Martin Larhammar

Mutations in DMRT3 affect locomotion in horses and spinal circuit function in mice

Locomotion in mammals relies on a central pattern-generating circuitry of spinal interneurons established during development that coordinates limb movement. These networks produce left–right alternation of limbs as well as coordinated activation of flexor and extensor muscles. Here we show that a premature stop codon in the DMRT3 gene has a major effect on the pattern of locomotion in horses. The mutation is permissive for the ability to perform alternate gaits and has a favourable effect on harness racing performance. Examination of wild-type and Dmrt3-null mice demonstrates that Dmrt3 is expressed in the dI6 subdivision of spinal cord neurons, takes part in neuronal specification within this subdivision, and is critical for the normal development of a coordinated locomotor network controlling limb movements. Our discovery positions Dmrt3 in a pivotal role for configuring the spinal circuits controlling stride in vertebrates. The DMRT3 mutation has had a major effect on the diversification of the domestic horse, as the altered gait characteristics of a number of breeds apparently require this mutation.

ZBED6, a novel transcription factor derived from a domesticated DNA transposon regulates IGF2 expression and muscle growth.

This study identifies a previously uncharacterized protein, encoded by a domesticated DNA transposon, called ZBED6 that regulates the expression of the insulin-like growth factor 2 (IGF2) gene, and possibly numerous others, in all placental mammals including human.

Alterations in the motor neuron–renshaw cell circuit in the Sod1G93A mouse model

Motor neurons become hyperexcitable during progression of amyotrophic lateral sclerosis (ALS). This abnormal firing behavior has been explained by changes in their membrane properties, but more recently it has been suggested that changes in premotor circuits may also contribute to this abnormal activity. The specific circuits that may be altered during development of ALS have not been investigated. Here we examined the Renshaw cell recurrent circuit that exerts inhibitory feedback control on motor neuron firing. Using two markers for Renshaw cells (calbindin and cholinergic nicotinic receptor subunit alpha2 [Chrna2]), two general markers for motor neurons (NeuN and vesicular acethylcholine transporter [VAChT]), and two markers for fast motor neurons (Chondrolectin and calcitonin‐related polypeptide alpha [Calca]), we analyzed the survival and connectivity of these cells during disease progression in the Sod1G93A mouse model. Most calbindin‐immunoreactive (IR) Renshaw cells survive to end stage but downregulate postsynaptic Chrna2 in presymptomatic animals. In motor neurons, some markers are downregulated early (NeuN, VAChT, Chondrolectin) and others at end stage (Calca). Early downregulation of presynaptic VAChT and Chrna2 was correlated with disconnection from Renshaw cells as well as major structural abnormalities of motor axon synapses inside the spinal cord. Renshaw cell synapses on motor neurons underwent more complex changes, including transitional sprouting preferentially over remaining NeuN‐IR motor neurons. We conclude that the loss of presynaptic motor axon input on Renshaw cells occurs at early stages of ALS and disconnects the recurrent inhibitory circuit, presumably resulting in diminished control of motor neuron firing. J. Comp. Neurol. 521:1449–1469, 2013.

The synaptic protein encoded by the gene Slc10A4 suppresses epileptiform activity and regulates sensitivity to cholinergic chemoconvulsants

The expanding number of disease-causing dysfunctions of synaptic proteins illustrates the importance of investigating newly discovered proteins involved in neuronal transmission. The gene Slc10A4 encodes a recently described carrier protein present in pre-synaptic terminals of cholinergic and monoaminergic neurons. The biological significance of this recently described transporter protein is currently unknown. We here investigated whether absence of the Slc10a4 protein has any impact on function of the cholinergic system. We first investigated the sensitivity of Slc10a4 null mice to cholinergic stimulus in vitro. In contrast to wild type mice, gamma oscillations occurred spontaneously in hippocampal slices from Slc10a4 null mice. Furthermore, moderate treatment of Slc10a4 null slices with the cholinergic agonist carbachol induced epileptiform activity. In vivo, 3-channel EEG measurements in freely behaving mice revealed that Slc10a4 null mice had frequent epileptiform spike-activity before treatment, and developed epileptic seizures, detected by EEG and accompanied by observable behavioral components, more rapidly after injection of the cholinergic agonist pilocarpine. Similar results were obtained on non-operated mice, as evaluated by behavioral seizures and post mortem c-Fos immunohistochemistry. Importantly, Slc10a4 null mice and wild type control mice were equally sensitive to the glutamatergic chemoconvulsant kainic acid, demonstrating that absence of Slc10a4 led to a selective cholinergic hypersensitivity. In summary, we report that absence of the recently discovered synaptic vesicle protein Slc10a4 results in increased sensitivity to cholinergic stimulation.

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 (RCsα2) in the mouse spinal cord. Immunohistochemistry and electrophysiological characterization of passive and active RCsα2 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 RCsα2 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 RCsα2, contributing to membrane repolarization but not to the resting membrane potential in neonatal mice. Additionally, we found RCsα2 to express small calcium‐activated potassium currents (ISK) that, when blocked by apamin, resulted in a complete attenuation of the afterhyperpolarisation potential, increasing cellular firing frequency. We conclude that RCsα2 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.

SLC10A4 is a vesicular amine-associated transporter modulating dopamine homeostasis.

BACKGROUND The neuromodulatory transmitters, biogenic amines, have profound effects on multiple neurons and are essential for normal behavior and mental health. Here we report that the orphan transporter SLC10A4, which in the brain is exclusively expressed in presynaptic vesicles of monoaminergic and cholinergic neurons, has a regulatory role in dopamine homeostasis. METHODS We used a combination of molecular and behavioral analyses, pharmacology, and in vivo amperometry to assess the role of SLC10A4 in dopamine-regulated behaviors. RESULTS We show that SLC10A4 is localized on the same synaptic vesicles as either vesicular acetylcholine transporter or vesicular monoamine transporter 2. We did not find evidence for direct transport of dopamine by SLC10A4; however, synaptic vesicle preparations lacking SLC10A4 showed decreased dopamine vesicular uptake efficiency. Furthermore, we observed an increased acidification in synaptic vesicles isolated from mice overexpressing SLC10A4. Loss of SLC10A4 in mice resulted in reduced striatal serotonin, noradrenaline, and dopamine concentrations and a significantly higher dopamine turnover ratio. Absence of SLC10A4 led to slower dopamine clearance rates in vivo, which resulted in accumulation of extracellular dopamine. Finally, whereas SLC10A4 null mutant mice were slightly hypoactive, they displayed hypersensitivity to administration of amphetamine and tranylcypromine. CONCLUSIONS Our results demonstrate that SLC10A4 is a vesicular monoaminergic and cholinergic associated transporter that is important for dopamine homeostasis and neuromodulation in vivo. The discovery of SLC10A4 and its role in dopaminergic signaling reveals a novel mechanism for neuromodulation and represents an unexplored target for the treatment of neurological and mental disorders.

Functional and Histological Outcome after Focal Traumatic Brain Injury Is Not Improved in Conditional EphA4 Knockout Mice

We investigated the role of the axon guidance molecule EphA4 following traumatic brain injury (TBI) in mice. Neutralization of EphA4 improved motor function and axonal regeneration following experimental spinal cord injury (SCI). We hypothesized that genetic absence of EphA4 could improve functional and histological outcome following TBI. Using qRT-PCR in wild-type (WT) mice, we evaluated the EphA4 mRNA levels following controlled cortical impact (CCI) TBI or sham injury and found it to be downregulated in the hippocampus (p<0.05) but not the cortex ipsilateral to the injury at 24 h post-injury. Next, we evaluated the behavioral and histological outcome following CCI using WT mice and Emx1-Cre-driven conditional knockout (cKO) mice. In cKO mice, EphA4 was completely absent in the hippocampus and markedly reduced in the cortical regions from embryonic day 16, which was confirmed using Western blot analysis. EphA4 cKO mice had similar learning and memory abilities at 3 weeks post-TBI compared to WT controls, although brain-injured animals performed worse than sham-injured controls (p<0.05). EphA4 cKO mice performed similarly to WT mice in the rotarod and cylinder tests of motor function up to 29 days post-injury. TBI increased cortical and hippocampal astrocytosis (GFAP immunohistochemistry, p<0.05) and hippocampal sprouting (Timm stain, p<0.05) and induced a marked loss of hemispheric tissue (p<0.05). EphA4 cKO did not alter the histological outcome. Although our results may argue against a beneficial role for EphA4 in the recovery process following TBI, further studies including post-injury pharmacological neutralization of EphA4 are needed to define the role for EphA4 following TBI.

Developmental disruption of recurrent inhibitory feedback results in compensatory adaptation in the Renshaw cell-motor neuron circuit

When activating muscles, motor neurons in the spinal cord also activate Renshaw cells, which provide recurrent inhibitory feedback to the motor neurons. The tight coupling with motor neurons suggests that Renshaw cells have an integral role in movement, a role that is yet to be elucidated. Here we used the selective expression of the nicotinic cholinergic receptor α2 (Chrna2) in mice to genetically target the vesicular inhibitory amino acid transporter (VIAAT) in Renshaw cells. Loss of VIAAT from Chrna2Cre-expressing Renshaw cells did not impact any aspect of drug-induced fictive locomotion in the neonatal mouse or change gait, motor coordination, or grip strength in adult mice of both sexes. However, motor neurons from neonatal mice lacking VIAAT in Renshaw cells received spontaneous inhibitory synaptic input with a reduced frequency, showed lower input resistance, and had an increased number of proprioceptive glutamatergic and calbindin-labeled putative Renshaw cell synapses on their soma and proximal dendrites. Concomitantly, Renshaw cells developed with increased excitability and a normal number of cholinergic motor neuron synapses, indicating a compensatory mechanism within the recurrent inhibitory feedback circuit. Our data suggest an integral role for Renshaw cell signaling in shaping the excitability and synaptic input to motor neurons. SIGNIFICANCE STATEMENT We here provide a deeper understanding of spinal cord circuit formation and the repercussions for the possible role for Renshaw cells in speed and force control. Our results suggest that while Renshaw cells are not directly required as an integral part of the locomotor coordination machinery, the development of their electrophysiological character is dependent on vesicular inhibitory amino acid transporter-mediated signaling. Further, Renshaw cell signaling is closely associated with the molding of motor neuron character proposing the existence of a concerted maturation process, which seems to endow this particular spinal cord circuit with the plasticity to compensate for loss of the Renshaw cell in adult circuit function.