Roles of ETS Genes ER81 and PEA3 in the Development of the Monosynaptic Stretch Reflex Circuit

The monosynaptic stretch reflex circuit consists of two neural cell types, sensory neurons and alpha-motoneurons. Ia afferents form synaptic connections with motoneurons projecting to the same or synergistic muscles, but not with motoneurons projecting to unrelated muscles. These synaptic connections form appropriately from the outset suggesting that they may be controlled by expression of specific adhesion molecules in matching sensory and motor neurons. Recently, two ETS-family transcription factors (Er81 and PEA3) were shown to be expressed in subsets of motoneurons and muscle sensory neurons. The expression patterns of these factors suggested that ETS genes might regulate the formation of synaptic connections between Ia afferents and motoneurons. This thesis explores the roles of Er81 and PEA3 in the formation of the stretch reflex circuit inferred from a study of Er81 and PEA3 null-mutant mice.Analysis of Er81 null-mutant mice revealed that Er81 controls a late step in Ia afferent axon guidance. Ia afferents induce the development of muscle spindles in the periphery and project axons into the spinal cord, but fail to grow axon collaterals into the ventral spinal cord where normally strong monosynaptic connections are formed with motoneurons. Consequently, monosynaptic Ia afferent inputs to motoneurons are greatly reduced in these mice. This severe phenotype precluded determination of whether or not the pattern of remaining Ia afferent inputs was normal.Intracellular recordings from quadriceps and obturator motoneurons in PEA3 null-mutants, however, revealed that functionally appropriate patterns of Ia afferent input to motoneurons develop normally in the absence of PEA3. PEA3 mutant mice demonstrated a role for PEA3 in the formation of a specific motor pool. Cutaneous maximus muscle motoneurons normally express PEA3. In PEA3 mutants, the majority of these motoneurons fail to migrate and coalesce appropriately into a discrete motor pool. These motoneurons also fail to project axons into the c. maximus muscle. Consequently, the muscle is atrophic.Thus, Er81 and PEA3 contribute to key developmental stages in the formation of the stretch reflex circuit: the growth of Ia afferents axons toward motoneurons and the formation of appropriate motor pool targets

The Anti-Inflammatory Agent Bindarit Attenuates the Impairment of Neural Development through Suppression of Microglial Activation in a Neonatal Hydrocephalus Mouse Model

Neonatal hydrocephalus presents with various degrees of neuroinflammation and long-term neurologic deficits in surgically treated patients, provoking a need for additional medical treatment. We previously reported elevated neuroinflammation and severe periventricular white matter damage in the progressive hydrocephalus (prh) mutant which contains a point mutation in the Ccdc39 gene, causing loss of cilia-mediated unidirectional CSF flow. In this study, we identified cortical neuropil maturation defects such as impaired excitatory synapse maturation and loss of homeostatic microglia, and swimming locomotor defects in early postnatal prh mutant mice. Strikingly, systemic application of the anti-inflammatory small molecule bindarit significantly supports healthy postnatal cerebral cortical development in the prh mutant. While bindarit only mildly reduced the ventricular volume, it significantly improved the edematous appearance and myelination of the corpus callosum. Moreover, the treatment attenuated thinning in cortical Layers II–IV, excitatory synapse formation, and interneuron morphogenesis, by supporting the ramified-shaped homeostatic microglia from excessive cell death. Also, the therapeutic effect led to the alleviation of a spastic locomotor phenotype of the mutant. We found that microglia, but not peripheral monocytes, contribute to amoeboid-shaped activated myeloid cells in prh mutants’ corpus callosum and the proinflammatory cytokines expression. Bindarit blocks nuclear factor (NF)-kB activation and its downstream proinflammatory cytokines, including monocyte chemoattractant protein-1, in the prh mutant. Collectively, we revealed that amelioration of neuroinflammation is crucial for white matter and neuronal maturation in neonatal hydrocephalus. Future studies of bindarit treatment combined with CSF diversion surgery may provide long-term benefits supporting neuronal development in neonatal hydrocephalus.

Characterization of Calbindin Positive Interneurons within the Ventral Horn of the Mouse Spinal Cord

Sensory-motor circuits in the spinal cord integrate sensory feedback from muscles and modulate locomotor behavior. Although we know how the sensory-motor system generally works, the main issue lies in identifying all neurons involved and understanding their interrelationships. Many interneurons contribute to sensory-motor circuits and have been well studied. For example, Renshaw cells (RC) are inhibitory interneurons that prevent motor neurons from over-activity. A distinguishing feature of RCs is that they are the only interneurons within the ventral-most region of the spinal cord expressing the calcium binding protein calbindin (CB). Recent studies have found other subpopulations of ventral horn interneurons outside of the RC area that express CB, but knowledge regarding the function and connectivity of these neurons is limited. We hypothesize CB expression serves a functional purpose for ventral horn interneurons and as well as identifying RCs. Here we compare known characteristics of RCs with other ventral horn interneurons that express CB. We analyze anatomical location; cellular density; expression of neurotransmitters; motor neuron and sensory afferent contacts; expression of calcium binding proteins CB, calretinin and parvalbumin; and premotor neuron identification.https://corescholar.libraries.wright.edu/urop_celebration/1010/thumbnail.jp

Loss of ETV1/ER81 in motor neurons leads to reduced monosynaptic inputs from proprioceptive sensory neurons

Presynaptic inputs determine the pattern of activation of postsynaptic neurons in a neural circuit. Molecular and genetic pathways that regulate the selective formation of subsets of presynaptic inputs are largely unknown, despite significant understanding of the general process of synaptogenesis. In this study, we have begun to identify such factors using the spinal monosynaptic stretch reflex circuit as a model system. In this neuronal circuit, Ia proprioceptive afferents establish monosynaptic connections with spinal motor neurons that project to the same muscle (termed homonymous connections) or muscles with related or synergistic function. However, monosynaptic connections are not formed with motor neurons innervating muscles with antagonistic functions. The ETS transcription factor ER81 (also known as ETV1) is expressed by all proprioceptive afferents, but only a small set of motor neuron pools in the lumbar spinal cord of the mouse. Here we use conditional mouse genetic techniques to eliminate Er81 expression selectively from motor neurons. We find that ablation of Er81 in motor neurons reduces synaptic inputs from proprioceptive afferents conveying information from homonymous and synergistic muscles, with no change observed in the connectivity pattern from antagonistic proprioceptive afferents. In summary, these findings suggest a role for ER81 in defined motor neuron pools to control the assembly of specific presynaptic inputs and thereby influence the profile of activation of these motor neurons

The Role of the ETS Gene PEA3 in the Development of Motor and Sensory Neurons

The ETS family of transcription factors includes two members, ER81 and PEA3, which are expressed in groups of sensory and motor neurons supplying individual muscles. To investigate a possible role of these genes in determining sensory and/or motor neuron phenotype, we studied mice in which each of these genes was deleted. In contrast to the deletion of ER81, which blocks the formation of projections from muscle sensory neurons to motor neurons in the spinal cord, deletion of PEA3 causes no obvious effects on sensory neurons or on their synaptic connections with motor neurons. PEA3 does play a major role in the formation of some brachial motoneurons however. Motoneurons innervating the cutaneous maximus muscle, which are normally PEA3+, fail to develop normally so that postnatally the muscle is innervated by few motoneurons and is severely atrophic. Other studies suggest that these motoneurons initially appear during development but fail to contact their normal muscle targets

Calcium Homeostasis in Parvalbumin Drg Neurons Is Altered After Sciatic Nerve Crush and Sciatic Nerve Transection Injuries

Reflex abnormalities mediated by proprioceptive sensory neurons after peripheral nerve injury (PNI) can limit functional improvement, leaving patients with disability that affects their quality of life. We examined postinjury calcium transients in a subpopulation of dorsal root ganglion (DRG) neurons consisting primarily of proprioceptors to determine whether alterations in calcium homeostasis are present in proprioceptors, as has been documented in other DRG neurons after PNI. Using transgenic mice, we restricted expression of the calcium indicator GCaMP6s to DRG neurons containing parvalbumin (PV). Mice of both sexes were randomly assigned to sham, sciatic nerve crush, or sciatic nerve transection and resuture conditions. Calcium transients were recorded from ex vivo preparations of animals at one of three postsurgery time points: 1–3 days, 7–11 days, and after 60 days of recovery. Results demonstrated that the post-PNI calcium transients of PV DRG neurons are significantly different than sham. Abnormalities were not present during the acute response to injury (1–3 days), but transients were significantly different than sham at the recovery stage where axon regeneration is thought to be underway (7–11 days). During late-stage recovery (60 days postinjury), disturbances in the decay time course of calcium transients in transection animals persisted, whereas parameters of transients from crush animals returned to normal. These findings identify a deficit in calcium homeostasis in proprioceptive neurons, which may contribute to the failure to fully recover proprioceptive reflexes after PNI. Significant differences in the calcium transients of crush versus transection animals after reinnervation illustrate calcium homeostasis alterations are distinctive to injury type. NEW & NOTEWORTHY This study examines calcium homeostasis after peripheral nerve injury in dorsal root ganglion (DRG) neurons expressing parvalbumin, a group of large-diameter afferents primarily consisting of proprioceptors, using two-photon calcium imaging in the intact DRG. Our findings identify aberrant calcium homeostasis as an additional source of sensory neuron dysfunction following peripheral nerve injury, uncover differences between two injury models, and track how these changes develop and resolve over the course of recovery

Facilitation of Antagonist Motor Output Through Short-Latency Sensory Pathways During Postnatal Development in the Mouse

Reciprocal inhibition of motor neurons via Ia inhibitory interneurons recruited by stimulation of proprioceptive afferents supplying antagonist muscles has been well described. Changes in the efficacy of inhibition, and sometimes even a switch from inhibition to facilitation, have been reported in the literature after disruption of descending pathways. We sought to test whether such facilitation could be expressed in normal animals by evaluating the presence of facilitation in acute preparations from uninjured animals. Using an isolated spinal cord preparation from neonatal mice, changes in the monosynaptic stretch reflex response in knee flexor motor neurons (posterior biceps semitendinosus; PBST) were monitored following conditioning stimulation of proprioceptive sensory afferents in other muscle nerves. As expected for reciprocal inhibition, conditioning by stimulation of quadriceps (knee extensors and PBST antagonists) sensory afferents resulted in inhibition of the stretch reflex response. Facilitation, however, of the stretch reflex response by quadriceps conditioning stimulation was observed when the glycinergic reciprocal inhibitory pathway was blocked by application of strychnine. Facilitation was elicited by low-threshold proprioceptive afferents and occurred at latencies consistent with a disynaptic circuit. The magnitude of facilitation was larger at birth than at one week postnatal. Our results also suggest reciprocal facilitation is restricted to antagonist muscle pairs, as facilitation of PBST responses was not observed when conditioned with the obturator nerve supplying the adductor muscles. Overall, these data suggest the efficacy of facilitation is modulated during the first postnatal week, while the specificity of facilitation is already established by birth

Facilitation of Antagonist Motor Output Through Short-Latency Sensory Pathways During Postnatal Development in the Mouse

Reciprocal inhibition of motor neurons via Ia inhibitory interneurons recruited by stimulation of proprioceptive afferents supplying antagonist muscles has been well described. Changes in the efficacy of inhibition, and sometimes even a switch from inhibition to facilitation, have been reported in the literature after disruption of descending pathways. We sought to test whether such facilitation could be expressed in normal animals by evaluating the presence of facilitation in acute preparations from uninjured animals. Using an isolated spinal cord preparation from neonatal mice, changes in the monosynaptic stretch reflex response in knee flexor motor neurons (posterior biceps semitendinosus; PBST) were monitored following conditioning stimulation of proprioceptive sensory afferents in other muscle nerves. As expected for reciprocal inhibition, conditioning by stimulation of quadriceps (knee extensors and PBST antagonists) sensory afferents resulted in inhibition of the stretch reflex response. Facilitation, however, of the stretch reflex response by quadriceps conditioning stimulation was observed when the glycinergic reciprocal inhibitory pathway was blocked by application of strychnine. Facilitation was elicited by low-threshold proprioceptive afferents and occurred at latencies consistent with a disynaptic circuit. The magnitude of facilitation was larger at birth than at one week postnatal. Our results also suggest reciprocal facilitation is restricted to antagonist muscle pairs, as facilitation of PBST responses was not observed when conditioned with the obturator nerve supplying the adductor muscles. Overall, these data suggest the efficacy of facilitation is modulated during the first postnatal week, while the specificity of facilitation is already established by birth

Characterization of Calbindin D28k Expressing Interneurons in the Ventral Horn of the Mouse Spinal Cord

Background: Expression of the calcium binding protein, calbindin (CB), is well established as a hallmark of Renshaw cells, a class of interneurons found in spatially restricted areas in the ventral spinal cord that directly modulate motor neuron activity. CB expression, however, is not restricted only to Renshaw cells in the ventral horn, and within this population other interneuron subtypes may be identifiable on the basis of cell position and the potential for coexpression of other calcium binding proteins. Results: Here we have quantified the changing CB expression pattern in the ventral spinal cord across postnatal development in the mouse. Fewer neurons express CB as postnatal development progresses, and those neurons frequently coexpress other calcium binding proteins (calretinin and parvalbumin) in subpopulations with distinct spatial distributions. We also found a significant portion of CB‐expressing interneurons receive putative synaptic contacts from primary sensory afferents. Conclusions: These findings suggest CB labels a heterogeneous group of interneurons in the ventral horn, some of which may process sensory information. Based on cellular position, CB expression may be a shared feature of subsets of interneurons arising from multiple ventral progenitor domains