V1 Interneurons: The Spinal Cord’s Hidden Conductors

Most neuroscience coverage focuses on the brain. That makes sense — the brain is where perception, memory, and thought live. But walking, running, and even the simple act of reaching for a coffee cup happen largely because of circuits that never consult the brain at all. The spinal cord is not just a cable connecting the brain to the body. It is a computational device in its own right, and embedded within it is a rich population of interneurons that orchestrate nearly everything your muscles do.

This post is about one particularly important class of those interneurons — the V1 interneuron — and what my colleagues and I learned about them in our study published in 2024 in eLife.

What is an interneuron, and why does the spinal cord need so many?

Your spinal cord has two basic types of neurons you may have heard of. Motor neurons are the output cells — they send long axons out to muscles and make them contract. Sensory neurons carry signals from the body (touch, stretch, pain) into the spinal cord which then relay to the rest of the brain. But between those two populations there is a third category, far more numerous and far less famous: interneurons. These cells talk only to other neurons. They integrate information from sensory inputs and from the brain and then shape the activity of motor neurons and of each other.

Most of the computation that makes movement elegant — the smooth coordination between your hamstrings and quadriceps as you walk, the automatic adjustment of grip force when you pick up something unexpectedly heavy — is done by interneurons before your brain even registers it.

Inhibition is not the absence of movement, it is the sculptor of it

When people think about neural signals, they usually think about activation: neurons fire, muscles activate. But equally important is inhibition — suppressing activity. Inhibitory interneurons dampen motor neuron activity, and this dampening is as critical to coordinated movement as excitation.

Think about flexing your elbow. The biceps has to contract, but at the same time the triceps — the antagonist muscle on the other side — has to relax. If it did not, you would be fighting against yourself. The spinal cord handles this automatically through reciprocal inhibition: when signals arrive that activate one muscle group, inhibitory interneurons simultaneously suppress the antagonist motor neurons. The result is smooth, efficient movement.

Without inhibition, movement becomes rigid, uncoordinated, and violent. Patients with conditions like tetanus (which blocks inhibitory synaptic transmission) develop the characteristic muscle spasms and locked-jaw posture precisely because inhibitory interneurons can no longer do their job.

V1 interneurons are exclusively inhibitory. Every synapse they make onto other neurons is a suppressive one.

What defines a V1 interneuron?

For most of neuroscience history, interneuron types were identified by where they were located, what they looked like, to whom they were connected, and how they fired. But these criteria are imprecise: many different cell types can occupy the same neighborhood and look similar under a microscope.

Modern spinal cord biology has moved to a genetic definition. During embryonic development, the early spinal cord is organized into strips called progenitor domains, each producing a specific class of neurons. V1 interneurons arise from the p1 progenitor domain and express a transcription factor — a protein that controls which genes are switched on or off inside a cell — called Engrailed-1 (En1) during their development. This genetic fingerprint is what defines them — not their location, not their shape, and not the specific muscles they influence.

This distinction matters because it lets experimenters use genetic tools to identify and specifically manipulate V1 interneurons. You can genettically engineer mice so that only V1 interneurons carry a fluorescent label, making them glow a particular color when viewed under a microscope. You can express light-sensitive proteins in V1 cells and turn them on or off with a laser. You can even permanently delete or silence them to see what movement looks like without them.

When investigators probe V1 function directly — for example, by optogenetically activating V1 interneurons in isolated neonatal spinal cord preparations that generate fictive locomotion — the timing and coordination between flexor and extensor motor outputs is strongly altered. Genetic studies in which both V1 and a related inhibitory class (V2b) are eliminated together show that the normal alternating flexor-extensor pattern required for limbed walking is severely disrupted. V1 interneurons are essential, not optional.

The four clades: V1s are not one cell type, but a family

Here is where the story gets more interesting, and where my research comes in.

V1 interneurons are not a single uniform population. They are defined by a shared genetic origin (the En1 lineage), but within that lineage there is enormous diversity. A 2016 study led by Jay Bikoff, previous members of my lab, and colleagues at Columbia University used molecular profiling to identify at least four major subgroups of V1s, called clades, each defined by a unique combination of transcription factors expressed in the postnatal period:

• Renshaw cells — defined by the transcription factors MafA/MafB. These are the best-characterized V1 subtype, discussed briefly in my H-reflex post. They receive direct input from motor axons and send inhibitory feedback back to motor neurons, forming a recurrent feedback loop.
• Pou6f2-V1s — defined by the POU domain transcription factor Pou6f2. Relatively understudied, and as my work showed, not strongly connected to motor neuron cell bodies.
• Foxp2-V1s — defined by Foxp2, a transcription factor also known for its role in speech and language in the human brain. This is by far the largest clade, and the most complex. More on this below.
• Sp8-V1s — defined by the zinc-finger transcription factor Sp8. Like Pou6f2-V1s, these cells appear to play their role in premotor networks of interneurons rather than directly on motor neurons.

The existence of these four clades suggested that V1 interneurons are not a uniform inhibitory cell type but rather a structured family of functional subtypes. What was not known was whether these molecular differences translate into real differences in development or circuit organization: when the cells are born, where they end up, and which neurons they actually connect to.

That is the question my colleagues and I set out to answer.

When neurons are born determines what they become

One of the central findings of our study concerns neurogenesis — the timing of when neurons are born from their progenitor stem cells during embryonic development. We used a technique called birthdating, in which a chemical marker is introduced to mouse embryos at a precise time during their development. Cells that are dividing at that exact moment incorporate the marker into their DNA. By examining the spinal cord later and asking which V1 cells contain the marker, we can reconstruct a timeline of when each clade was generated.

What we found was that the four clades are born at different times:

• Renshaw cells are the first V1 interneurons born, during early embryogenesis.
• Pou6f2-V1s are also early-born, with a birthdate window that overlaps Renshaw cells.
• Foxp2-V1s are born later, with a peak around the midpoint of V1 neurogenesis.
• Sp8-V1s are the latest-born, arriving at the tail end of V1 production.

This sequential neurogenesis is not just a developmental curiosity. In the nervous system, when a neuron is born often predicts where it ends up and what it connects to. Neurons born earlier tend to settle in deeper (more ventral and lateral) positions; later-born neurons migrate to more dorsal positions. The physical location of a neuron in the spinal cord strongly influences which axons can reach it and which cells its own axons can target.

In other words, birth timing is part of how the spinal cord wires itself.

Connectivity to motor neurons: not all clades are equal

The next major question was whether these four clades differ in how they connect to motor neurons. The spinal cord is organized into laminae (layers), and motor neurons reside in a deep region called lamina IX. Interneurons that want to directly inhibit a motor neuron need to project their axons into that region and make synapses on the motor neuron’s cell body or dendrites.

We used genetic tools to label each V1 clade with a fluorescent protein, then used high-resolution fluorescent confocal imaging to count synapses from each clade onto different types of motor neurons.

The results were striking. Renshaw cells and Foxp2-V1 interneurons account for a large proportion of direct inhibitory input to motor neuron cell bodies. Pou6f2-V1 and Sp8-V1 interneurons, despite belonging to the same V1 family and being present in similar anatomical regions, contribute far fewer direct synapses to motor neuron somata. They appear to act primarily on premotor networks — inhibiting interneurons that then influence motor neurons, rather than acting on motor neurons directly.

This is a meaningful distinction. Synapses on the cell body and proximal dendrites are the most powerful — they are close to where action potentials are generated, so they have the most direct influence on whether a motor neuron fires. Foxp2-V1 interneurons, in particular, make a very high density of these proximal synapses on the motor neurons that control limb muscles.

The lateral motor column (LMC), which controls the muscles of the arms and legs, receives proportionally more Foxp2-V1 input than the motor columns controlling axial trunk muscles. And the total number of Foxp2-V1 interneurons scales with the size of the LMC across spinal cord segments — roughly a 2:1 to 3:1 ratio of Foxp2-V1s to motor neurons wherever limb control is concentrated. This tight numerical relationship suggests a specific functional relationship between this inhibitory clade and the neural machinery of limb movements.

Foxp2-V1s contain multitudes

The Foxp2-V1 clade deserves special attention because it is, by a wide margin, the largest V1 subgroup. Genetic lineage tracing — a technique that permanently labels all cells descended from a Foxp2-expressing progenitor, even if those cells later downregulate Foxp2 — revealed that this clade comprises more than half of all V1 interneurons in the lumbar spinal cord. Previous estimates based on counting cells that actively express Foxp2 at a given developmental time point systematically undercount this group because many cells turn off Foxp2 expression as they mature.

Within the Foxp2-V1 clade, there is further internal structure. We identified four subgroups based on the combinatorial expression of additional transcription factors (Otp, Foxp4) and on physical location in the spinal cord. These subgroups also differ in birthdate: each arises during a slightly different embryonic window and settles at a characteristic position.

One subgroup in particular — the cells that express Otp postnatally and cluster near the lateral motor column — has the profile expected of Ia inhibitory interneurons (IaINs). These are the cells responsible for reciprocal inhibition: they receive input from sensory afferents reporting stretch in one muscle and project to the motor neurons controlling the opposing muscle, suppressing contraction in the antagonist. Their existence was predicted from physiology decades ago, but which specific genetic population they belong to remained unclear.

Our anatomical evidence — showing that Otp-expressing Foxp2-V1 cells receive dense input from proprioceptive sensory fibers and are interposed in circuits linking flexor sensory input to extensor motor output — puts Foxp2-V1 interneurons at the heart of reciprocal inhibition. Direct circuit tracing using viral tools confirmed that some of these cells receive input from ankle flexor afferents and project to ankle extensor motor neurons, exactly the connectivity expected of IaINs.

That connectivity is exactly what you would expect to see reflected in the kind of experiments I described in my H-reflex post, where conditioning a spinal reflex with afferent input from the antagonist muscle reveals the inhibitory circuit connecting them. The anatomy puts Foxp2-V1 interneurons at the center of that circuit; whether they are causally responsible for producing reciprocal inhibition in a behaving animal is what I am currently testing in my dissertation.

Why this matters: ALS and motor neuron disease

Beyond the basic science of how healthy spinal circuits work, V1 interneurons have become a focus of research in amyotrophic lateral sclerosis (ALS), the fatal disease in which motor neurons progressively die.

Studies over the past decade have shown that, in mouse models of ALS, V1 synapses on motor neurons are lost before the motor neurons themselves die. By the time a motor neuron starts to degenerate, it has already been deprived of much of the inhibitory input it normally receives. This suggests that the death of motor neurons in ALS is not simply a cell-autonomous failure — the collapse of the inhibitory circuit around them may contribute to their eventual death.

Among V1 clades, Foxp2-V1 interneurons appear to be particularly vulnerable in ALS models, with higher rates of cell loss compared to Renshaw cells and other subtypes. Given that Foxp2-V1s provide the largest portion of direct inhibitory input to limb motor neurons, their early loss would leave motor neurons abnormally disinhibited — firing too readily, perhaps accelerating their own degeneration through excitotoxic mechanisms.

Understanding which specific V1 subtype is lost, when, and from which motor columns is important for designing interventions. A therapy that preserves Renshaw cells but not Foxp2-V1s, or vice versa, would have very different effects depending on which motor neurons are most affected in a given patient. The more we understand about the specific circuit elements involved, the better we can target them therapeutically.

The technical challenge: tracking cells across development

One of the most technically demanding parts of this kind of work is simply establishing who is who. Molecular markers that define a clade are often expressed transiently — present in embryos or early postnatally but downregulated by adulthood. Conversely, some cells begin expressing a given marker only after a developmental delay. This creates a moving target.

We addressed this by using intersectional genetic labeling — a strategy in which a cell receives a permanent fluorescent mark only if it meets two independent criteria at any point in its history (for example, expressing En1 at any point in its lineage and expressing Foxp2 at some point). Because each criterion triggers an irreversible recombination event, the mark is retained even if neither marker is still expressed in the adult. These tools allow you to count and map cells based on their developmental history, not just their current molecular state. The discrepancy between clade sizes estimated by postnatal protein expression versus genetic lineage tracing — the Foxp2-V1 clade is roughly twice as large by lineage as by protein counting — illustrates why this matters.

We also used custom-built software for the cell density mapping in this paper. The contour plots used to visualize where different V1 subpopulations settle in the spinal cord were generated with a MATLAB tool we adapted for this project, make_contours, which is openly available.

What remains unknown

Understanding V1 interneuron diversity is an ongoing project with many open questions.

First, the functional roles of Pou6f2-V1 and Sp8-V1 interneurons remain obscure. They clearly do not directly drive motor neuron inhibition in the way Renshaw cells and Foxp2-V1s do, but their targets in premotor circuits — and the behavioral consequences of disrupting them — are not yet known.

Second, within the Foxp2-V1 clade, the exact mapping between genetic subgroups and specific motor behaviors has not been established. The evidence that Otp-expressing Foxp2-V1s are IaINs is anatomically compelling but not yet functionally confirmed. Doing so requires new genetic tools that can selectively target those cells and either silence them or record from them during actual movement.

Third, these studies were done almost entirely in mice. The four-clade organization of V1 interneurons appears to be conserved across mammals, and V1-like interneurons are found in all vertebrates — from fish (where they help coordinate the body-wave undulations of swimming) to frogs, birds, and mammals. But whether the specific subtype diversity described here scales with limb complexity in the same way across species is an open and fascinating question.

Finally, and perhaps most importantly for disease: we do not yet know whether the Foxp2-V1 vulnerability observed in ALS models is a cause or a consequence of motor neuron degeneration. Separating those possibilities will require tools that can selectively protect or deplete specific V1 clades while leaving others intact. See work from labs like Illary Allodi’s for exciting progress in this area.

Summary

V1 interneurons are not a uniform population — they are a family of at least four genetically defined clades with different developmental origins, positions in the spinal cord, and connections to motor neurons. Renshaw cells and Foxp2-V1 interneurons are the two clades most directly coupled to motor neuron output; the other two clades act on premotor networks upstream. The Foxp2-V1 clade is by far the largest and most internally diverse, containing subpopulations that include the long-sought reciprocal Ia inhibitory interneurons governing antagonist muscle coordination during movement. These same interneurons are among the most vulnerable in animal models of ALS, pointing toward the inhibitory circuit — not just the motor neuron itself — as relevant to the disease process.

The spinal cord has been studied for well over a century, yet we are still learning the identities of its most fundamental circuit elements. Modern molecular genetics has transformed this field, moving us from descriptions of “an inhibitory interneuron somewhere in lamina VII” to a precise catalogue with developmental histories, anatomical addresses, and circuit roles. There is still a long road before that catalogue is anything like complete. But knowing where the pieces are, and that they are not interchangeable, is an important start.

This post relates to our 2024 publication: Worthy AE, Anderson JT, Lane AR, Gomez-Perez LJ, Wang AA, Griffith RW, Rivard AF, Bikoff JB, Alvarez FJ. Spinal V1 inhibitory interneuron clades differ in birthdate, projections to motoneurons, and heterogeneity. eLife 13:RP95172.