Effect of size on expression of bistability in mouse spinal motoneurons.

Bistability in spinal motoneurons supports tonic spike activity in the absence of excitatory drive. Earlier work in adult preparations suggested that smaller motoneurons innervating slow antigravity muscle fibers are more likely to generate bistability for postural maintenance. However, whether large motoneurons innervating fast-fatigable muscle fibers display bistability is still controversial. To address this, we examined the relationship between soma size and bistability in lumbar (L4-L5) ventrolateral α-motoneurons of choline acetyltransferase (ChAT)-green fluorescent protein (GFP) and Hb9-GFP mice during the first 4 wk of life. We found that as neuron size increases, the prevalence of bistability rises. Smaller α-motoneurons lack bistability, whereas larger fast α-motoneurons [matrix metalloproteinase-9 (MMP-9)+/Hb9+] with a soma area ≥ 400 µm2 exhibit significantly higher bistability. Ionic currents associated with bistability, including the persistent Nav1.6 current, the thermosensitive Trpm5 Ca2+-activated Na+ current, and the slowly inactivating Kv1.2 current, also scale with cell size. Serotonin evokes full bistability in large motoneurons with partial bistable properties but not in small motoneurons. Our study provides important insights into the neural mechanisms underlying bistability and how motoneuron size correlates with bistability in mice.NEW & NOTEWORTHY Bistability is not a common feature of all mouse spinal motoneurons. It is absent in small, slow motoneurons but present in most large, fast motoneurons. This difference results from differential expression of ionic currents that enable bistability, which are highly expressed in large motoneurons but small or absent in small motoneurons. These results support a possible role for fast motoneurons in maintenance of tonic posture in addition to their known roles in fast movements.

A size principle for bistability in mouse spinal motoneurons.

Bistability in spinal motoneurons supports tonic spike activity in the absence of excitatory drive. Earlier work in adult preparations suggested that smaller motoneurons innervating slow antigravity muscle fibers are more likely to generate bistability for postural maintenance. However, whether large motoneurons innervating fast-fatigable muscle fibers display bistability related to postural tone is still controversial. To address this, we examined the relationship between soma size and bistability in lumbar ventrolateral α-motoneurons of ChAT-GFP and Hb9-GFP mice across different developmental stages: neonatal (P2-P7), young (P7-P14) and mature (P21-P25). We found that as neuron size increases, the prevalence of bistability rises. Smaller α-motoneurons lack bistability, while larger fast α-motoneurons (MMP-9 + /Hb9 + ) with a soma area ≥ 400µm 2 exhibit significantly higher bistability. Ionic currents associated with bistability, including the persistent Nav1.6 current, thermosensitive Trpm5 Ca 2+ -activated Na + current and the slowly inactivating Kv1.2 current, also scale with cell size. Serotonin evokes full bistability in large motoneurons with partial bistable properties, but not in small motoneurons. Our study provides important insights into the neural mechanisms underlying bistability and how motoneuron size dictates this process. New and Noteworthy: Bistability is not a common feature of all mouse spinal motoneurons. It is absent in small, slow motoneurons but present in most large, fast motoneurons. This difference results from differential expression of ionic currents that enable bistability, which are highly expressed in large motoneurons but small or absent in small motoneurons. These results support a possible role for fast motoneurons in maintenance of tonic posture in addition to their known roles in fast movements.

Persistent Nav1.1 and Nav1.6 currents drive spinal locomotor functions through nonlinear dynamics.

Persistent sodium current (INaP) in the spinal locomotor network promotes two distinct nonlinear firing patterns: a self-sustained spiking triggered by a brief excitation in bistable motoneurons and bursting oscillations in interneurons of the central pattern generator (CPG). Here, we identify the NaV channels responsible for INaP and their role in motor behaviors. We report the axonal Nav1.6 as the main molecular player for INaP in lumbar motoneurons. The inhibition of Nav1.6, but not of Nav1.1, in motoneurons impairs INaP, bistability, postural tone, and locomotor performance. In interneurons of the rhythmogenic CPG region, both Nav1.6 and Nav1.1 equally mediate INaP. Inhibition of both channels is required to abolish oscillatory bursting activities and the locomotor rhythm. Overall, Nav1.6 plays a significant role both in posture and locomotion by governing INaP-dependent bistability in motoneurons and working in tandem with Nav1.1 to provide INaP-dependent rhythmogenic properties of the CPG.

Astrocytic Kir4.1 channels regulate locomotion by orchestrating neuronal rhythmicity in the spinal network.

Neuronal rhythmogenesis in the spinal cord is correlated with variations in extracellular K+ levels ([K+ ]e ). Astrocytes play important role in [K+ ]e homeostasis and compute neuronal information. Yet it is unclear how neuronal oscillations are regulated by astrocytic K+ homeostasis. Here we identify the astrocytic inward-rectifying K+ channel Kir4.1 (a.k.a. Kcnj10) as a key molecular player for neuronal rhythmicity in the spinal central pattern generator (CPG). By combining two-photon calcium imaging with electrophysiology, immunohistochemistry and genetic tools, we report that astrocytes display Ca2+ transients before and during oscillations of neighboring neurons. Inhibition of astrocytic Ca2+ transients with BAPTA decreases the barium-sensitive Kir4.1 current responsible of K+ clearance. Finally, we show in mice that Kir4.1 knockdown in astrocytes progressively prevents neuronal oscillations and alters the locomotor pattern resulting in lower motor performances in challenging tasks. These data identify astroglial Kir4.1 channels as key regulators of neuronal rhythmogenesis in the CPG driving locomotion.

Altered action potential waveform and shorter axonal initial segment in hiPSC-derived motor neurons with mutations in VRK1.

We recently described new pathogenic variants in VRK1, in patients affected with distal Hereditary Motor Neuropathy associated with upper motor neurons signs. Specifically, we provided evidences that hiPSC-derived Motor Neurons (hiPSC-MN) from these patients display Cajal Bodies (CBs) disassembly and defects in neurite outgrowth and branching. We here focused on the Axonal Initial Segment (AIS) and the related firing properties of hiPSC-MNs from these patients. We found that the patient's Action Potential (AP) was smaller in amplitude, larger in duration, and displayed a more depolarized threshold while the firing patterns were not altered. These alterations were accompanied by a decrease in the AIS length measured in patients' hiPSC-MNs. These data indicate that mutations in VRK1 impact the AP waveform and the AIS organization in MNs and may ultimately lead to the related motor neuron disease.

Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice.

Bistable motoneurons of the spinal cord exhibit warmth-activated plateau potential driven by Na+ and triggered by a brief excitation. The thermoregulating molecular mechanisms of bistability and their role in motor functions remain unknown. Here, we identify thermosensitive Na+-permeable Trpm5 channels as the main molecular players for bistability in mouse motoneurons. Pharmacological, genetic or computational inhibition of Trpm5 occlude bistable-related properties (slow afterdepolarization, windup, plateau potentials) and reduce spinal locomotor outputs while central pattern generators for locomotion operate normally. At cellular level, Trpm5 is activated by a ryanodine-mediated Ca2+ release and turned off by Ca2+ reuptake through the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump. Mice in which Trpm5 is genetically silenced in most lumbar motoneurons develop hindlimb paresis and show difficulties in executing high-demanding locomotor tasks. Overall, by encoding bistability in motoneurons, Trpm5 appears indispensable for producing a postural tone in hindlimbs and amplifying the locomotor output.

Kv1.2 Channels Promote Nonlinear Spiking Motoneurons for Powering Up Locomotion.

Spinal motoneurons are endowed with nonlinear spiking behaviors manifested by a spike acceleration whose functional significance remains uncertain. Here, we show in rodent lumbar motoneurons that these nonlinear spiking properties do not rely only on activation of dendritic nifedipine-sensitive L-type Ca2+ channels, as assumed for decades, but also on the slow inactivation of a nifedipine-sensitive K+ current mediated by Kv1.2 channels that are highly expressed in axon initial segments. Specifically, the pharmacological and computational inhibition of Kv1.2 channels occluded the spike acceleration of rhythmically active motoneurons and the correlated slow buildup of rhythmic motor output recorded at the onset of locomotor-like activity. This study demonstrates that slow inactivation of Kv1.2 channels provides a potent gain control mechanism in mammalian spinal motoneurons and has a behavioral role in enhancing locomotor drive during the transition from immobility to steady-state locomotion.

Role for Visual Experience in the Development of Direction-Selective Circuits.

Visually guided behavior can depend critically on detecting the direction of object movement. This computation is first performed in the retina where direction is encoded by direction-selective ganglion cells (DSGCs) that respond strongly to an object moving in the preferred direction and weakly to an object moving in the opposite, or null, direction (reviewed in [1]). DSGCs come in multiple types that are classified based on their morphologies, response properties, and targets in the brain. This study focuses on two types-ON and ON-OFF DSGCs. Though animals can sense motion in all directions, the preferred directions of DSGCs in adult retina cluster along distinct directions that we refer to as the cardinal axes. ON DSGCs have three cardinal axes-temporal, ventral, and dorsonasal-while ON-OFF DSGCs have four-nasal, temporal, dorsal, and ventral. How these preferred directions emerge during development is still not understood. Several studies have demonstrated that ON [2] and ON-OFF DSGCs are well tuned at eye-opening, and even a few days prior to eye-opening, in rabbits [3], rats [4], and mice [5-8], suggesting that visual experience is not required to produce direction-selective tuning. However, here we show that at eye-opening the preferred directions of both ON and ON-OFF DSGCs are diffusely distributed and that visual deprivation prevents the preferred directions from clustering along the cardinal axes. Our findings indicate a critical role for visual experience in shaping responses in the retina.

Neuron-glia signaling in developing retina mediated by neurotransmitter spillover.

Neuron-glia interactions play a critical role in the maturation of neural circuits; however, little is known about the pathways that mediate their communication in the developing CNS. We investigated neuron-glia signaling in the developing retina, where we demonstrate that retinal waves reliably induce calcium transients in Müller glial cells (MCs). During cholinergic waves, MC calcium transients were blocked by muscarinic acetylcholine receptor antagonists, whereas during glutamatergic waves, MC calcium transients were inhibited by ionotropic glutamate receptor antagonists, indicating that the responsiveness of MCs changes to match the neurotransmitter used to support retinal waves. Using an optical glutamate sensor we show that the decline in MC calcium transients is caused by a reduction in the amount of glutamate reaching MCs. Together, these studies indicate that neurons and MCs exhibit correlated activity during a critical period of retinal maturation that is enabled by neurotransmitter spillover from retinal synapses.

Visual stimulation switches the polarity of excitatory input to starburst amacrine cells.

Direction-selective ganglion cells (DSGCs) are tuned to motion in one direction. Starburst amacrine cells (SACs) are thought to mediate this direction selectivity through precise anatomical wiring to DSGCs. Nevertheless, we previously found that visual adaptation can reverse DSGCs's directional tuning, overcoming the circuit anatomy. Here we explore the role of SACs in the generation and adaptation of direction selectivity. First, using pharmacogenetics and two-photon calcium imaging, we validate that SACs are necessary for direction selectivity. Next, we demonstrate that exposure to an adaptive stimulus dramatically alters SACs' synaptic inputs. Specifically, after visual adaptation, On-SACs lose their excitatory input during light onset but gain an excitatory input during light offset. Our data suggest that visual stimulation alters the interactions between rod- and cone-mediated inputs that converge on the terminals of On-cone BCs. These results demonstrate how the sensory environment can modify computations performed by anatomically defined neuronal circuits.

Activation of 5-HT2A receptors upregulates the function of the neuronal K-Cl cotransporter KCC2.

In healthy adults, activation of γ-aminobutyric acid (GABA)(A) and glycine receptors inhibits neurons as a result of low intracellular chloride concentration ([Cl(-)](i)), which is maintained by the potassium-chloride cotransporter KCC2. A reduction of KCC2 expression or function is implicated in the pathogenesis of several neurological disorders, including spasticity and chronic pain following spinal cord injury (SCI). Given the critical role of KCC2 in regulating the strength and robustness of inhibition, identifying tools that may increase KCC2 function and, hence, restore endogenous inhibition in pathological conditions is of particular importance. We show that activation of 5-hydroxytryptamine (5-HT) type 2A receptors to serotonin hyperpolarizes the reversal potential of inhibitory postsynaptic potentials (IPSPs), E(IPSP), in spinal motoneurons, increases the cell membrane expression of KCC2 and both restores endogenous inhibition and reduces spasticity after SCI in rats. Up-regulation of KCC2 function by targeting 5-HT(2A) receptors, therefore, has therapeutic potential in the treatment of neurological disorders involving altered chloride homeostasis. However, these receptors have been implicated in several psychiatric disorders, and their effects on pain processing are controversial, highlighting the need to further investigate the potential systemic effects of specific 5-HT(2A)R agonists, such as (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2).

Glucose is an adequate energy substrate for the depolarizing action of GABA and glycine in the neonatal rat spinal cord in vitro.

In vitro studies have repeatedly demonstrated that the neurotransmitters γ-aminobutyric acid (GABA) and glycine depolarize immature neurons in many areas of the CNS, including the spinal cord. This widely accepted phenomenon was recently challenged by experiments showing that the depolarizing action of GABA on neonatal hippocampus and neocortex in vitro was prevented by adding energy substrates (ES), such as the ketone body metabolite dl-ß-hydroxybutyric acid (DL-BHB), lactate, or pyruvate to the artificial cerebrospinal fluid (ACSF). It was suggested that GABA-induced depolarizations in vitro might be an artifact due to inadequate energy supply when glucose is the sole energy source, consistent with the energy metabolism of neonatal rat brain being largely dependent on ESs other than glucose. Here we examined the effects of these ESs (DL-BHB, lactate, pyruvate) on inhibitory postsynaptic potentials (IPSPs) recorded from neonatal rat lumbar spinal cord motoneurons (MNs), in vitro. We report that supplementing the ACSF with physiologic concentrations of DL-BHB, lactate, or pyruvate does not alter the reversal potential of IPSPs (E(IPSP)). Only high concentrations of pyruvate hyperpolarized E(IPSP). In addition, the depolarizing action of GABA on primary afferent terminals was not affected by supplementing the ACSF with ES at physiologic concentrations. We conclude that depolarizing IPSPs in immature MNs and the primary afferent depolarizations are not caused by inadequate energy supply. Glucose at its standard concentration appears to be an adequate ES for the neonatal spinal cord in vitro.

Primary afferent terminals acting as excitatory interneurons contribute to spontaneous motor activities in the immature spinal cord.

Patterned, spontaneous activity plays a critical role in the development of neuronal networks. A robust spontaneous activity is observed in vitro in spinal cord preparations isolated from immature rats. The rhythmic ventral root discharges rely mainly on the depolarizing/excitatory action of GABA and glycine early during development, whereas at later stages glutamate drive is primarily responsible for the rhythmic activity and GABA/glycine are thought to play an inhibitory role. However, rhythmic discharges mediated by the activation of GABA(A) receptors are recorded from dorsal roots (DRs). In the present study, we used the in vitro spinal cord preparation of neonatal rats to identify the relationship between discharges that are conducted antidromically along DRs and the spontaneous activity recorded from lumbar motoneurons. We show that discharges in DRs precede those in ventral roots and that primary afferent depolarizations (PADs) start earlier than EPSPs in motoneurons. EPSP-triggered averaging revealed that the action potentials propagate not only antidromically in the DR but also centrally and trigger EPSPs in motoneurons. Potentiating GABAergic antidromic discharges by diazepam increased the EPSPs recorded from motoneurons; conversely, blocking DR bursts markedly reduced these EPSPs. High intracellular concentrations of chloride are maintained in primary afferent terminals by the sodium-potassium-chloride cotransporter NKCC1. Blocking these cotransporters by bumetanide decreased both dorsal and ventral root discharges. We conclude that primary afferent fibers act as excitatory interneurons and that GABA, through PADs reaching firing threshold, is still playing a key role in promoting spontaneous activity in neonates.

Chapter 1--importance of chloride homeostasis in the operation of rhythmic motor networks.

GABA and glycine are classically called "inhibitory" amino acids, despite the fact that their action can rapidly switch from inhibition to excitation and vice versa. The postsynaptic action depends on the intracellular concentration of chloride ions ([Cl(-)](i)), which is regulated by proteins in the plasma membrane: the K(+)-Cl(-) cotransporter KCC2 and the Na(+)-K(+)-Cl(-) cotransporter NKCC1, which extrude and intrude Cl(-) ions, respectively. A high [Cl(-)](i) leads to a depolarizing (excitatory) action of GABA and glycine, as observed in mature dorsal root ganglion neurons and in motoneurons both early during development and in several pathological conditions, such as following spinal cord injury. Here, we review some recent data regarding chloride homeostasis in the spinal cord and its contribution to network operation involved in locomotion.

Down-regulation of the potassium-chloride cotransporter KCC2 contributes to spasticity after spinal cord injury.

Hyperexcitability of spinal reflexes and reduced synaptic inhibition are commonly associated with spasticity after spinal cord injury (SCI). In adults, the activation of gamma-aminobutyric acid(A) (GABAA) and glycine receptors inhibits neurons as a result of low intracellular chloride (Cl-) concentration, which is maintained by the potassium-chloride cotransporter KCC2 (encoded by Slc12a5). We show that KCC2 is downregulated after SCI in rats, particularly in motoneuron membranes, thereby depolarizing the Cl- equilibrium potential and reducing the strength of postsynaptic inhibition. Blocking KCC2 in intact rats reduces the rate-dependent depression (RDD) of the Hoffmann reflex, as is observed in spasticity. RDD is also decreased in KCC2-deficient mice and in intact rats after intrathecal brain-derived neurotrophic factor (BDNF) injection, which downregulates KCC2. The early decrease in KCC2 after SCI is prevented by sequestering BDNF at the time of SCI. Conversely, after SCI, BDNF upregulates KCC2 and restores RDD. Our results open new perspectives for the development of therapeutic strategies to alleviate spasticity.