Differential Loss of Spinal Interneurons in a Mouse Model of ALS.

Amyotrophic lateral sclerosis (ALS) leads to a loss of specific motor neuron populations in the spinal cord and cortex. Emerging evidence suggests that interneurons may also be affected, but a detailed characterization of interneuron loss and its potential impacts on motor neuron loss and disease progression is lacking. To examine this issue, the fate of V1 inhibitory neurons during ALS was assessed in the ventral spinal cord using the SODG93A mouse model. The V1 population makes up ∼30% of all ventral inhibitory neurons, ∼50% of direct inhibitory synaptic contacts onto motor neuron cell bodies, and is thought to play a key role in modulating motor output, in part through recurrent and reciprocal inhibitory circuits. We find that approximately half of V1 inhibitory neurons are lost in SODG93A mice at late disease stages, but that this loss is delayed relative to the loss of motor neurons and V2a excitatory neurons. We further identify V1 subpopulations based on transcription factor expression that are differentially susceptible to degeneration in SODG93A mice. At an early disease stage, we show that V1 synaptic contacts with motor neuron cell bodies increase, suggesting an upregulation of inhibition before V1 neurons are lost in substantial numbers. These data support a model in which progressive changes in V1 synaptic contacts early in disease, and in select V1 subpopulations at later stages, represent a compensatory upregulation and then deleterious breakdown of specific interneuron circuits within the spinal cord.

Role of muscle spindle feedback in regulating muscle activity strength during walking at different speed in mice.

Terrestrial animals increase their walking speed by increasing the activity of the extensor muscles. However, the mechanism underlying how this speed-dependent amplitude modulation is achieved remains obscure. Previous studies have shown that group Ib afferent feedback from Golgi tendon organs that signal force is one of the major regulators of the strength of muscle activity during walking in cats and humans. In contrast, the contribution of group Ia/II afferent feedback from muscle spindle stretch receptors that signal angular displacement of leg joints is unclear. Some studies indicate that group II afferent feedback may be important for amplitude regulation in humans, but the role of muscle spindle feedback in regulation of muscle activity strength in quadrupedal animals is very poorly understood. To examine the role of feedback from muscle spindles, we combined in vivo electrophysiology and motion analysis with mouse genetics and gene delivery with adeno-associated virus. We provide evidence that proprioceptive sensory feedback from muscle spindles is important for the regulation of the muscle activity strength and speed-dependent amplitude modulation. Furthermore, our data suggest that feedback from the muscle spindles of the ankle extensor muscles, the triceps surae, is the main source for this mechanism. In contrast, muscle spindle feedback from the knee extensor muscles, the quadriceps femoris, has no influence on speed-dependent amplitude modulation. We provide evidence that proprioceptive feedback from ankle extensor muscles is critical for regulating muscle activity strength as gait speed increases. NEW & NOTEWORTHY Animals upregulate the activity of extensor muscles to increase their walking speed, but the mechanism behind this is not known. We show that this speed-dependent amplitude modulation requires proprioceptive sensory feedback from muscle spindles of ankle extensor muscle. In the absence of muscle spindle feedback, animals cannot walk at higher speeds as they can when muscle spindle feedback is present.

Origin and Segmental Diversity of Spinal Inhibitory Interneurons.

Motor output varies along the rostro-caudal axis of the tetrapod spinal cord. At limb levels, ∼60 motor pools control the alternation of flexor and extensor muscles about each joint, whereas at thoracic levels as few as 10 motor pools supply muscle groups that support posture, inspiration, and expiration. Whether such differences in motor neuron identity and muscle number are associated with segmental distinctions in interneuron diversity has not been resolved. We show that select combinations of nineteen transcription factors that specify lumbar V1 inhibitory interneurons generate subpopulations enriched at limb and thoracic levels. Specification of limb and thoracic V1 interneurons involves the Hox gene Hoxc9 independently of motor neurons. Thus, early Hox patterning of the spinal cord determines the identity of V1 interneurons and motor neurons. These studies reveal a developmental program of V1 interneuron diversity, providing insight into the organization of inhibitory interneurons associated with differential motor output.

Spinal Inhibitory Interneuron Diversity Delineates Variant Motor Microcircuits.

Animals generate movement by engaging spinal circuits that direct precise sequences of muscle contraction, but the identity and organizational logic of local interneurons that lie at the core of these circuits remain unresolved. Here, we show that V1 interneurons, a major inhibitory population that controls motor output, fractionate into highly diverse subsets on the basis of the expression of 19 transcription factors. Transcriptionally defined V1 subsets exhibit distinct physiological signatures and highly structured spatial distributions with mediolateral and dorsoventral positional biases. These positional distinctions constrain patterns of input from sensory and motor neurons and, as such, suggest that interneuron position is a determinant of microcircuit organization. Moreover, V1 diversity indicates that different inhibitory microcircuits exist for motor pools controlling hip, ankle, and foot muscles, revealing a variable circuit architecture for interneurons that control limb movement.

Neuronal Ig/Caspr recognition promotes the formation of axoaxonic synapses in mouse spinal cord.

Inhibitory microcircuits are wired with a precision that underlies their complex regulatory roles in neural information processing. In the spinal cord, one specialized class of GABAergic interneurons (GABApre) mediates presynaptic inhibitory control of sensory-motor synapses. The synaptic targeting of these GABAergic neurons exhibits an absolute dependence on proprioceptive sensory terminals, yet the molecular underpinnings of this specialized axoaxonic organization remain unclear. Here, we show that sensory expression of an NB2 (Contactin5)/Caspr4 coreceptor complex, together with spinal interneuron expression of NrCAM/CHL1, directs the high-density accumulation of GABAergic boutons on sensory terminals. Moreover, genetic elimination of NB2 results in a disproportionate stripping of inhibitory boutons from high-density GABApre-sensory synapses, suggesting that the preterminal axons of GABApre neurons compete for access to individual sensory terminals. Our findings define a recognition complex that contributes to the assembly and organization of a specialized GABAergic microcircuit.

Production and characterization of monoclonal antibodies sensitive to conformation in the 5HT2c serotonin receptor.

mAbs that are sensitive to protein conformation can be helpful in studies of protein structure and function; in particular, mAb fragments are useful reagents in membrane protein crystallization. We immunized mice with the rat 5HT2c serotonin receptor and derived clonal hybridoma cells, which we tested for specific antigen reactivity by using the complementarity of purified protein from bacteria and receptor-embedded mammalian cell membranes. Nine mAbs met our criteria for specificity, affinity, and sensitivity to conformational features. Epitopes were mapped in various additional tests. Five of the nine mAbs have cytoplasmic epitopes, and two of these are sensitive to the ligand state of the receptor. These properties should be useful both for structural analysis and in probes of function.

A Hox regulatory network establishes motor neuron pool identity and target-muscle connectivity.

Spinal motor neurons acquire specialized "pool" identities that determine their ability to form selective connections with target muscles in the limb, but the molecular basis of this striking example of neuronal specificity has remained unclear. We show here that a Hox transcriptional regulatory network specifies motor neuron pool identity and connectivity. Two interdependent sets of Hox regulatory interactions operate within motor neurons, one assigning rostrocaudal motor pool position and a second directing motor pool diversity at a single segmental level. This Hox regulatory network directs the downstream transcriptional identity of motor neuron pools and defines the pattern of target-muscle connectivity.