Activity of Spinal Interneurons during Forward and Backward Locomotion.

Higher vertebrates are capable not only of forward but also backward and sideways locomotion. Also, single steps in different directions are generated for postural corrections. While the networks responsible for the control of forward walking (FW) have been studied in considerable detail, the networks controlling steps in other directions are mostly unknown. Here, to characterize the operation of the spinal locomotor network during FW and backward walking (BW), we recorded the activity of individual spinal interneurons from L4 to L6 during both FW and BW evoked by epidural stimulation (ES) of the spinal cord at L5-L6 in decerebrate cats of either sex. Three groups of neurons were revealed. Group 1 (45%) had a similar phase of modulation during both FW and BW. Group 2 (27%) changed the phase of modulation in the locomotor cycle depending on the direction of locomotion. Group 3 neurons were modulated during FW only (Group 3a, 21%) or during BW only (Group 3b, 7%). We suggest that Group 1 neurons belong to the network generating the vertical component of steps (the limb elevation and lowering) because it should operate similarly during locomotion in any direction, while Groups 2 and 3 neurons belong to the networks controlling the direction of stepping. Results of this study provide new insights into the organization of the spinal locomotor circuits, advance our understanding of ES therapeutic effects, and can potentially be used for the development of novel strategies for recuperation of impaired balance control, which requires the generation of corrective steps in different directions.SIGNIFICANCE STATEMENT Animals and humans can perform locomotion in different directions in relation to the body axis (forward, backward, sideways). While the networks that control forward walking have been studied in considerable detail, the networks controlling steps in other directions are unknown. Here, by recording the activity of the same spinal neurons during forward and backward walking, we revealed three groups of neurons forming, respectively, the network operating similarly during stepping in different directions, the network changing its operation with a change in the direction of stepping, and the network operating only during locomotion in a specific direction. These networks presumably control different aspects of the step. The obtained results provide new insights into the organization of the spinal locomotor networks.

Comparison of operation of spinal locomotor networks activated by supraspinal commands and by epidural stimulation of the spinal cord in cats.

KEY POINTS: Epidural electrical stimulation (ES) of the spinal cord restores/improves locomotion in patients. ES-evoked locomotor movements differ to some extent from the normal ones. Operation of the locomotor network during ES is unknown. We compared the activity of individual spinal neurons during locomotion initiated by signals from the brainstem and by ES. We demonstrated that the spinal network generating locomotion under each of the two conditions is formed by the same neurons. A part of this network operates similarly under the two conditions, suggesting that it is essential for generation of locomotion under both conditions. Another part of this network operates differently under the two conditions, suggesting that it is responsible for differences in the movement kinematics observed under the two conditions. ABSTRACT: Locomotion is a vital motor function for both animals and humans. Epidural electrical stimulation (ES) of the spinal cord is used to restore/improve locomotor movements in patients. However, operation of locomotor networks during ES has never been studied. Here we compared the activity of individual spinal neurons recorded in decerebrate cats of either sex during locomotion initiated by supraspinal commands (caused by stimulation of the mesencephalic locomotor region, MLR) and by ES. We found that under both conditions, the same neurons had modulation of their activity related to the locomotor rhythm, suggesting that the network generating locomotion under the two conditions is formed by the same neurons. About 40% of these neurons had stable modulation (i.e. small dispersion of their activity phase in sequential cycles), as well as a similar phase and shape of activity burst in MLR- and ES-evoked locomotor cycles. We suggest that these neurons form a part of the locomotor network that operates similarly under the two conditions, and are critical for generation of locomotion. About 23% of the modulated neurons had stable modulation only during MLR-evoked locomotion. We suggest that these neurons are responsible for some differences in kinematics of MLR- and ES-evoked locomotor movements. Finally, 25% of the modulated neurons had unstable modulation during both MLR- and ES-evoked locomotion. One can assume that these neurons contribute to maintenance of the excitability level of locomotor networks necessary for generation of stepping, or belong to postural networks, activated simultaneously with locomotor networks by both MLR stimulation and ES.

Limb and trunk mechanisms for balance control during locomotion in quadrupeds.

In quadrupeds, the most critical aspect of postural control during locomotion is lateral stability. However, neural mechanisms underlying lateral stability are poorly understood. Here, we studied lateral stability in decerebrate cats walking on a treadmill with their hindlimbs. Two destabilizing factors were used: a brief lateral push of the cat and a sustained lateral tilt of the treadmill. It was found that the push caused considerable trunk bending and twisting, as well as changes in the stepping pattern, but did not lead to falling. Due to postural reactions, locomotion with normal body configuration was restored in a few steps. It was also found that the decerebrate cat could keep balance during locomotion on the laterally tilted treadmill. This postural adaptation was based on the transformation of the symmetrical locomotor pattern into an asymmetrical one, with different functional lengths of the right and left limbs. Then, we analyzed limb and trunk neural mechanisms contributing to postural control during locomotion. It was found that one of the limb mechanisms operates in the transfer phase and secures a standard (relative to the trunk) position for limb landing. Two other limb mechanisms operate in the stance phase; they counteract distortions of the locomotor pattern by regulating the limb stiffness. The trunk configuration mechanism controls the body shape on the basis of sensory information coming from trunk afferents. We suggest that postural reactions generated by these four mechanisms are integrated, thus forming a response of the whole system to perturbation of balance during locomotion.

Spinal and supraspinal control of the direction of stepping during locomotion.

Most bipeds and quadrupeds, in addition to forward walking, are also capable of backward and sideward walking. The direction of walking is determined by the direction of stepping movements of individual limbs in relation to the front-to-rear body axis. Our goal was to assess the functional organization of the system controlling the direction of stepping. Experiments were performed on decerebrate cats walking on the treadmill with their hindlimbs, whereas the head and trunk were rigidly fixed. Different directions of the treadmill motion relative to the body axis were used (0, ± 45, ± 90, and 180°). For each direction, we compared locomotion evoked from the brainstem (by stimulation of the mesencephalic locomotor region, MLR) with locomotion evoked by epidural stimulation of the spinal cord (SC). It was found that SC stimulation evoked well coordinated stepping movements at different treadmill directions. The direction of steps was opposite to the treadmill motion, suggesting that this direction was determined by sensory input from the limb during stance. Thus, SC stimulation activates limb controllers, which are able to generate stepping movements in different directions. By contrast, MLR stimulation evoked well coordinated stepping movements only if the treadmill was moving in the front-to-rear direction. One can conclude that supraspinal commands (caused by MLR stimulation) select one of the numerous forms of operation of the spinal limb controllers, namely, the forward walking. The MLR can thus be considered as a command center for forward locomotion, which is the main form of progression in bipeds and quadrupeds.

Postnatal growth of the lumbosacral spinal segments in cat: Their lengths and positions in relation to vertebrae.

Cat is a prominent model for investigating neural networks of the lumbosacral spinal cord that control locomotor and visceral activity. We previously proposed an integral function, establishing the topographical relationship between the spinal cord segments and vertebrae in adult animals. Here, we investigated the dynamic of this topographical relationship through early and middle periods of development in kittens. We calculated the length of each vertebra relative to the total length of the region from 13th thoracic (T) to the 7th lumbar (L) vertebrae (V) as well as the length of each segment relative to the total region from T13 to the three-dimensional sacral (S) segment. As in our previous work, the length and position of VL2 were used to establish relationships between the characteristics of the segments and vertebrae. Cubic regression reliably approximates the lengths of segments relative to VL2 length. As the cat aged, the relative length of VT13 and VL1 decreased while the relative length of VL5 increased. The relative length of the T13 and L3 segments increased while the relative length of the S1-S2 segments decreased. The T13-L2 segments are descended monotonically relative to the VL1-VL2 border. The L3-S1 segments are also descended, though with more complex dynamics. The positions of the S2-S3 segments remained unchanged. To conclude, different spinal segments displayed different developmental dynamics. The revealed relationship between vertebrae and lumbosacral spinal segments may be helpful for clearly defining stimulation regions to invoke particular functions, both in experimental studies on the spinal cord and clinical treatment.

Alteration of Postural Reactions in Rats with Different Levels of Dopamine Depletion.

Dopamine (DA) is the critical neurotransmitter involved in the unconscious control of muscle tone and body posture. We evaluated the general motor capacities and muscle responses to postural disturbance in three conditions: normal DA level (wild-type rats, WT), mild DA deficiency (WT after administration of α-methyl-p-tyrosine-AMPT, that blocks DA synthesis), and severe DA depletion (DAT-KO rats after AMPT). The horizontal displacements in WT rats elicited a multi-component EMG corrective response in the flexor and extensor muscles. Similar to the gradual progression of DA-related diseases, we observed different degrees of bradykinesia, rigidity, and postural instability after AMPT. The mild DA deficiency impaired the initiation pattern of corrective responses, specifically delaying the extensor muscles' activity ipsilaterally to displacement direction and earlier extensor activity from the opposite side. DA depletion in DAT-KO rats after AMPT elicited tremors, general stiffness, and akinesia, and caused earlier response to horizontal displacements in the coactivated flexor and extensor muscles bilaterally. The data obtained show the specific role of DA in postural reactions and suggest that this experimental approach can be used to investigate sensorimotor control in different dopamine-deficient states and to model DA-related diseases.

Trace Amine-Associated Receptor 2 Is Expressed in the Limbic Brain Areas and Is Involved in Dopamine Regulation and Adult Neurogenesis.

Trace amines are a group of biogenic amines that are structurally and functionally close to classical monoamine neurotransmitters. Trace amine-associated receptors (TAARs) are emerging as promising targets for treating neuropsychiatric disorders. It has been documented that all TAARs, apart from TAAR1, function as olfactory receptors involved in sensing innate odors encoded by volatile amines. However, recently, brain expression and function of TAAR5 were also demonstrated. In this study, we assessed the behavior, brain neurochemistry, and electrophysiology changes in knock-out mice lacking Trace amine-associated receptor 2 (TAAR2) but expressing beta-Galactosidase mapping expression of TAAR2 receptors. As expected, we detected beta-Galactosidase staining in the glomerular layer of the olfactory bulb. However, we also found staining in the deeper layers of the olfactory bulb and several brain regions, including the hippocampus, cerebellum, cortex, raphe nuclei, hypothalamus, and habenula, indicating that TAAR2 receptors are not only expressed in the olfactory system but are also present in the limbic brain areas that receive olfactory input. In behavioral experiments, TAAR2 knock-out (TAAR2-KO) mice showed increased locomotor activity and less immobility in the forced swim test, with no changes in anxiety level. Furthermore, TAAR2-KO mice showed alterations in brain electrophysiological activity-particularly, decreased spectral power of the cortex and striatum in the 0, 9-20 Hz range. TAAR2-KO mice also had elevated tissue dopamine levels in the striatum and an increased dopaminergic neuron number in the Substantia Nigra. In addition, an increased brain-derived neurotrophic factor (BDNF) mRNA level in the striatum and Monoamine Oxidase B (MAO-B) mRNA level in the striatum and midbrain was found in TAAR2-KO mice. Importantly, TAAR2-KO mice demonstrated an increased neuroblast-like and proliferating cell number in the subventricular and subgranular zone, indicating increased adult neurogenesis. These data indicate that in addition to its role in the innate olfaction of volatile amines, TAAR2 is expressed in limbic brain areas and regulates the brain dopamine system, neuronal electrophysiological activity, and adult neurogenesis. These findings further corroborated observations in TAAR1-KO and TAAR5-KO mice, indicating common for TAAR family pattern of expression in limbic brain areas and role in regulating monoamine levels and adult neurogenesis, but with variable involvement of each subtype of TAAR receptors in these functions.

Effects of Alpha-2 Adrenergic Agonist Mafedine on Brain Electrical Activity in Rats after Traumatic Brain Injury.

The search for and development of new neuroprotective (or cerebroprotective) drugs, as well as suitable methods for their preclinical efficacy evaluation, are priorities for current biomedical research. Alpha-2 adrenergic agonists, such as mafedine and dexmedetomidine, are a highly appealing group of drugs capable of reducing neurological deficits which result from brain trauma and vascular events in both experimental animals and human patients. Thus, our aim was to assess the effects of mafedine and dexmedetomidine on the brain's electrical activity in a controlled cortical-impact model of traumatic brain injury (TBI) in rats. The functional status of the animals was assessed by electrocorticography (ECoG), using ECoG electrodes which were chronically implanted in different cortical regions. The administration of intraperitoneal mafedine sodium at 2.5 mg∙kg-1 at 1 h after TBI induction, and daily for the following 6 days, restored interhemispheric connectivity in remote brain regions and intrahemispheric connections within the unaffected hemisphere at post-TBI day 7. Animals that had received mafedine sodium also demonstrated an improvement in cortical responses to photic and somatosensory stimulation. Dexmedetomidine at 25 µg∙kg-1 did not affect the brain's electrical activity in brain-injured rats. Our results confirm the previously described neuroprotective effects of mafedine sodium and suggest that ECoG registration and analysis are a viable method evaluating drug efficacy in experimental animal models of TBI.

Increased dopamine transmission and adult neurogenesis in trace amine-associated receptor 5 (TAAR5) knockout mice.

Trace amine-associated receptors (TAARs) are a class of sensory G protein-coupled receptors that detect biogenic amines, products of decarboxylation of amino acids. The majority of TAARs (TAAR2-TAAR9) have been described mainly in the olfactory epithelium and considered to be olfactory receptors sensing innate odors. However, there is recent evidence that one of the members of this family, TAAR5, is expressed also in the limbic brain areas receiving projection from the olfactory system and involved in the regulation of emotions. In this study, we further characterized a mouse line lacking TAAR5 (TAAR5 knockout, TAAR5-KO mice) that express beta-galactosidase mapping TAAR5 expression. We found that in TAAR5-KO mice the number of dopamine neurons, the striatal levels of dopamine and its metabolites, as well as striatal levels of GDNF mRNA, are elevated indicating a potential increase in dopamine neuron proliferation. Furthermore, an analysis of TAAR5 beta-galactosidase expression revealed that TAAR5 is present in the major neurogenic areas of the brain such as the subventricular zone (SVZ), the subgranular zone (SGZ) and the less characterized potentially neurogenic zone surrounding the 3rd ventricle. Direct analysis of neurogenesis by using specific markers doublecortin (DCX) and proliferating cell nuclear antigen (PCNA) revealed at least 2-fold increase in the number of proliferating neurons in the SVZ and SGZ of TAAR5-KO mice, but no such markers were detected in mutant or control mice in the areas surrounding the 3rd ventricle. These observations indicate that TAAR5 involved not only in regulation of emotional status but also adult neurogenesis and dopamine transmission. Thus, future TAAR5 antagonists may exert not only antidepressant and/or anxiolytic action but may also provide new treatment opportunity for neurodegenerative disorders such as Parkinson's disease.

Decoding the role of zebrafish neuroglia in CNS disease modeling.

Neuroglia, including microglia and astrocytes, is a critical component of the central nervous system (CNS) that interacts with neurons to modulate brain activity, development, metabolism and signaling pathways. Thus, a better understanding of the role of neuroglia in the brain is critical. Complementing clinical and rodent data, the zebrafish (Danio rerio) is rapidly becoming an important model organism to probe the role of neuroglia in brain disorders. With high genetic and physiological similarity to humans and rodents, zebrafish possess some common (shared), as well as some specific molecular biomarkers and features of neuroglia development and functioning. Studying these common and zebrafish-specific aspects of neuroglia may generate important insights into key brain mechanisms, including neurodevelopmental, neurodegenerative, neuroregenerative and neurological processes. Here, we discuss the biology of neuroglia in humans, rodents and fish, its role in various CNS functions, and further directions of translational research into the role of neuroglia in CNS disorders using zebrafish models.

Nervous mechanisms of locomotion in different directions.

Locomotion, that is active propulsive movement of the body in space, is a vital motor function. Intensive studies of the main, for the majority of living beings, form of locomotion, forward locomotion, have revealed essential features of the organization and operation of underlying neural mechanisms. However, animals and humans are capable to locomote not only forward but also in other directions in relation to the body axis, e.g. backward, sideways, etc. Single steps in different directions are also used for postural corrections during locomotion and during standing. Recent studies of mechanisms underlying control of locomotion in different directions have greatly expanded our knowledge about locomotor system and can contribute to improvement of rehabilitation strategies aimed at restoration of locomotion and balance control in patients. This review outlines recent advances in the studies of locomotion in different directions in lower and higher vertebrates, with special attention given to the neuronal locomotor mechanisms.

Pharmacological screening of a new alpha-2 adrenergic receptor agonist, mafedine, in zebrafish.

Pharmacological agents acting at alpha-2 adrenergic receptors are widely used in physiology and neuroscience research. Mounting evidence of their potential utility in clinical and experimental psychopharmacology, necessitates new models and novel model organisms for their screening. Here, we characterize behavioral effects of mafedine (6-oxo-1-phenyl-2- (phenylamino)-1,6-dihydropyrimidine-4-sodium olate), a novel drug with alpha-2 adrenergic receptor agonistic effects, in adult zebrafish (Danio rerio) in the novel tank test of anxiety and activity. Following an acute 20-min exposure, mafedine at 60 mg/L produced a mild psychostimulant action with some anxiogenic-like effects. Repeated acute 20-min/day administration of mafedine for 7 consecutive days at 1, 5 and 10 mg/L had a similar action on fish behavior as an acute exposure to 60 mg/L. Since mafedine demonstrated robust behavioral effects in zebrafish - a sensitive vertebrate aquatic model, it is likely that it may modulate rodent and human behavior as well. Thus, further studies are needed to explore this possibility in detail, and whether it may foster clinical application of mafedine and related alpha-2 adrenergic agents.

Prediction Algorithm of the Cat Spinal Segments Lengths and Positions in Relation to the Vertebrae.

Detailed knowledge of the topographic organization and precise access to the spinal cord segments is crucial for the neurosurgical manipulations as well as in vivo neurophysiological investigations of the spinal networks involved in sensorimotor and visceral functions. Because of high individual variability, accurate identification of particular portion of the lumbosacral enlargement is normally possible only during postmortem dissection. Yet, it is often necessary to determine the precise location of spinal segments prior to in vivo investigation, targeting spinal cord manipulations, neurointerface implantations, and neuronal activity recordings. To solve this problem, we have developed an algorithm to predict spinal segments locations based on their relation to vertebral reference points. The lengths and relative positions of the spinal cord segments (T13-S3) and the vertebrae (VT13-VL7) were measured in 17 adult cats. On the basis of these measurements, we elaborated the estimation procedure: the cubic regression of the ratio of the segment's length to the lengths of the VL2 vertebra was used for the determination of segment's length; and the quadratic regression of the ratio of their positions in relation to the VL2 rostral part was used to determine the position of the segments. The coefficients of these regressions were calculated at the training sample (nine cats) and were then confirmed at the testing sample (eight cats). Although the quality of the prediction is decreased in the caudal direction, we found high correlations between the regressions and real data. The proposed algorithm can be further translated to other species including human. Anat Rec, 302:1628-1637, 2019. © 2018 American Association for Anatomy.

Zebrafish models relevant to studying central opioid and endocannabinoid systems.

The endocannabinoid and opioid systems are two interplaying neurotransmitter systems that modulate drug abuse, anxiety, pain, cognition, neurogenesis and immune activity. Although they are involved in such critical functions, our understanding of endocannabinoid and opioid physiology remains limited, necessitating further studies, novel models and new model organisms in this field. Zebrafish (Danio rerio) is rapidly emerging as one of the most effective translational models in neuroscience and biological psychiatry. Due to their high physiological and genetic homology to humans, zebrafish may be effectively used to study the endocannabinoid and opioid systems. Here, we discuss current models used to target the endocannabinoid and opioid systems in zebrafish, and their potential use in future translational research and high-throughput drug screening. Emphasizing the high degree of conservation of the endocannabinoid and opioid systems in zebrafish and mammals, we suggest zebrafish as an excellent model organism to study these systems and to search for the new drugs and therapies targeting their evolutionarily conserved mechanisms.

Activation of spinal locomotor circuits in the decerebrated cat by spinal epidural and/or intraspinal electrical stimulation.

The present study was designed to further compare the stepping-like movements generated via epidural (ES) and/or intraspinal (IS) stimulation. We examined the ability to generate stepping-like movements in response to ES and/or IS of spinal lumbar segments L1-L7 in decerebrate cats. ES (5-10 Hz) of the dorsal surface of the spinal cord at L3-L7 induced hindlimb stepping-like movements on a moving treadmill belt, but with no rhythmic activity in the forelimbs. IS (60 Hz) of the dorsolateral funiculus at L1-L3 (depth of 0.5-1.0mm from the dorsal surface of the spinal cord) induced quadrupedal stepping-like movements. Forelimb movements appeared first, followed by stepping-like movements in the hindlimbs. ES and IS simultaneously enhanced the rhythmic performance of the hindlimbs more robustly than ES or IS alone. The differences in the stimulation parameters, site of stimulation, and motor outputs observed during ES vs. IS suggest that different neural mechanisms were activated to induce stepping-like movements. The effects of ES may be mediated more via dorsal structures in the lumbosacral region of the spinal cord, whereas the effects of IS may be mediated via more ventral propriospinal networks and/or brainstem locomotor areas. Furthermore, the more effective facilitation of the motor output during simultaneous ES and IS may reflect some convergence of pathways on the same interneuronal populations involved in the regulation of locomotion.