Serotonergic dysfunction impairs locomotor coordination in spinal muscular atrophy.

Neuromodulation by serotonin regulates the activity of neuronal networks responsible for a wide variety of essential behaviours. Serotonin (or 5-HT) typically activates metabotropic G protein-coupled receptors, which in turn initiate second messenger signalling cascades and induce short and long-lasting behavioural effects. Serotonin is intricately involved in the production of locomotor activity and gait control for different motor behaviours. Although dysfunction of serotonergic neurotransmission has been associated with mood disorders and spasticity after spinal cord injury, whether and to what extent such dysregulation is implicated in movement disorders has not been firmly established. Here, we investigated whether serotonergic neuromodulation is affected in spinal muscular atrophy (SMA), a neurodegenerative disease caused by ubiquitous deficiency of the SMN protein. The hallmarks of SMA are death of spinal motor neurons, muscle atrophy and impaired motor control, both in human patients and mouse models of disease. We used a severe mouse model of SMA, that closely recapitulates the severe symptoms exhibited by type I SMA patients, the most common and most severe form of the disease. Together, with mouse genetics, optogenetics, physiology, morphology and behavioural analysis, we report severe dysfunction of serotonergic neurotransmission in the spinal cord of SMA mice, both at early and late stages of the disease. This dysfunction is followed by reduction of 5-HT synapses on vulnerable motor neurons. We demonstrate that motor neurons innervating axial and trunk musculature are preferentially affected, suggesting a possible cause for the proximo-distal progression of disease, and raising the possibility that it may underlie scoliosis in SMA patients. We also demonstrate that the 5-HT dysfunction is caused by SMN deficiency in serotonergic neurons in the raphe nuclei of the brainstem. The behavioural significance of the dysfunction in serotonergic neuromodulation is underlined by inter-limb discoordination in SMA mice, which is ameliorated when selective restoration of SMN in 5-HT neurons is achieved by genetic means. Our study uncovers an unexpected dysfunction of serotonergic neuromodulation in SMA and indicates that, if normal function is to be restored under disease conditions, 5-HT neuromodulation should be a key target for therapeutic approaches.

Bioluminescence calcium imaging of network dynamics and their cholinergic modulation in slices of cerebral cortex from male rats.

The activity of neuronal ensembles was monitored in neocortical slices from male rats using wide-field bioluminescence imaging of a calcium sensor formed with the fusion of green fluorescent protein and aequorin (GA) and expressed through viral transfer. GA expression was restricted to pyramidal neurons and did not conspicuously alter neuronal morphology or neocortical cytoarchitecture. Removal of extracellular magnesium or addition of GABA receptor antagonists triggered epileptiform flashes of variable amplitude and spatial extent, indicating that the excitatory and inhibitory networks were functionally preserved in GA-expressing slices. We found that agonists of muscarinic acetylcholine receptors largely increased the peak bioluminescence response to local electrical stimulation in layer I or white matter, and gave rise to a slowly decaying response persisting for tens of seconds. The peak increase involved layers II/III and V and did not result in marked alteration of response spatial properties. The persistent response involved essentially layer V and followed the time course of the muscarinic afterdischarge depolarizing plateau in layer V pyramidal cells. This plateau potential triggered spike firing in layer V, but not layer II/III pyramidal cells, and was accompanied by recurrent synaptic excitation in layer V. Our results indicate that wide-field imaging of GA bioluminescence is well suited to monitor local and global network activity patterns, involving different mechanisms of intracellular calcium increase, and occurring on various timescales.

Reduced sensory synaptic excitation impairs motor neuron function via Kv2.1 in spinal muscular atrophy.

Behavioral deficits in neurodegenerative diseases are often attributed to the selective dysfunction of vulnerable neurons via cell-autonomous mechanisms. Although vulnerable neurons are embedded in neuronal circuits, the contributions of their synaptic partners to disease process are largely unknown. Here we show that, in a mouse model of spinal muscular atrophy (SMA), a reduction in proprioceptive synaptic drive leads to motor neuron dysfunction and motor behavior impairments. In SMA mice or after the blockade of proprioceptive synaptic transmission, we observed a decrease in the motor neuron firing that could be explained by the reduction in the expression of the potassium channel Kv2.1 at the surface of motor neurons. Chronically increasing neuronal activity pharmacologically in vivo led to a normalization of Kv2.1 expression and an improvement in motor function. Our results demonstrate a key role of excitatory synaptic drive in shaping the function of motor neurons during development and the contribution of its disruption to a neurodegenerative disease.

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.

Early functional impairment of sensory-motor connectivity in a mouse model of spinal muscular atrophy.

To define alterations of neuronal connectivity that occur during motor neuron degeneration, we characterized the function and structure of spinal circuitry in spinal muscular atrophy (SMA) model mice. SMA motor neurons show reduced proprioceptive reflexes that correlate with decreased number and function of synapses on motor neuron somata and proximal dendrites. These abnormalities occur at an early stage of disease in motor neurons innervating proximal hindlimb muscles and medial motor neurons innervating axial muscles, but only at end-stage disease in motor neurons innervating distal hindlimb muscles. Motor neuron loss follows afferent synapse loss with the same temporal and topographical pattern. Trichostatin A, which improves motor behavior and survival of SMA mice, partially restores spinal reflexes, illustrating the reversibility of these synaptic defects. Deafferentation of motor neurons is an early event in SMA and may be a primary cause of motor dysfunction that is amenable to therapeutic intervention.

Calcium imaging in single neurons from brain slices using bioluminescent reporters.

Responses of three bioluminescent Ca(2+) sensors were studied in vitro and in neurons from brain slices. These sensors consisted of tandem fusions of green fluorescent protein (GFP) with the photoproteins aequorin, obelin, or a mutant aequorin with high Ca(2+) sensitivity. Kinetics of GFP-obelin responses to a saturating Ca(2+) concentration were faster than those of GFP-aequorin at all Mg(2+) concentrations tested, whereas GFP-mutant aequorin responses were the slowest. GFP-photoproteins were efficiently expressed in pyramidal neurons following overnight incubation of acute neocortical slices with recombinant Sindbis viruses. Expression of GFP-photoproteins did not result in conspicuous modification of morphological or electrophysiological properties of layer V pyramidal cells. The three sensors allowed the detection of Ca(2+) transients associated with action potential discharge in single layer V pyramidal neurons. In these neurons, depolarizing steps of increasing amplitude elicited action potential discharge of increasing frequency. Bioluminescent responses of the three sensors were similar in several respects: detection thresholds, an exponential increase with stimulus intensity, photoprotein consumptions, and kinetic properties. These responses, which were markedly slower than kinetics measured in vitro, increased linearly during the action potential discharge and decayed exponentially at the end of the discharge. Onset slopes increased with stimulus intensity, whereas decay kinetics remained constant. Dendritic light emission contributed to whole-field responses, but the spatial resolution of bioluminescence imaging was limited to the soma and proximal apical dendrite. Nonetheless, the high signal-to-background ratio of GFP-photoproteins allowed the detection of Ca(2+) transients associated with 5 action potentials in single neurons upon whole-field bioluminescence recordings.

A simple method for short-term controlled anesthesia in newborn mice.

In this study, we describe a simple and inexpensive method for inducing short-term anesthesia and rapid recovery in newborn mice. Litters of Swiss mice pups were randomly allocated to testing on postnatal days 2, 5, and 8. Anesthesia was induced by placing the pup in a syringe and adding a volume of isoflurane-saturated gas that produced an estimated level of 32% isoflurane. Exposure to isoflurane lasted 30 s. All the pups survived the anesthesia. At all study ages, this method abolished the nociceptive response to tail clamp without inducing mortality, thus showing effective anesthesia. Recovery from anesthesia was assessed immediately after isoflurane exposure, based on two nonnoxious behavioral tests: the defensive response to a drop of water (10 tests, 1 min apart) and 10 min later the righting reflex, i.e., the time to recovery of the prone position (five tests, 10 min apart). The water drop test scores increased during the recovery phase toward the control values in all age groups. Treatment and time had no significant effect on righting reflex scores. The initial volume in the syringe, the volume of added isoflurane-saturated gas, and the duration of exposure may be adjusted according to postnatal age and specific strains or species (e.g., rats). This method is well suited to behavioral or physiological phenotype studies in developing mice, in which noxious procedures must precede functional testing, making rapid recovery from anesthesia a key requirement.