the locomotor and animal behavioral fields, we can now examine context-specific control of locomotion, with an emphasis on descending modulation arising from various regions of the brainstem. Here we examine approach and avoidance behaviors and the circuits that lead to the production and arrest of locomotion.

Decerebrate mouse model for studies of the spinal cord circuits.

The adult decerebrate mouse model (a mouse with the cerebrum removed) enables the study of sensory-motor integration and motor output from the spinal cord for several hours without compromising these functions with anesthesia. For example, the decerebrate mouse is ideal for examining locomotor behavior using intracellular recording approaches, which would not be possible using current anesthetized preparations. This protocol describes the steps required to achieve a low-blood-loss decerebration in the mouse and approaches for recording signals from spinal cord neurons with a focus on motoneurons. The protocol also describes an example application for the protocol: the evocation of spontaneous and actively driven stepping, including optimization of these behaviors in decerebrate mice. The time taken to prepare the animal and perform a decerebration takes ∼2 h, and the mice are viable for up to 3-8 h, which is ample time to perform most short-term procedures. These protocols can be modified for those interested in cardiovascular or respiratory function in addition to motor function and can be performed by trainees with some previous experience in animal surgery.

Modulation of Rhythmic Activity in Mammalian Spinal Networks Is Dependent on Excitability State.

Neuromodulators play an important role in activating rhythmically active motor networks; however, what remains unclear are the network interactions whereby neuromodulators recruit spinal motor networks to produce rhythmic activity. Evidence from invertebrate systems has demonstrated that the effect of neuromodulators depends on the pre-existing state of the network. We explored how network excitation state affects the ability of dopamine to evoke rhythmic locomotor activity in the neonatal mouse isolated spinal cord. We found that dopamine can evoke unique patterns of motor activity that are dependent on the excitability state of motor networks. Different patterns of motor activity ranging from tonic, nonrhythmic activity to multirhythmic, nonlocomotor activity to locomotor activity were produced by altering global motor network excitability through manipulations of the extracellular potassium and bath NMDA concentration. A similar effect was observed when network excitation was manipulated during an unstable multirhythm evoked by a low concentration (15 µm) of 5-HT, suggesting that our results are not neuromodulator specific. Our data show in vertebrate systems that modulation is a two-way street and that modulatory actions are largely influenced by the network state. The level of network excitation can account for variability between preparations and is an additional factor to be considered when circuit elements are removed from the network.

AlphaB-crystallin regulates remyelination after peripheral nerve injury.

AlphaB-crystallin (αBC) is a small heat shock protein that is constitutively expressed by peripheral nervous system (PNS) axons and Schwann cells. To determine what role this crystallin plays after peripheral nerve damage, we found that loss of αBC impaired remyelination, which correlated with a reduced presence of myelinating Schwann cells and increased numbers of nonmyelinating Schwann cells. The heat shock protein also seems to regulate the cross-talk between Schwann cells and axons, because expected changes in neuregulin levels and ErbB2 receptor expression after PNS injury were disrupted in the absence of αBC. Such dysregulations led to defects in conduction velocity and motor and sensory functions that could be rescued with therapeutic application of the heat shock protein in vivo. Altogether, these findings show that αBC plays an important role in regulating Wallerian degeneration and remyelination after PNS injury.

Dopaminergic modulation of locomotor network activity in the neonatal mouse spinal cord.

Dopamine is now well established as a modulator of locomotor rhythms in a variety of developing and adult vertebrates. However, in mice, while all five dopamine receptor subtypes are present in the spinal cord, it is unclear which receptor subtypes modulate the rhythm. Dopamine receptors can be grouped into two families-the D1/5 receptor group and the D2/3/4 group, which have excitatory and inhibitory effects, respectively. Our data suggest that dopamine exerts contrasting dose-dependent modulatory effects via the two receptor families. Our data show that administration of dopamine at concentrations >35 µM slowed and increased the regularity of a locomotor rhythm evoked by bath application of 5-hydroxytryptamine (5-HT) and N-methyl-d(l)-aspartic acid (NMA). This effect was independent of the baseline frequency of the rhythm that was manipulated by altering the NMA concentration. We next examined the contribution of the D1- and D2-like receptor families on the rhythm. Our data suggest that the D1-like receptor contributes to enhancement of the stability of the rhythm. Overall, the D2-like family had a pronounced slowing effect on the rhythm; however, quinpirole, the D2-like agonist, also enhanced rhythm stability. These data indicate a receptor-dependent delegation of the modulatory effects of dopamine on the spinal locomotor pattern generator.