Functional specialization of hippocampal somatostatin-expressing interneurons.

Hippocampal somatostatin-expressing (Sst) GABAergic interneurons (INs) exhibit considerable anatomical and functional heterogeneity. Recent single-cell transcriptome analyses have provided a comprehensive Sst-IN subpopulations census, a plausible molecular ground truth of neuronal identity whose links to specific functionality remain incomplete. Here, we designed an approach to identify and access subpopulations of Sst-INs based on transcriptomic features. Four mouse models based on single or combinatorial Cre- and Flp- expression differentiated functionally distinct subpopulations of CA1 hippocampal Sst-INs that largely tiled the morpho-functional parameter space of the Sst-INs superfamily. Notably, the Sst;;Tac1 intersection revealed a population of bistratified INs that preferentially synapsed onto fast-spiking interneurons (FS-INs) and were sufficient to interrupt their firing. In contrast, the Ndnf;;Nkx2-1 intersection identified a population of oriens lacunosum-moleculare INs that predominantly targeted CA1 pyramidal neurons, avoiding FS-INs. Overall, our results provide a framework to translate neuronal transcriptomic identity into discrete functional subtypes that capture the diverse specializations of hippocampal Sst-INs.

A flowchart for adequate controls in virus-based monosynaptic tracing experiments identified Cre-independent leakage of the TVA receptor in RΦGT mice.

BACKGROUND: A pseudotyped modified rabies virus lacking the rabies glycoprotein (G-protein), which is crucial for transsynaptic spread, can be used for monosynaptic retrograde tracing. By coupling the pseudotyped virus with transgene expression of the G-protein and the avian leukosis and sarcoma virus subgroup A receptor (TVA), which is necessary for cell entry of the virus, researchers can investigate specific neuronal populations. Responder mouse lines, like the RΦGT mouse line, carry the genes encoding the G-protein and TVA under Cre-dependent expression. These mouse lines are valuable tools because they reduce the number of viral injections needed compared to when using helper viruses. Since RΦGT mice do not express Cre themselves, introducing the pseudotyped rabies virus into their brain should not result in viral cell entry or spread. RESULTS: We present a straightforward flowchart for adequate controls in tracing experiments, which we employed to demonstrate Cre-independent expression of TVA in RΦGT mice. CONCLUSIONS: Our observations revealed TVA leakage, indicating that RΦGT mice should be used with caution for transgene expression of TVA. Inaccurate tracing outcomes may occur if TVA is expressed in the absence of Cre since background leakage leads to nonspecific cell entry. Moreover, conducting appropriate control experiments can identify the source of potential caveats in virus-based neuronal tracing experiments.

Unraveling the role of Slc10a4 in auditory processing and sensory motor gating: Implications for neuropsychiatric disorders?

BACKGROUND: Psychiatric disorders, such as schizophrenia, are complex and challenging to study, partly due to the lack of suitable animal models. However, the absence of the Slc10a4 gene, which codes for a monoaminergic and cholinergic associated vesicular transporter protein, in knockout mice (Slc10a4-/-), leads to the accumulation of extracellular dopamine. A major challenge for studying schizophrenia is the lack of suitable animal models that accurately represent the disorder. We sought to overcome this challenge by using Slc10a4-/- mice as a potential model, considering their altered dopamine levels. This makes them a potential animal model for schizophrenia, a disorder known to be associated with altered dopamine signaling in the brain. METHODS: The locomotion, auditory sensory filtering and prepulse inhibition (PPI) of Slc10a4-/- mice were quantified and compared to wildtype (WT) littermates. Intrahippocampal electrodes were used to record auditory event-related potentials (aERPs) for quantifying sensory filtering in response to paired-clicks. The channel above aERPs phase reversal was chosen for reliably comparing results between animals, and aERPs amplitude and latency of click responses were quantified. WT and Slc10a4-/- mice were also administered subanesthetic doses of ketamine to provoke psychomimetic behavior. RESULTS: Baseline locomotion during auditory stimulation was similar between Slc10a4-/- mice and WT littermates. In WT animals, normal auditory processing was observed after i.p saline injections, and it was maintained under the influence of 5 mg/kg ketamine, but disrupted by 20 mg/kg ketamine. On the other hand, Slc10a4-/- mice did not show significant differences between N40 S1 and S2 amplitude responses in saline or low dose ketamine treatment. Auditory gating was considered preserved since the second N40 peak was consistently suppressed, but with increased latency. The P80 component showed higher amplitude, with shorter S2 latency under saline and 5 mg/kg ketamine treatment in Slc10a4-/- mice, which was not observed in WT littermates. Prepulse inhibition was also decreased in Slc10a4-/- mice when the longer interstimulus interval of 100 ms was applied, compared to WT littermates. CONCLUSION: The Slc10a4-/- mice responses indicate that cholinergic and monoaminergic systems participate in the PPI magnitude, in the temporal coding (response latency) of the auditory sensory gating component N40, and in the amplitude of aERPs P80 component. These results suggest that Slc10a4-/- mice can be considered as potential models for neuropsychiatric conditions.

The alpha2 nicotinic acetylcholine receptor, a subunit with unique and selective expression in inhibitory interneurons associated with principal cells.

Nicotinic acetylcholine receptors (nAChRs) play crucial roles in various human disorders, with the α7, α4, α6, and α3-containing nAChR subtypes extensively studied in relation to conditions such as Alzheimer's disease, Parkinson's disease, nicotine dependence, mood disorders, and stress disorders. In contrast, the α2-nAChR subunit has received less attention due to its more restricted expression and the scarcity of specific agonists and antagonists for studying its function. Nevertheless, recent research has shed light on the unique expression pattern of the Chrna2 gene, which encodes the α2-nAChR subunit, and its involvement in distinct populations of inhibitory interneurons. This review highlights the structure, pharmacology, localization, function, and disease associations of α2-containing nAChRs and points to the unique expression pattern of the Chrna2 gene and its role in different inhibitory interneuron populations. These populations, including the oriens lacunosum moleculare (OLM) cells in the hippocampus, Martinotti cells in the neocortex, and Renshaw cells in the spinal cord, share common features and contribute to recurrent inhibitory microcircuits. Thus, the α2-nAChR subunit's unique expression pattern in specific interneuron populations and its role in recurrent inhibitory microcircuits highlight its importance in various physiological processes. Further research is necessary to uncover the comprehensive functionality of α2-containing nAChRs, delineate their specific contributions to neuronal circuits, and investigate their potential as therapeutic targets for related disorders.

Cortical somatostatin interneuron subtypes form cell-type-specific circuits.

The cardinal classes are a useful simplification of cortical interneuron diversity, but such broad subgroupings gloss over the molecular, morphological, and circuit specificity of interneuron subtypes, most notably among the somatostatin interneuron class. Although there is evidence that this diversity is functionally relevant, the circuit implications of this diversity are unknown. To address this knowledge gap, we designed a series of genetic strategies to target the breadth of somatostatin interneuron subtypes and found that each subtype possesses a unique laminar organization and stereotyped axonal projection pattern. Using these strategies, we examined the afferent and efferent connectivity of three subtypes (two Martinotti and one non-Martinotti) and demonstrated that they possess selective connectivity with intratelecephalic or pyramidal tract neurons. Even when two subtypes targeted the same pyramidal cell type, their synaptic targeting proved selective for particular dendritic compartments. We thus provide evidence that subtypes of somatostatin interneurons form cell-type-specific cortical circuits.

Functional specialization of hippocampal somatostatin-expressing interneurons.

Hippocampal somatostatin-expressing (Sst) GABAergic interneurons (INs) exhibit considerable anatomical and functional heterogeneity. Recent single cell transcriptome analyses have provided a comprehensive Sst-IN subtype census, a plausible molecular ground truth of neuronal identity whose links to specific functionality remain incomplete. Here, we designed an approach to identify and access subpopulations of Sst-INs based on transcriptomic features. Four mouse models based on single or combinatorial Cre- and Flp- expression differentiated functionally distinct subpopulations of CA1 hippocampal Sst-INs that largely tiled the morpho-functional parameter space of the Sst-INs superfamily. Notably, the Sst;;Tac1 intersection revealed a population of bistratified INs that preferentially synapsed onto fast-spiking interneurons (FS-INs) and were both necessary and sufficient to interrupt their firing. In contrast, the Ndnf;;Nkx2-1 intersection identified a population of oriens lacunosum-moleculare (OLM) INs that predominantly targeted CA1 pyramidal neurons, avoiding FS-INs. Overall, our results provide a framework to translate neuronal transcriptomic identity into discrete functional subtypes that capture the diverse specializations of hippocampal Sst-INs.

Adult spinal Dmrt3 neurons receive direct somatosensory inputs from ipsi- and contralateral primary afferents and from brainstem motor nuclei.

In the spinal cord, sensory-motor circuits controlling motor activity are situated in the dorso-ventral interface. The neurons identified by the expression of the transcription factor Doublesex and mab-3 related transcription factor 3 (Dmrt3) have previously been associated with the coordination of locomotion in horses (Equus caballus, Linnaeus, 1758), mice (Mus musculus, Linnaeus, 1758), and zebrafish (Danio rerio, F. Hamilton, 1822). Based on earlier studies, we hypothesized that, in mice, these neurons may be positioned to receive sensory and central inputs to relay processed commands to motor neurons. Thus, we investigated the presynaptic inputs to spinal Dmrt3 neurons using monosynaptic retrograde replication-deficient rabies tracing. The analysis showed that lumbar Dmrt3 neurons receive inputs from intrasegmental neurons, and intersegmental neurons from the cervical, thoracic, and sacral segments. Some of these neurons belong to the excitatory V2a interneurons and to plausible Renshaw cells, defined by the expression of Chx10 and calbindin, respectively. We also found that proprioceptive primary sensory neurons of type Ia2, Ia3, and Ib, defined by the expression of calbindin, calretinin, and Brn3c, respectively, provide presynaptic inputs to spinal Dmrt3 neurons. In addition, we demonstrated that Dmrt3 neurons receive inputs from brain areas involved in motor regulation, including the red nucleus, primary sensory-motor cortex, and pontine nuclei. In conclusion, adult spinal Dmrt3 neurons receive inputs from motor-related brain areas as well as proprioceptive primary sensory neurons and have been shown to connect directly to motor neurons. Dmrt3 neurons are thus positioned to provide sensory-motor control and their connectivity is suggestive of the classical reflex pathways present in the spinal cord.

Hop Mice Display Synchronous Hindlimb Locomotion and a Ventrally Fused Lumbar Spinal Cord Caused by a Point Mutation in Ttc26.

Identifying the spinal circuits controlling locomotion is critical for unravelling the mechanisms controlling the production of gaits. Development of the circuits governing left-right coordination relies on axon guidance molecules such as ephrins and netrins. To date, no other class of proteins have been shown to play a role during this process. Here, we have analyzed hop mice, which walk with a characteristic hopping gait using their hindlimbs in synchrony. Fictive locomotion experiments suggest that a local defect in the ventral spinal cord contributes to the aberrant locomotor phenotype. Hop mutant spinal cords had severe morphologic defects, including the absence of the ventral midline and a poorly defined border between white and gray matter. The hop mice represent the first model where, exclusively found in the lumbar domain, the left and right components of the central pattern generators (CPGs) are fused with a synchronous hindlimb gait as a functional consequence. These defects were associated with abnormal developmental processes, including a misplaced notochord and reduced induction of ventral progenitor domains. Whereas the underlying mutation in hop mice has been suggested to lie within the Ttc26 gene, other genes in close vicinity have been associated with gait defects. Mouse embryos carrying a CRISPR replicated point mutation within Ttc26 displayed an identical morphologic phenotype. Thus, our data suggest that the assembly of the lumbar CPG network is dependent on fully functional TTC26 protein.

Cortical disinhibitory circuits: cell types, connectivity and function.

The concept of a dynamic excitation/inhibition balance tuned by circuit disinhibition, which can shape information flow during complex behavioral tasks, has arisen as an important and conserved information-processing motif. In cortical circuits, different subtypes of GABAergic inhibitory interneurons are connected to each other, offering an anatomical foundation for disinhibitory processes. Moreover, a subpopulation of GABAergic cells that express vasoactive intestinal polypeptide (VIP) preferentially innervates inhibitory interneurons, highlighting their central role in disinhibitory modulation. We discuss inhibitory neuron subtypes involved in disinhibition, with a focus on local circuits and long-range synaptic connections that drive disinhibitory function. We highlight multiple layers of disinhibition across cortical circuits that regulate behavior and serve to maintain an excitation/inhibition balance.

A loss-of-function mutation in RORB disrupts saltatorial locomotion in rabbits.

Saltatorial locomotion is a type of hopping gait that in mammals can be found in rabbits, hares, kangaroos, and some species of rodents. The molecular mechanisms that control and fine-tune the formation of this type of gait are unknown. Here, we take advantage of one strain of domesticated rabbits, the sauteur d'Alfort, that exhibits an abnormal locomotion behavior defined by the loss of the typical jumping that characterizes wild-type rabbits. Strikingly, individuals from this strain frequently adopt a bipedal gait using their front legs. Using a combination of experimental crosses and whole genome sequencing, we show that a single locus containing the RAR related orphan receptor B gene (RORB) explains the atypical gait of these rabbits. We found that a splice-site mutation in an evolutionary conserved site of RORB results in several aberrant transcript isoforms incorporating intronic sequence. This mutation leads to a drastic reduction of RORB-positive neurons in the spinal cord, as well as defects in differentiation of populations of spinal cord interneurons. Our results show that RORB function is required for the performance of saltatorial locomotion in rabbits.

Using Cortical Neuron Markers to Target Cells in the Dorsal Cochlear Nucleus.

The dorsal cochlear nucleus (DCN) is a region of particular interest for auditory and tinnitus research. However, lack of useful genetic markers for in vivo manipulations hinders elucidation of the DCN contribution to tinnitus pathophysiology. This work assesses whether adeno-associated viral vectors (AAV) containing the calcium/calmodulin-dependent protein kinase 2α (CaMKIIα) promoter and a mouse line of nicotinic acetylcholine receptor α2 subunit (Chrna2)-Cre can target specific DCN populations. We found that CaMKIIα cannot be used to target excitatory fusiform DCN neurons as labeled cells showed diverse morphology indicating they belong to different classes of DCN neurons. Light stimulation after driving Channelrhodopsin2 (ChR2) by the CaMKIIα promoter generated spikes in some units but firing rate decreased when light stimulation coincided with sound. Expression and activation of CaMKIIα-eArchaerhodopsin3.0 in the DCN produced inhibition in some units but sound-driven spikes were delayed by concomitant light stimulation. We explored the existence of Cre+ cells in the DCN of Chrna2-Cre mice by hydrogel embedding technique (CLARITY). There were almost no Cre+ cell bodies in the DCN; however, we identified profuse projections arising from the ventral cochlear nucleus (VCN). Anterograde labeling in the VCN revealed projections to the ipsilateral superior olive and contralateral medial nucleus of the trapezoid body (MNTB; bushy cells), and a second bundle terminating in the DCN, suggesting the latter to be excitatory Chrna2+ T-stellate cells. Exciting Chrna2+ cells increased DCN firing. This work shows that cortical molecular tools may be useful for manipulating the DCN especially for tinnitus studies.

Single Cell Transcriptomic Analysis of Spinal Dmrt3 Neurons in Zebrafish and Mouse Identifies Distinct Subtypes and Reveal Novel Subpopulations Within the dI6 Domain.

The spinal locomotor network is frequently used for studies into how neuronal circuits are formed and how cellular activity shape behavioral patterns. A population of dI6 interneurons, marked by the Doublesex and mab-3 related transcription factor 3 (Dmrt3), has been shown to participate in the coordination of locomotion and gaits in horses, mice and zebrafish. Analyses of Dmrt3 neurons based on morphology, functionality and the expression of transcription factors have identified different subtypes. Here we analyzed the transcriptomes of individual cells belonging to the Dmrt3 lineage from zebrafish and mice to unravel the molecular code that underlies their subfunctionalization. Indeed, clustering of Dmrt3 neurons based on their gene expression verified known subtypes and revealed novel populations expressing unique markers. Differences in birth order, differential expression of axon guidance genes, neurotransmitters, and their receptors, as well as genes affecting electrophysiological properties, were identified as factors likely underlying diversity. In addition, the comparison between fish and mice populations offers insights into the evolutionary driven subspecialization concomitant with the emergence of limbed locomotion.

A prefrontal-paraventricular thalamus circuit requires juvenile social experience to regulate adult sociability in mice.

Juvenile social isolation reduces sociability in adulthood, but the underlying neural circuit mechanisms are poorly understood. We found that, in male mice, 2 weeks of social isolation immediately following weaning leads to a failure to activate medial prefrontal cortex neurons projecting to the posterior paraventricular thalamus (mPFC→pPVT) during social exposure in adulthood. Chemogenetic or optogenetic suppression of mPFC→pPVT activity in adulthood was sufficient to induce sociability deficits without affecting anxiety-related behaviors or preference toward rewarding food. Juvenile isolation led to both reduced excitability of mPFC→pPVT neurons and increased inhibitory input drive from low-threshold-spiking somatostatin interneurons in adulthood, suggesting a circuit mechanism underlying sociability deficits. Chemogenetic or optogenetic stimulation of mPFC→pPVT neurons in adulthood could rescue the sociability deficits caused by juvenile isolation. Our study identifies a pair of specific medial prefrontal cortex excitatory and inhibitory neuron populations required for sociability that are profoundly affected by juvenile social experience.

EphA4 Is Required for Neural Circuits Controlling Skilled Reaching.

Skilled forelimb movements are initiated by feedforward motor commands conveyed by supraspinal motor pathways. The accuracy of reaching and grasping relies on internal feedback pathways that update ongoing motor commands. In mice lacking the axon guidance molecule EphA4, axonal misrouting of the corticospinal tract and spinal interneurons is manifested, leading to a hopping gait in hindlimbs. Moreover, mice with a conditional forebrain deletion of EphA4, display forelimb hopping in adaptive locomotion and exploratory reaching movements. However, it remains unclear how loss of EphA4 signaling disrupts function of forelimb motor circuit and skilled reaching and grasping movements. Here we investigated how neural circuits controlling skilled reaching were affected by the loss of EphA4. Both male and female C57BL/6 wild-type, heterozygous EphA4+/-, and homozygous EphA4-/- mice were used in behavioral and in vivo electrophysiological investigations. We found that EphA4 knock-out (-/-) mice displayed impaired goal-directed reaching movements. In vivo intracellular recordings from forelimb motor neurons demonstrated increased corticoreticulospinal excitation, decreased direct reticulospinal excitation, and reduced direct propriospinal excitation in EphA4 knock-out mice. Cerebellar surface recordings showed a functional perturbation of the lateral reticular nucleus-cerebellum internal feedback pathway in EphA4 knock-out mice. Together, our findings provide in vivo evidence at the circuit level that loss of EphA4 disrupts the function of both feedforward and feedback motor pathways, resulting in deficits in skilled reaching.SIGNIFICANCE STATEMENT The central advances of this study are the demonstration that null mutation in the axon guidance molecule EphA4 gene impairs the ability of mice to perform skilled reaching, and identification of how these behavioral deficits correlates with discrete neurophysiological changes in central motor pathways involved in the control of reaching. Our findings provide in vivo evidence at the circuit level that loss of EphA4 disrupts both feedforward and feedback motor pathways, resulting in deficits in skilled reaching. This analysis of motor circuit function may help to understand the pathophysiological mechanisms underlying movement disorders in humans.

Behavioral Characterization of dmrt3a Mutant Zebrafish Reveals Crucial Aspects of Vertebrate Locomotion through Phenotypes Related to Acceleration.

Vertebrate locomotion is orchestrated by spinal interneurons making up a central pattern generator. Proper coordination of activity, both within and between segments, is required to generate the desired locomotor output. This coordination is altered during acceleration to ensure the correct recruitment of muscles for the chosen speed. The transcription factor Dmrt3 has been proposed to shape the patterned output at different gaits in horses and mice. Here, we characterized dmrt3a mutant zebrafish, which showed a strong, transient, locomotor phenotype in developing larvae. During beat-and-glide swimming, mutant larvae showed fewer and shorter movements with decreased velocity and acceleration. Developmental compensation likely occurs as the analyzed behaviors did not differ from wild-type at older larval stages. However, analysis of maximum swim speed in juveniles suggests that some defects persist within the mature locomotor network of dmrt3a mutants. Our results reveal the pivotal role Dmrt3 neurons play in shaping the patterned output during acceleration in vertebrates.

Lessons, insights and newly developed tools emerging from behavioral phenotyping core facilities.

Scientific investigations, in general, and research in neuroscience, in particular, are becoming ever more complex and require the integration of different techniques. Behavioral assays, which are among the most frequently used methodologies in neuroscience, nowadays rely on advanced, sophisticated technologies that require proficient application. Therefore, behavioral core facilities are becoming essential support units, as they provide the specialized expert research services needed to conduct advanced neuroscience. We here review the lessons learned and insights gathered from managing behavioral core facilities in different academic research institutes. This review addresses several issues, including: the advantages of behavioral core facilities, considerations for establishing a behavioral core facility, and the methodological advances made through calibration and standardization of assay protocols and the development of new assays. Collectively, the review highlights the benefits of both working within and collaborating with behavioral core facility units and emphasizes the potential progress in neuro-phenotyping that such facilities provide.

The polyamine transporter Slc18b1(VPAT) is important for both short and long time memory and for regulation of polyamine content in the brain.

SLC18B1 is a sister gene to the vesicular monoamine and acetylcholine transporters, and the only known polyamine transporter, with unknown physiological role. We reveal that Slc18b1 knock out mice has significantly reduced polyamine content in the brain providing the first evidence that Slc18b1 is functionally required for regulating polyamine levels. We found that this mouse has impaired short and long term memory in novel object recognition, radial arm maze and self-administration paradigms. We also show that Slc18b1 KO mice have altered expression of genes involved in Long Term Potentiation, plasticity, calcium signalling and synaptic functions and that expression of components of GABA and glutamate signalling are changed. We further observe a partial resistance to diazepam, manifested as significantly lowered reduction in locomotion after diazepam treatment. We suggest that removal of Slc18b1 leads to reduction of polyamine contents in neurons, resulting in reduced GABA signalling due to long-term reduction in glutamatergic signalling.

Chrna2-OLM interneurons display different membrane properties and h-current magnitude depending on dorsoventral location.

The hippocampus is an extended structure displaying heterogeneous anatomical cell layers along its dorsoventral axis. It is known that dorsal and ventral regions show different integrity when it comes to functionality, innervation, gene expression, and pyramidal cell properties. Still, whether hippocampal interneurons exhibit different properties along the dorsoventral axis is not known. Here, we report electrophysiological properties of dorsal and ventral oriens lacunosum moleculare (OLM) cells from coronal sections of the Chrna2-cre mouse line. We found dorsal OLM cells to exhibit a significantly more depolarized resting membrane potential compared to ventral OLM cells, while action potential properties were similar between the two groups. We found ventral OLM cells to show a higher initial firing frequency in response to depolarizing current injections but also to exhibit a higher spike-frequency adaptation than dorsal OLM cells. Additionally, dorsal OLM cells displayed large membrane sags in response to negative current injections correlating with our results showing that dorsal OLM cells have more hyperpolarization-activated current (Ih ) compared to ventral OLM cells. Immunohistochemical examination indicates the h-current to correspond to hyperpolarization-activated cyclic nucleotide-gated subunit 2 (HCN2) channels. Computational studies suggest that Ih in OLM cells is essential for theta oscillations in hippocampal circuits, and here we found dorsal OLM cells to present a higher membrane resonance frequency than ventral OLM cells. Thus, our results highlight regional differences in membrane properties between dorsal and ventral OLM cells allowing this interneuron to differently participate in the generation of hippocampal theta rhythms depending on spatial location along the dorsoventral axis of the hippocampus.

EphA4-ADAM10 Interplay Patterns the Cochlear Sensory Epithelium through Local Disruption of Adherens Junctions.

The cochlear sensory epithelium contains a functionally important triangular fluid-filled space between adjacent pillar cells referred to as the tunnel of Corti. However, the molecular mechanisms leading to local cell-cell separation during development remain elusive. Here we show that EphA4 associates with ADAM10 to promote the destruction of E-cadherin-based adhesions between adjacent pillar cells. These cells fail to separate from each other, and E-cadherin abnormally persists at the pillar cell junction in EphA4 forward-signaling-deficient mice, as well as in the presence of ADAM10 inhibitor. Using immunolabeling and an in situ proximity ligation assay, we found that EphA4 forms a complex with E-cadherin and its sheddase ADAM10, which could be activated by ephrin-B2 across the pillar cell junction to trigger the cleavage of E-cadherin. Altogether, our findings provide a new molecular insight into the regulation of adherens junctions, which might be extended to a variety of physiological or pathological processes.

Characterization of Dmrt3-Derived Neurons Suggest a Role within Locomotor Circuits.

Neuronal networks within the spinal cord, collectively known as the central pattern generator (CPG), coordinate rhythmic movements underlying locomotion. The transcription factor doublesex and mab-3-related transcription factor 3 (DMRT3) is involved in the differentiation of the dorsal interneuron 6 class of spinal cord interneurons. In horses, a non-sense mutation in the Dmrt3 gene has major effects on gaiting ability, whereas mice lacking the Dmrt3 gene display impaired locomotor activity. Although the Dmrt3 gene is necessary for normal spinal network formation and function in mice, a direct role for Dmrt3-derived neurons in locomotor-related activities has not been demonstrated. Here we present the characteristics of the Dmrt3-derived spinal cord interneurons. Using transgenic mice of both sexes, we characterized interneurons labeled by their expression of Cre driven by the endogenous Dmrt3 promoter. We used molecular, retrograde tracing and electrophysiological techniques to examine the anatomical, morphological, and electrical properties of the Dmrt3-Cre neurons. We demonstrate that inhibitory Dmrt3-Cre neurons receive extensive synaptic inputs, innervate surrounding CPG neurons, intrinsically regulate CPG neuron's electrical activity, and are rhythmically active during fictive locomotion, bursting at frequencies independent to the ventral root output. The present study provides novel insights on the character of spinal Dmrt3-derived neurons, data demonstrating that these neurons participate in locomotor coordination.SIGNIFICANCE STATEMENT In this work, we provide evidence for a role of the Dmrt3 interneurons in spinal cord locomotor circuits as well as molecular and functional insights on the cellular and microcircuit level of the Dmrt3-expressing neurons in the spinal cord. Dmrt3 neurons provide the first example of an interneuron population displaying different oscillation frequencies. This study presents novel findings on an under-reported population of spinal cord neurons, which will aid in deciphering the locomotor network and will facilitate the design and development of therapeutics for spinal cord injury and motor disorders.