Ventral Horn Cells Research Articles

SnRNA-seq reveals the heterogeneity of spinal ventral horn and mechanism of motor neuron axon regeneration

Spinal motor neurons, the distinctive neurons of the central nervous system, extend into the peripheral nervous system and have outstanding ability of axon regeneration after injury. Here, we explored the heterogeneity of spinal ventral horn cells after rat sciatic nerve crush via single-nuclei RNA sequencing. Interestingly, regeneration mainly occurred in a Sncg+ and Anxa2+ motor neuron subtype (MN2) surrounded by a newly emerged microglia subtype (Mg6) after injury. Subsequently, microglia depletion slowed down the regeneration of sciatic nerve. OPCs were also involved into the regeneration process. Knockdown of Cacna2d2 invitro and systemic blocking of Cacna2d2 invivo improved the axon growth ability, hinting us the importance of Ca2+. Ultimately, we proposed three possible phases of motor neuron axon regeneration: preparation stage, early regeneration stage, and regeneration stage. Taken together, our study provided a resource for deciphering the underlying mechanism of motor neuron axon regeneration in a single cell dimension.

The histone demethylase Kdm6b regulates subtype diversification of mouse spinal motor neurons during development

How a single neuronal population diversifies into subtypes with distinct synaptic targets is a fundamental topic in neuroscience whose underlying mechanisms are unclear. Here, we show that the histone H3-lysine 27 demethylase Kdm6b regulates the diversification of motor neurons to distinct subtypes innervating different muscle targets during spinal cord development. In mouse embryonic motor neurons, Kdm6b promotes the medial motor column (MMC) and hypaxial motor column (HMC) fates while inhibiting the lateral motor column (LMC) and preganglionic motor column (PGC) identities. Our single-cell RNA-sequencing analyses reveal the heterogeneity of PGC, LMC, and MMC motor neurons. Further, our single-cell RNA-sequencing data, combined with mouse model studies, demonstrates that Kdm6b acquires cell fate specificity together with the transcription factor complex Isl1-Lhx3. Our study provides mechanistic insight into the gene regulatory network regulating neuronal cell-type diversification and defines a regulatory role of Kdm6b in the generation of motor neuron subtypes in the mouse spinal cord.

Coherence analysis of the calcium activity of putative astrocytic and neuronal cells on the L5 ventral horn and neural output in activated lumbar CPG networks

Astrocytes are thought to play a crucial role in providing structure to the spinal cord and maintaining efficient synaptic function and metabolism because their fine processes envelop the synapses of neurons and form many neuronal networks within the central nervous system (CNS). To investigate whether putative astrocytes and putative neurons distributed on the ventral horn play a role in the modulation of lumbar locomotor central pattern generator (CPG) networks, we used extracellular recording and optical imaging techniques and recorded the neural output from the left L5 ventral root and the calcium activity of putative astrocytes and neurons in the L5 ventral horn at the same time when activating an isolated L1–L5 spinal cord preparation from rats aged 0–2 days. Optical measurements detected cells that showed a fluorescence intensity change under all experimental conditions, namely, (1) 5-HT + NMDA, (2) TTX, and (3) TTX + Low K+. These cells were semiautomatically identified using an in-house MATLAB-based program, as putative astrocytes and neurons according to the cell classification, i.e., increased or decreased fluorescence intensity change (ΔF/F0), and subjective judgment based on their soma size. Coherence and its phase were calculated according to the calcium activity of the putative astrocytes and putative neurons, and neural output was calculated during fictive locomotion with in-house MATLAB-based programs. We found that the number of putative astrocytes activated by applying low K+ tends not to differ from that activated by applying the protease-activated receptor 1 (PAR1) selective agonist TFLLR-NH2 (TFLLR). Moreover, the calcium activity of several putative astrocytes and neurons synchronized with locomotor-like activity at a frequency range below 0.5 Hz and the time lag between peaks of cellular calcium activity and locomotor-like activity ranged from −1000 to + 1000 ms. These findings presumably indicates that these putative astrocytes and neurons in the left L5 ventral horn require −1000 to + 1000 ms to communicate with lumbar CPG networks and maintain efficient synaptic function and metabolism in activated lumbar CPG networks. This finding suggests the possibility that putative astrocytic and neuronal cells in the L5 ventral horn contribute to generating the rhythms and patterns of locomotor-like activity by activated CPG networks in the first to fifth lumbar spinal cord.

TDP-43 is essential for Eph receptor-class-specific spinal motor axon trajectory into the limb

During the neural circuit formation, neuronal growth cones must be guided precisely to their neuronal or muscle targets, which can be achieved by the activation of membrane-bound guidance receptors at the periphery. However, the mechanisms that regulate the temporal availability of these receptors remain largely unknown. TAR DNA binding protein-43 (TDP-43) has been proposed to bind with the mRNAs of guidance receptors, thus prompting us to investigate its role in axon guidance of the spinal lateral motor column (LMC) neurons into the limb mesenchyme. We first identified the TDP-43 expression in the LMC neurons at the stage of axons growth into the limb using in situ mRNA hybridization. The loss and gain of TDP-43 function in chick LMC neurons redirected their axon trajectory with opposite effects. In mice, a spinal motor neuron-specific TDP-43 deletion led to the misrouting of LMC axons. Further, ectopic TDP-43 expression increased EphB protein levels in LMC neurons, suggesting that TDP-43 mediates LMC pathfinding by regulating EphB expression. Finally, TDP-43 levels influenced the growth preference of LMC neurites against ephrin-B, but not Netrin-1 and Semaphorin ligands. Our results demonstrate that TDP-43 is essential for the ephrinB:EphB signaling-mediated axon trajectory selection of LMC subtypes into the limb.

Vav2 is required for Netrin-1 receptor-class-specific spinal motor axon guidance.

Proper guidance of neuronal axons to their targets is required to assemble neural circuits during the development of the nervous system. However, the mechanism by which the guidance of axonal growth cones is regulated by specific intermediaries activated by receptor signaling pathways to mediate cytoskeleton dynamics is unclear. Vav protein members have been proposed to mediate this process, prompting us to investigate their role in the limb selection of the axon trajectory of spinal lateral motor column (LMC) neurons. We found Vav2 and Vav3 expression in LMC neurons when motor axons grew into the limb. Vav2, but not Vav3, loss-of-function perturbed LMC pathfinding, while Vav2 gain-of-function exhibited the opposite effects, demonstrating that Vav2 plays an important role in motor axon growth. Vav2 knockdown also attenuated the redirectional phenotype of LMC axons induced by Dcc, but not EphA4, in vivo and lateral LMC neurite growth preference to Netrin-1 in vitro. This study showed that Vav2 knockdown and ectopic nonphosphorylable Vav2 mutant expression abolished the Src-induced stronger growth preference of lateral LMC neurites to Netrin-1, suggesting that Vav2 is downstream of Src in this context. Vav2 is essential for Netrin-1-regulated LMC motor axon pathfinding through Src interaction.

Paxillin Is Required for Proper Spinal Motor Axon Growth into the Limb.

To assemble the functional circuits of the nervous system, the neuronal axonal growth cones must be precisely guided to their proper targets, which can be achieved through cell-surface guidance receptor activation by ligand binding in the periphery. We investigated the function of paxillin, a focal adhesion protein, as an essential growth cone guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory selection in the limb mesenchyme. Using in situ mRNA detection, we first show paxillin expression in LMC neurons of chick and mouse embryos at the time of spinal motor axon extension into the limb. Paxillin loss-of-function and gain-of-function using in ovo electroporation in chick LMC neurons, of either sex, perturbed LMC axon trajectory selection, demonstrating an essential role of paxillin in motor axon guidance. In addition, a neuron-specific paxillin deletion in mice led to LMC axon trajectory selection errors. We also show that knocking down paxillin attenuates the growth preference of LMC neurites against ephrins in vitro, and erythropoietin-producing human hepatocellular (Eph)-mediated retargeting of LMC axons in vivo, suggesting paxillin involvement in Eph-mediated LMC motor axon guidance. Finally, both paxillin knockdown and ectopic expression of a nonphosphorylable paxillin mutant attenuated the retargeting of LMC axons caused by Src overexpression, implicating paxillin as a Src target in Eph signal relay in this context. In summary, our findings demonstrate that paxillin is required for motor axon guidance and suggest its essential role in the ephrin-Eph signaling pathway resulting in motor axon trajectory selection.SIGNIFICANCE STATEMENT During the development of neural circuits, precise connections need to be established among neurons or between neurons and their muscle targets. A protein family found in neurons, Eph, is essential at different stages of neural circuit formation, including nerve outgrowth and pathfinding, and is proposed to mediate the onset and progression of several neurodegenerative diseases, such as Alzheimer's disease. To investigate how Ephs relay their signals to mediate nerve growth, we investigated the function of a molecule called paxillin and found it important for the development of spinal nerve growth toward their muscle targets, suggesting its role as an effector of Eph signals. Our work could thus provide new information on how neuromuscular connectivity is properly established during embryonic development.

Histamine H3 Receptors Expressed in Ventral Horns Modulate Spinal Motor Output.

Motoneuron activity is modulated by histamine receptors. While H1 and H2 receptors have been widely explored, H3 histamine receptors (H3Rs) have not been sufficiently characterized. This paper targets the effects of the selective activation of H3Rs and their expression on the membranes of large ventral horn cells. The application of selective pharmacological agents to spinal cords isolated from neonatal rats was used to identify the presence of functional H3Rs on the membrane of physiologically identified lumbar motoneurons. Intra and extracellular recordings revealed that H3R agonist, α-methylhistamine, depolarized both single motoneurons and ventral roots, even in the presence of tetrodotoxin, an effect prevented by H3R antagonist, thioperamide. Finally, immunohistochemistry located the expression of H3Rs on a subpopulation of large cells in lamina IX. This study identifies H3Rs as a new exploitable pharmacological target against motor disturbances.

Simulated microgravity-cultured mesenchymal stem cells improve recovery following spinal cord ischemia in rats.

Spinal cord ischemia is a potential complication of thoracoabdominal aortic surgery that may induce irreversible motor disability. We investigated the therapeutic efficacy of simulated microgravity-cultured mesenchymal stem cell (MSC) injection following spinal cord ischemia-reperfusion injury. Sprague-Dawley rats were divided into sham, phosphate-buffered saline (PBS), normal gravity-cultured MSC (MSC-1 G), and simulated microgravity-cultured MSC (MSC-MG) groups. Spinal cord ischemia was induced by transient balloon occlusion of the thoracic aorta, which was followed immediately by PBS or MSC injection into the left carotid artery. Hindlimb motor function was evaluated by the Basso-Beattie-Bresnahan (BBB) scale. Spinal cords were removed 1, 3, or 7 days post-injury for immunohistochemical staining and Western blot analysis. One day post-injury, a few infiltrating inflammatory cells and small vacuoles were observed without significant group differences, followed over several days by progressive spinal cord degeneration. Glial fibrillary acidic protein (GFAP)-positive (reactive) astrocyte numbers were increased in all three groups, and brain-derived neurotrophic factor (BDNF) was colocalized with GFAP-positive cells in spinal ventral horn. Animals in the MSC-MG group demonstrated greater BDNF-positive astrocyte numbers, reduced caspase-3-positive cell numbers, and superior motor recovery. Microgravity-cultured MSC-based therapy may improve functional recovery following spinal ischemia-reperfusion injury by promoting astrocytic BDNF release, thereby preventing apoptosis.

Diameters of Perikarya in Different Regions of Rat Central Nervous System after Streptomycin and Kanamycin Intoxication

Introduction: Streptomycin and Kanamycin, an aminoglycosidic antibiotic, is known to destroy the ventral cochlear nuclei of brainstem in man. Ototoxicity is well known side effect of kanamycin, the effect on central nervous system in general and central auditory pathway in particular is still unclear. Subjects and Methods: Thirty albino rats were divided into three groups I, II and III of ten animals each. Group III was control. Group I and II received Streptomycin (30mg/Kg body weight) and kanamycin (400mg/kg body weight) intramuscular injections, daily for 3 weeks. Paraffin embedded sections of cerebellum, spinal cord, dorsal cochlear nucleus, inferior colliculus nucleus and auditory cortex were stained with haematoxylin and eosin stains and perikarya measured using slide and ocular micrometres. Results: Size of cerebellar Purkinje cells increased significantly in control rats for streptomycin and kanamycin intoxicated animals respectively. Ventral horn cells of spinal cord are affected highly significantly only by streptomycin. Vestibular nucleus also showed similar results i.e. neuronal body. Neurons of dorsal cochlear nucleus is affected significantly by both the drugs i.e. streptomycin and kanamycin. Conclusion: Streptomycin causes an increase in diameter of auditory cortical cells on other hand kanamycin led to highly significant decrement in size of cells of same region. Preferential affinity and differential effects were noticed. The latter throws some clues about mechanism of action.

Nck2 is essential for limb trajectory selection by spinal motor axons

The development of a functioning nervous system requires precise assembly of neuronal connections, which can be achieved by the guidance of axonal growth cones to their proper targets. How axons are guided by signals transmitted to the cytoskeleton through cell surface-expressed guidance receptors remains unclear. We investigated the function of Nck2 adaptor protein as an essential guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory into the limb. Nck2 mRNA and protein are preferentially expressed in the medial subgroups of chick LMC neurons during axon trajectory into the limb. Nck2 loss- and gain-of-function in LMC neurons using in ovo electroporation perturb LMC axon trajectory selection demonstrating an essential role of Nck2 in motor axon guidance. We also showed that Nck2 knockdown and overexpression perturb the growth preference of LMC neurites against ephrins in vitro and Eph-mediated redirection of LMC axons in vivo. Finally, the significant changes of LMC neurite growth preference against ephrins in the context of Nck2 and α2-chimaerin loss- and gain-of-function implicated Nck2 function to modulate α2-chimaerin activity. Here, we showed that Nck2 is required for Eph-mediated axon trajectory selection from spinal motor neurons through possible interaction with α2-chimaerin. Developmental Dynamics 247:1043-1056, 2018. © 2018 Wiley Periodicals, Inc.

T25. Changes in central motor conduction time and its implication in the pathogenesis of distal-type cervical spondylotic amyotrophy

The true pathogenesis of cervical spondylotic amyotrophy (CSA) remains controversial. One of the current hypotheses for the etiology of CSA mainly involves selective compression of the ventral nerve roots and/or anterior horn cells by a bony spur or herniated disc. Some scholars have proposed that CSA may be ascribed to ischemia of anterior horn cells caused by compressive injury of anterior spinal artery. Recently published studies demonstrated that chronic incomplete cervical cord compression can cause the loss of motor neurons in the spinal anterior horn caudal to the level of the compressive injury, which was described as trans-synaptic damage. Although CSA was previously considered a neurological disease involving only lower motor neurons (LMNs), some authors revealed that approximately 28.6–66.7% of distal-type CSA patients had significant lower limb hyperreflexia, which is consistent with upper motor neuron (UMN) lesions. Therefore, trans-synaptic damage may be involved in the pathogenesis of distal-type CSA. The present study has been undertaken to evaluate central motor conduction in distal-type CSA patients and to analyze the role of trans-synaptic damage in the pathogenesis of distal-type CSA. Central motor conduction time (CMCT) to both upper and lower limbs was measured in 28 distal-type CSA patients and 21 controls, and all subjects underwent further motor unit number estimation (MUNE), handgrip strength examination (HGS) and magnetic resonance imaging evaluation. Prolonged CMCT was recorded in 9/28 (32.1%) CSA patients, and both lower MUNE values and smaller HGS were observed in these 9 cases (P < 0.05). Imaging analysis demonstrated that 5 of these 9 cases had cervical compression both proximally and distally, and severe proximal cervical compression without distal selective compression was found in the other two cases (2/9, 22.2%). There was an inverse correlation between CMCT and both MUNE values and HGS on symptomatic side (P < 0.05), and a positive relationship between CMCT and amplitude of single motor unit potentials was found on less-symptomatic side (P < 0.05). Distal-type CSA patients may have substantial corticospinal tract lesions, and trans-synaptic damage caused by these lesions may be involved in the pathogenesis of distal-type CSA. Therefore, clinicians should be cautious when evaluating and managing distal-type CSA patients, especially the cases with additional proximal cervical compression, because severe muscular atrophy may be caused by both trans-synaptic damage and selective compression.

Ephexin1 Is Required for Eph-Mediated Limb Trajectory of Spinal Motor Axons.

The precise assembly of a functional nervous system relies on the guided migration of axonal growth cones, which is made possible by signals transmitted to the cytoskeleton by cell surface-expressed guidance receptors. We investigated the function of ephexin1, a Rho guanine nucleotide exchange factor, as an essential growth-cone guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory selection in the limb mesenchyme. Using in situ mRNA detection, we first show that ephexin1 is expressed in LMC neurons of chick and mouse embryos at the time of spinal motor axon extension into the limb. Ephexin1 loss of function and gain of function using in ovo electroporation in chick LMC neurons, of either sex, perturbed LMC axon trajectory selection, demonstrating an essential role of ephexin1 in motor axon guidance. In addition, ephexin1 loss in mice of either sex led to LMC axon trajectory selection errors. We also show that ephexin1 knockdown attenuates the growth preference of LMC neurites against ephrins in vitro and Eph receptor-mediated retargeting of LMC axons in vivo, suggesting that ephexin1 is required in Eph-mediated LMC motor axon guidance. Finally, both ephexin1 knockdown and ectopic expression of nonphosphorylatable ephexin1 mutant attenuated the retargeting of LMC axons caused by Src overexpression, implicating ephexin1 as an Src target in Eph signal relay in this context. In summary, our findings demonstrate that ephexin1 is essential for motor axon guidance and suggest an important role in relaying ephrin:Eph signals that mediate motor axon trajectory selection.SIGNIFICANCE STATEMENT The proper development of functioning neural circuits requires precise nerve connections among neurons or between neurons and their muscle targets. The Eph tyrosine kinase receptors expressed in neurons are important in many contexts during neural-circuit formation, such as axon outgrowth, axon guidance, and synaptic formation, and have been suggested to be involved in neurodegenerative disorders, including amyotrophic lateral sclerosis and Alzheimer's disease. To dissect the mechanism of Eph signal relay, we studied ephexin1 gain of function and loss of function and found ephexin1 essential for the development of limb nerves toward their muscle targets, concluding that it functions as an intermediary to relay Eph signaling in this context. Our work could thus shed new light on the molecular mechanisms controlling neuromuscular connectivity during embryonic development.

Yellow Laser Stimulation at GV2 Acupoint Mitigates Apoptosis, Oxidative Stress, Inflammation, and Motor Deficit in Spinal Cord Injury Rats

Currently, the suppression of oxidative stress and inflammation is considered as the treatment targets of spinal cord injury due to their roles on the hindrance of recovery process. Since laser acupuncture decreased oxidative stress and enhanced the survival of neurons from oxidative stress damage and GV2 stimulation was selected as one stimulated acupoint in order to enhance the recovery of spinal cord injury, we hypothesized that laser acupuncture at GV2 should enhance the recovery of spinal cord injury. To test this hypothesis, male Wistar rats were induced spinal cord injury at T10 level and they were exposed to a 10 minute-stimulation at GV2 by yellow laser. Laser acupuncture was performed at 0.25 and 1, 2, 6, and 12 hours after spinal cord injury. Then, the stimulation was performed once daily for 7 days. Locomotor assessment was carried out on days 3 and 7 after injury. At the end of study period, the densities of polymorphonuclear of leukocyte, Bax, Caspase-3, Bcl-2, and BDNF positive stained cells in ventral horn of spinal cord were determined. Cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and oxidative stress status was also assessed. The results showed that laser acupuncture at GV2 increased BBB score, gross motor score, and densities of Bcl-2 and BDNF positive stained cells but decreased density with polymorphonuclear leukocyte, the densities of Bax and Caspase-3 positive stained cells, COX-2 level, and oxidative stress status in ventral horn of the lesion spinal cord. The reduction of serum COX-2 was also decreased. Therefore, GV2 stimulation by yellow laser might enhance the recovery of spinal cord via the increase in BDNF and the decrease in inflammation, apoptosis, and oxidative stress status in the lesion spinal cord.

Motor neurons with limb-innervating character in the cervical spinal cord are sculpted by apoptosis based on the Hox code in chick embryo.

In the developing chick embryo, a certain population of motor neurons (MNs) in the non-limb-innervating cervical spinal cord undergoes apoptosis between embryonic days 4 and 5. However, the characteristics of these apoptotic MNs remain undefined. Here, by examining the spatiotemporal profiles of apoptosis and MN subtype marker expression in normal or apoptosis-inhibited chick embryos, we found that this apoptotic population is distinguishable by Foxp1 expression. When apoptosis was inhibited, the Foxp1+ MNs survived and showed characteristics of lateral motor column (LMC) neurons, which are of a limb-innervating subtype, suggesting that cervical Foxp1+ MNs are the rostral continuation of the LMC. Knockdown and misexpression of Foxp1 did not affect apoptosis progression, but revealed the role of Foxp1 in conferring LMC identity on the cervical MNs. Furthermore, ectopic expression of Hox genes that are normally expressed in the brachial region prevented apoptosis, and directed Foxp1+ MNs to LMC neurons at the cervical level. These results indicate that apoptosis in the cervical spinal cord plays a role in sculpting Foxp1+ MNs committed to LMC neurons, depending on the Hox expression pattern.

SPACEFLIGHT EFFECTS ON GLIAL CELLS OF M4USE B0ONEMARROW.

Immunohistochmistrry was used to analyze lumbar enlarements in C57BL/76N mice on day 30 of a space flight with and w/o 7 day deadaptation to Earth. Space flown and non-deadpted mice had a increased number of S100B+ -cells in the cental channel zone and decreased population of GFAP astrocytes in ventral horns, Olig2+ cells in the corticospinal tract, PO+ -cells in vental horns and dorsal roots entry zone and Krox24+ -cells in ventral horns, dorsal roots entry zone and central channel zone, and reduced OSP fluorescence density in ventral channels. Elevated expression of myelin-producing Olig2, PO and Krox24 markers on day 7 of deadaptation implies recovery, significance of immunocytochemical changes in the myelin-producing cells in space flight, and suggests portentially rapid normalization of pathologic deviations in myelin fibers in consequence of sthe hypogravity motor syndrome.

The Potential Role of Motor Unit Number Estimation as an Additional Diagnostic and Prognostic Value in Canine Neurology.

Motor unit number estimation (MUNE) is an electrophysiological technique to assess the number of motor units innervating a single muscle or muscle group of interest. It may quantify axonal loss in any disease involving injury or degeneration of ventral horn cells or motor axons. Since MUNE has rarely been used in veterinary medicine, our study aimed to evaluate its potential role as an additional diagnostic and prognostic parameter in canine neurology. Therefore, we examined five healthy dogs and seven dogs suffering from diseases that necessitated general anesthesia for further diagnostics and treatment and that were not expected to interfere with the results of electrodiagnostic testing. By using the incremental technique to study MUNE in the cranial tibial muscle, we determined the number of motor units, the size of the compound muscle action potential, and the mean size of individual motor unit potentials of each dog as well as the mean values for each group. Moreover, we studied the correlation between these parameters. Taking the results into consideration, we addressed the difficulties and limitations of this technique. We, furthermore, pointed out possible fields of application for MUNE in canine neurology, and emphasized several aspects that future studies should focus on when applying MUNE to canine patients.

Calbindin-Immunoreactive Interneurons in the Intermediate Zone and Ventral Horn of the Gray Matter of the SC in White Rats

The aim of the present work was to study the topography and structure of 28-kDa calbindin (CB)-containing interneurons in the ventral horn and intermediate zone of the gray matter at levels TII and LIV of the spinal cord (SC). Experiments were performed in adult female Wistar rats (n = 4) using immunohistochemical and morphometric methods. Cryostat transverse sections of the SC of thickness 14 μm were studied. The intermediate zone of the SC was found to contain two subpopulations of CB-immunoreactive (IR) interneurons in lamina VII in both segments: sympathetic preganglionic neurons and “partition” cells. The ventral horn also contained two subpopulations of CB-IR interneurons: Renshaw cells in lamina IX in TII and lamina VII in LIV and large interneurons in lamina VIII in TII and lamina VII in LIV. The only differences between segments were in the larger numbers of Renshaw cells and partition cells in LIV. The mean cross-sectional areas of CB-IR Renshaw cells and autonomic preganglionic neurons were greater in TII, while large interneurons of the ventral horn and partition cells were larger in LIV.

Firing properties of Renshaw cells defined by Chrna2 are modulated by hyperpolarizing and small conductance ion currents Ih and ISK.

Renshaw cells in the spinal cord ventral horn regulate motoneuron output through recurrent inhibition. Renshaw cells can be identified in vitro using anatomical and cellular criteria; however, their functional role in locomotion remains poorly defined because of the difficulty of functionally isolating Renshaw cells from surrounding motor circuits. Here we aimed to investigate whether the cholinergic nicotinic receptor alpha2 (Chrna2) can be used to identify Renshaw cells (RCs(α2)) in the mouse spinal cord. Immunohistochemistry and electrophysiological characterization of passive and active RCs(α2) properties confirmed that neurons genetically marked by the Chrna2-Cre mouse line together with a fluorescent reporter mouse line are Renshaw cells. Whole-cell patch-clamp recordings revealed that RCs(α2) constitute an electrophysiologically stereotyped population with a resting membrane potential of -50.5 ± 0.4 mV and an input resistance of 233.1 ± 11 MΩ. We identified a ZD7288-sensitive hyperpolarization-activated cation current (Ih) in all RCs(α2), contributing to membrane repolarization but not to the resting membrane potential in neonatal mice. Additionally, we found RCs(α2) to express small calcium-activated potassium currents (I(SK)) that, when blocked by apamin, resulted in a complete attenuation of the afterhyperpolarisation potential, increasing cellular firing frequency. We conclude that RCs(α2) can be genetically targeted through their selective Chrna2 expression and that they display currents known to modulate rebound excitation and firing frequency. The genetic identification of Renshaw cells and their electrophysiological profile is required for genetic and pharmacological manipulation as well as computational simulations with the aim to understand their functional role.

Emergence of Motor Circuit Activity

In the developing nervous system, ordered neuronal activity patterns can occur even in the absence of sensory input and to investigate how these arise, we have used the model system of the embryonic chicken spinal motor circuit, focusing on motor neurons of the lateral motor column (LMC). At the earliest stages of their molecular differentiation, we can detect differences between medial and lateral LMC neurons in terms of expression of neurotransmitter receptor subunits, including CHRNA5, CHRNA7, GRIN2A, GRIK1, HTR1A and HTR1B, as well as the KCC2 transporter. Using patch-clamp recordings we also demonstrate that medial and lateral LMC motor neurons have subtly different activity patterns that reflect the differential expression of neurotransmitter receptor subunits. Using a combination of patch-clamp recordings in single neurons and calcium-imaging of motor neuron populations, we demonstrate that inhibition of nicotinic, muscarinic or GABA-ergic activity, has profound effects of motor circuit activity during the initial stages of neuromuscular junction formation. Finally, by analysing the activity of large populations of motor neurons at different developmental stages, we show that the asynchronous, disordered neuronal activity that occurs at early stages of circuit formation develops into organised, synchronous activity evident at the stage of LMC neuron muscle innervation. In light of the considerable diversity of neurotransmitter receptor expression, activity patterns in the LMC are surprisingly similar between neuronal types, however the emergence of patterned activity, in conjunction with the differential expression of transmitter systems likely leads to the development of near-mature patterns of locomotor activity by perinatal ages.

Distribution of Serotonin 4(a) Receptors in the juvenile Rat Brain and Spinal Cord

In the last 20 years there have been many studies investigating the distribution of 5-HT4-receptors in the brain of different species. Most studies are methodically based on RT-PCR or in situ hybridization and have analysed the receptors at the mRNA level.Furthermore there have been some autoradiographic studies using specific 5-HT4-receptor antagonists like [3H]GR113808, [3H]BIMU-1 or [125I]SB207710.This study investigates the topographical distribution of the 5-HT4(a)-receptor in the juvenile rat brain and spinal cord, which is important for neuromodulation of cellular excitability and could be involved in various developmental processes of the central nervous system. We analysed the 5-HT4(a)-receptor at protein level with a monospecific polyclonal antibody by using an immunohistochemical staining.We saw an intensive staining in some areas of the cortex, in the olfactory bulb, in most areas of the cerebellum, in hippocampal areas like the dentate gyrus and in several different areas of the brainstem, especially in the motor nuclei. Overall we have shown comparable results in accordance with the results of other studies investigating the distribution of 5-HT4(a)-receptors.Some areas like the islands of Calleja, the preoptic nucleus or the medial habenular nucleus showed a lower intensity of 5-HT4(a)-receptors in comparison with the results of other studies. As a novel result we found a higher intensity of 5-HT4(a)-receptor in several brain areas associated with motor function than was shown by other studies, especially in the motor cortex, in different areas of the cerebellum, in the red nucleus, in the motor nuclei of the brainstem or in the ventral horn cells of the spinal cord.We conclude that the 5-HT4(a)-receptor may play a more prominent role in the modulation of motor cortico-ponto-cerebellar, cortico-spinal, rubro-spinal, vestibulo-spinal and cortico-nuclear tracts during juvenile development.