Excitatory Spinal Lhx9-Derived Interneurons Modulate Locomotor Frequency in Mice.

Locomotion allows us to move and interact with our surroundings. Spinal networks that control locomotion produce rhythm and left-right and flexor-extensor coordination. Several glutamatergic populations, Shox2 non-V2a, Hb9-derived interneurons, and, recently, spinocerebellar neurons have been proposed to be involved in the mouse rhythm generating networks. These cells make up only a smaller fraction of the excitatory cells in the ventral spinal cord. Here, we set out to identify additional populations of excitatory spinal neurons that may be involved in rhythm generation or other functions in the locomotor network. We use RNA sequencing from glutamatergic, non-glutamatergic, and Shox2 cells in the neonatal mice from both sexes followed by differential gene expression analyses. These analyses identified transcription factors that are highly expressed by glutamatergic spinal neurons and differentially expressed between Shox2 neurons and glutamatergic neurons. From this latter category, we identified the Lhx9-derived neurons as having a restricted spinal expression pattern with no Shox2 neuron overlap. They are purely glutamatergic and ipsilaterally projecting. Ablation of the glutamatergic transmission or acute inactivation of the neuronal activity of Lhx9-derived neurons leads to a decrease in the frequency of locomotor-like activity without change in coordination pattern. Optogenetic activation of Lhx9-derived neurons promotes locomotor-like activity and modulates the frequency of the locomotor activity. Calcium activities of Lhx9-derived neurons show strong left-right out-of-phase rhythmicity during locomotor-like activity. Our study identifies a distinct population of spinal excitatory neurons that regulates the frequency of locomotor output with a suggested role in rhythm-generation in the mouse alongside other spinal populations.

Dual-mode operation of neuronal networks involved in left-right alternation.

All forms of locomotion are repetitive motor activities that require coordinated bilateral activation of muscles. The executive elements of locomotor control are networks of spinal neurons that determine gait pattern through the sequential activation of motor-neuron pools on either side of the body axis. However, little is known about the constraints that link left-right coordination to locomotor speed. Recent advances have indicated that both excitatory and inhibitory commissural neurons may be involved in left-right coordination. But the neural underpinnings of this, and a possible causal link between these different groups of commissural neurons and left-right alternation, are lacking. Here we show, using intersectional mouse genetics, that ablation of a group of transcriptionally defined commissural neurons--the V0 population--leads to a quadrupedal hopping at all frequencies of locomotion. The selective ablation of inhibitory V0 neurons leads to a lack of left-right pattern at low frequencies, mixed coordination at medium frequencies, and alternation at high locomotor frequencies. When ablation is targeted to excitatory V0 neurons, left-right alternation is present at low frequencies, and hopping is restricted to medium and high locomotor frequencies. Therefore, the intrinsic logic of the central control of locomotion incorporates a modular organization, with two subgroups of V0 neurons required for the existence of left-right alternating modes at different speeds of locomotion. The two molecularly distinct sets of commissural neurons may constrain species-related naturally occurring frequency-dependent coordination and be involved in the evolution of different gaits.

Neuronal calcium-binding proteins 1/2 localize to dorsal root ganglia and excitatory spinal neurons and are regulated by nerve injury.

Neuronal calcium (Ca(2+))-binding proteins 1 and 2 (NECAB1/2) are members of the phylogenetically conserved EF-hand Ca(2+)-binding protein superfamily. To date, NECABs have been explored only to a limited extent and, so far, not at all at the spinal level. Here, we describe the distribution, phenotype, and nerve injury-induced regulation of NECAB1/NECAB2 in mouse dorsal root ganglia (DRGs) and spinal cord. In DRGs, NECAB1/2 are expressed in around 70% of mainly small- and medium-sized neurons. Many colocalize with calcitonin gene-related peptide and isolectin B4, and thus represent nociceptors. NECAB1/2 neurons are much more abundant in DRGs than the Ca(2+)-binding proteins (parvalbumin, calbindin, calretinin, and secretagogin) studied to date. In the spinal cord, the NECAB1/2 distribution is mainly complementary. NECAB1 labels interneurons and a plexus of processes in superficial layers of the dorsal horn, commissural neurons in the intermediate area, and motor neurons in the ventral horn. Using CLARITY, a novel, bilaterally connected neuronal system with dendrites that embrace the dorsal columns like palisades is observed. NECAB2 is present in cell bodies and presynaptic boutons across the spinal cord. In the dorsal horn, most NECAB1/2 neurons are glutamatergic. Both NECAB1/2 are transported into dorsal roots and peripheral nerves. Peripheral nerve injury reduces NECAB2, but not NECAB1, expression in DRG neurons. Our study identifies NECAB1/2 as abundant Ca(2+)-binding proteins in pain-related DRG neurons and a variety of spinal systems, providing molecular markers for known and unknown neuron populations of mechanosensory and pain circuits in the spinal cord.

Optogenetic dissection reveals multiple rhythmogenic modules underlying locomotion.

Neural networks in the spinal cord known as central pattern generators produce the sequential activation of muscles needed for locomotion. The overall locomotor network architectures in limbed vertebrates have been much debated, and no consensus exists as to how they are structured. Here, we use optogenetics to dissect the excitatory and inhibitory neuronal populations and probe the organization of the mammalian central pattern generator. We find that locomotor-like rhythmic bursting can be induced unilaterally or independently in flexor or extensor networks. Furthermore, we show that individual flexor motor neuron pools can be recruited into bursting without any activity in other nearby flexor motor neuron pools. Our experiments differentiate among several proposed models for rhythm generation in the vertebrates and show that the basic structure underlying the locomotor network has a distributed organization with many intrinsically rhythmogenic modules.

Spinal glutamatergic neurons defined by EphA4 signaling are essential components of normal locomotor circuits.

EphA4 signaling is essential for the spatiotemporal organization of neuronal circuit formation. In mice, deletion of this signaling pathway causes aberrant midline crossing of axons from both brain and spinal neurons and the complete knock-outs (KOs) exhibit a pronounced change in motor behavior, where alternating gaits are replaced by a rabbit-like hopping gait. The neuronal mechanism that is responsible for the gait switch in these KO mice is not known. Here, using intersectional genetics, we demonstrate that a spinal cord-specific deletion of EphA4 signaling is sufficient to generate the overground hopping gait. In contrast, selective deletion of EphA4 signaling in forebrain neurons, including the corticospinal tract neurons, did not result in a change in locomotor pattern. The gait switch was attributed to the loss of EphA4 signaling in excitatory Vglut2+ neurons, which is accompanied by an increased midline crossing of Vglut2+ neurons in the ventral spinal cord. Our findings functionally define spinal EphA4 signaling in excitatory Vglut2+ neurons as required for proper organization of the spinal locomotor circuitry, and place these cells as essential components of the mammalian locomotor network.

Change in the balance of excitatory and inhibitory midline fiber crossing as an explanation for the hopping phenotype in EphA4 knockout mice.

Neuronal networks in the spinal cord termed central pattern generators (CPGs) are responsible for the generation of rhythmic movements, such as walking. The axon guidance molecule EphA4 has been suggested to play a role in the configuration of spinal CPG networks in mammals. In EphA4 knockout (EphA4-KO) mice, the normal alternating walking pattern is replaced by a rabbit-like hopping gait, which can be reproduced when locomotor-like activity is induced in the isolated spinal cord. This hopping phenotype has been explained by an abnormal midline crossing of ipsilateral axons. Here, we investigated the nature of this overcrossing in heterozygous EphA4 (EphA4(lacZ/+) ) mice that showed normal alternating gait and homozygous EphA4 (EphA4(lacZ/lacZ) ) mice with hopping gait. Localized lesions showed that the hopping phenotype is maintained by fibers crossing in the ventral commissure. Using transgenic mouse lines in which glutamatergic, GABAergic and glycinergic neurons are intrinsically labeled, we showed a significant increase in the number of crossing excitatory ß-galactosidase-positive neurons and a decrease in the number of inhibitory neurons crossing the midline in EphA4(lacZ/lacZ) mice compared with EphA4(lacZ/+) mice. These results show that the hopping phenotype is the result of a change in the balance between excitatory and inhibitory signals across the midline and that EphA4-positive neurons play an essential role in the mammalian CPG.

A transgenic mouse line for molecular genetic analysis of excitatory glutamatergic neurons.

Excitatory glutamatergic neurons are part of most of the neuronal circuits in the mammalian nervous system. We have used BAC-technology to generate a BAC-Vglut2::Cre mouse line where Cre expression is driven by the vesicular glutamate transporter 2 (Vglut2) promotor. This BAC-Vglut2::Cre mouse line showed specific expression of Cre in Vglut2 positive cells in the spinal cord with no ectopic expression in GABAergic or glycinergic neurons. This mouse line also showed specific Cre expression in Vglut2 positive structures in the brain such as thalamus, hypothalamus, superior colliculi, inferior colliculi and deep cerebellar nuclei together with nuclei in the midbrain and hindbrain. Cre-mediated recombination was restricted to Cre expressing cells in the spinal cord and brain and occurred as early as E 12.5. Known Vglut2 positive neurons showed normal electrophysiological properties in the BAC-Vglut2::Cre transgenic mice. Altogether, this BAC-Vglut2::Cre mouse line provides a valuable tool for molecular genetic analysis of excitatory neuronal populations throughout the mouse nervous system.

Probing spinal circuits controlling walking in mammals.

Locomotion in mammals is a complex motor act that involves the activation of a large number of muscles in a well-coordinated pattern. Understanding the network organization of the intrinsic spinal networks that control the locomotion, the central pattern generators, has been a challenge to neuroscientists. However, experiments using the isolated rodent spinal cord and combining electrophysiology and molecular genetics to dissect the locomotor network have started to shed new light on the network structure. In the present review, we will discuss findings that have revealed the role of designated populations of neurons for the key network functions including coordinating muscle activity and generating rhythmic activity. These findings are summarized in proposed organizational principles for the mammalian segmental CPG.

Excitatory components of the mammalian locomotor CPG.

Locomotion in mammals is to a large degree controlled directly by intrinsic spinal networks, called central pattern generators (CPGs). The overall function of these networks is governed by interaction between inhibitory and excitatory neurons. In the present review, we will discuss recent findings addressing the role of excitatory synaptic transmission for network function including the role of specific excitatory neuronal populations in coordinating muscle activity and in generating rhythmic activity.

Neuronal atlas of the dorsal horn defines its architecture and links sensory input to transcriptional cell types.

The dorsal horn of the spinal cord is critical to processing distinct modalities of noxious and innocuous sensation, but little is known of the neuronal subtypes involved, hampering efforts to deduce principles governing somatic sensation. Here we used single-cell RNA sequencing to classify sensory neurons in the mouse dorsal horn. We identified 15 inhibitory and 15 excitatory molecular subtypes of neurons, equaling the complexity in cerebral cortex. Validating our classification scheme in vivo and matching cell types to anatomy of the dorsal horn by spatial transcriptomics reveals laminar enrichment for each of the cell types. Neuron types, when combined, define a multilayered organization with like neurons layered together. Employing our scheme, we find that heat and cold stimuli activate discrete sets of both excitatory and inhibitory neuron types. This work provides a systematic and comprehensive molecular classification of spinal cord sensory neurons, enabling functional interrogation of sensory processing.

Ca2+-binding protein NECAB2 facilitates inflammatory pain hypersensitivity.

Pain signals are transmitted by multisynaptic glutamatergic pathways. Their first synapse between primary nociceptors and excitatory spinal interneurons gates the sensory load. In this pathway, glutamate release is orchestrated by Ca2+-sensor proteins, with N-terminal EF-hand Ca2+-binding protein 2 (NECAB2) being particular abundant. However, neither the importance of NECAB2+ neuronal contingents in dorsal root ganglia (DRGs) and spinal cord nor the function determination by NECAB2 has been defined. A combination of histochemical analyses and single-cell RNA-sequencing showed NECAB2 in small- and medium-sized C- and Aδ D-hair low-threshold mechanoreceptors in DRGs, as well as in protein kinase C γ excitatory spinal interneurons. NECAB2 was downregulated by peripheral nerve injury, leading to the hypothesis that NECAB2 loss of function could limit pain sensation. Indeed, Necab2-/- mice reached a pain-free state significantly faster after peripheral inflammation than did WT littermates. Genetic access to transiently activated neurons revealed that a mediodorsal cohort of NECAB2+ neurons mediates inflammatory pain in the mouse spinal dorsal horn. Here, besides dampening excitatory transmission in spinal interneurons, NECAB2 limited pronociceptive brain-derived neurotrophic factor (BDNF) release from sensory afferents. Hoxb8-dependent reinstatement of NECAB2 expression in Necab2-/- mice then demonstrated that spinal and DRG NECAB2 alone could control inflammation-induced sensory hypersensitivity. Overall, we identify NECAB2 as a critical component of pronociceptive pain signaling, whose inactivation offers substantial pain relief.

Spinal Hb9::Cre-derived excitatory interneurons contribute to rhythm generation in the mouse.

Rhythm generating neurons are thought to be ipsilaterally-projecting excitatory neurons in the thoracolumbar mammalian spinal cord. Recently, a subset of Shox2 interneurons (Shox2 non-V2a INs) was found to fulfill these criteria and make up a fraction of the rhythm-generating population. Here we use Hb9::Cre mice to genetically manipulate Hb9::Cre-derived excitatory interneurons (INs) in order to determine the role of these INs in rhythm generation. We demonstrate that this line captures a consistent population of spinal INs which is mixed with respect to neurotransmitter phenotype and progenitor domain, but does not overlap with the Shox2 non-V2a population. We also show that Hb9::Cre-derived INs include the comparatively small medial population of INs which continues to express Hb9 postnatally. When excitatory neurotransmission is selectively blocked by deleting Vglut2 from Hb9::Cre-derived INs, there is no difference in left-right and/or flexor-extensor phasing between these cords and controls, suggesting that excitatory Hb9::Cre-derived INs do not affect pattern generation. In contrast, the frequencies of locomotor activity are significantly lower in cords from Hb9::Cre-Vglut2Δ/Δ mice than in cords from controls. Collectively, our findings indicate that excitatory Hb9::Cre-derived INs constitute a distinct population of neurons that participates in the rhythm generating kernel for spinal locomotion.

Medial septal dysfunction by Aß-induced KCNQ channel-block in glutamatergic neurons.

Amyloid ß (Aß) peptides play a central role in the pathophysiology of Alzheimer's disease (AD). The cellular mechanisms underlying Aß toxicity, however, are poorly understood. Here we show that Aß(25-35) and Aß(1-40) acutely and differentially affect the characteristics of 3 classes of medial septum (MS) neurons in mice. In glutamatergic neurons Aß increases firing frequency and blocks the A- and the M-current (I(A) and I(M), respectively). While the I(A) block is similar in other MS neuron classes, the block of I(M) is specific to glutamatergic neurons. I(M) block and a simulated Aß block mimic the Aß-induced increase in spontaneous firing in glutamatergic neurons. Calcium imaging shows that under control conditions glutamatergic neurons rarely fire while nonglutamatergic neurons fire coherently at theta frequencies. Aß increases the firing rate of glutamatergic neurons while nonglutamatergic neurons lose theta firing coherence. Our results demonstrate that Aß-induced dysfunction of glutamatergic neurons via I(M) decrease diminishes MS rhythmicity, which may negatively affect hippocampal rhythmogenesis and underlie the memory loss observed in Alzheimer's disease.

Identification of minimal neuronal networks involved in flexor-extensor alternation in the mammalian spinal cord.

Neural networks in the spinal cord control two basic features of locomotor movements: rhythm generation and pattern generation. Rhythm generation is generally considered to be dependent on glutamatergic excitatory neurons. Pattern generation involves neural circuits controlling left-right alternation, which has been described in great detail, and flexor-extensor alternation, which remains poorly understood. Here, we use a mouse model in which glutamatergic neurotransmission has been ablated in the locomotor region of the spinal cord. The isolated in vitro spinal cord from these mice produces locomotor-like activity-when stimulated with neuroactive substances-with prominent flexor-extensor alternation. Under these conditions, unlike in control mice, networks of inhibitory interneurons generate the rhythmic activity. In the absence of glutamatergic synaptic transmission, the flexor-extensor alternation appears to be generated by Ia inhibitory interneurons, which mediate reciprocal inhibition from muscle proprioceptors to antagonist motor neurons. Our study defines a minimal inhibitory network that is needed to produce flexor-extensor alternation during locomotion.

Activation of groups of excitatory neurons in the mammalian spinal cord or hindbrain evokes locomotion.

Central pattern generators (CPGs) are spinal neuronal networks required for locomotion. Glutamatergic neurons have been implicated as being important for intrinsic rhythm generation in the CPG and for the command signal for initiating locomotion, although this has not been demonstrated directly. We used a newly generated vesicular glutamate transporter 2-channelrhodopsin2-yellow fluorescent protein (Vglut2-ChR2-YFP) mouse to directly examine the functional role of glutamatergic neurons in rhythm generation and initiation of locomotion. This mouse line expressed ChR2-YFP in the spinal cord and hindbrain. ChR2-YFP was reliably expressed in Vglut2-positive cells and YFP-expressing cells could be activated by light. Photo-stimulation of either the lumbar spinal cord or the caudal hindbrain was sufficient to both initiate and maintain locomotor-like activity. Our results indicate that glutamatergic neurons in the spinal cord are critical for initiating or maintaining the rhythm and that activation of hindbrain areas containing the locomotor command regions is sufficient to directly activate the spinal locomotor network.

Neuropilin1 is a direct downstream target of Nurr1 in the developing brain stem.

The orphan nuclear receptor Nurr1 is expressed in the developing and adult central nervous system. Previous studies have shown that Nurr1 is essential for the generation of midbrain dopamine neurons. Furthermore, Nurr1 is critical for respiratory functions associated with the brain stem. Very few Nurr1 regulated genes have been identified and it remains unclear how Nurr1 influences the function and development of neurons. To identify novel Nurr1 target genes we have searched for regulated genes in the dopaminergic MN9D cell line. These experiments identified Neuropilin-1 (Nrp1), a receptor protein involved in axon guidance and angiogenesis, as a novel Nurr1 target gene. Nrp1 expression was rapidly up-regulated by Nurr1 in MN9D cells and in situ hybridization analysis showed that Nrp1 was coexpressed with Nurr1 in the brain stem dorsal motor nucleus. Importantly, Nrp1 expression was down-regulated in this area in Nurr1 null mice. Moreover, two functional Nurr1 binding sites were identified in the Nrp1 promoter and Nurr1 was found to be recruited to these sites in MN9D cells, further supporting that Nrp1 is a direct downstream target of Nurr1. Taken together, our findings suggest that Nurr1 might influence the processes of axon guidance and/or angiogenesis via the regulation of Nrp1 expression.

Glucocorticoid signaling is perturbed by the atypical orphan receptor and corepressor SHP.

SHP (NROB2) is an atypical orphan nuclear receptor that lacks a DNA-binding domain but contains a putative ligand-binding domain. Previous studies have revealed that SHP interacts with a variety of nuclear receptors and inhibits their transcriptional activity, thereby acting as a corepressor. In this report we identify the glucocorticoid receptor (GR) as a novel downstream target receptor for SHP inhibition. SHP potently inhibits dexamethasone-induced transcriptional GR activity in mammalian cells, and the inhibition involves a functional second NR-box within SHP. Interestingly, this motif shows a high homology with the NR-box in the glucocorticoid and cAMP-inducible GR coactivator PGC-1, indicating similar binding specificity and shared target receptors. We show that SHP antagonizes PGC-1 coactivation and, in addition, we identify the PGC- 1-regulated phospho(enol)pyruvate carboxykinase (PEPCK) promoter as a novel target promoter for SHP inhibition. This implies a physiologically relevant role for SHP in modulating hepatic glucocorticoid action. Furthermore, when coexpressing green fluorescent protein-tagged GR together with SHP, an intranuclear redistribution of GR was observed. As inhibition-deficient SHP mutants were unable to induce this redistribution, intranuclear tethering of target receptors may represent yet another, previously uncovered, aspect of SHP inhibition.