Amyotrophic lateral sclerosis-related VAPB P56S mutation differentially affects the function and survival of corticospinal and spinal motor neurons.

The substitution of Proline with Serine at residue 56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) has been linked to an atypical autosomal dominant form of familial amyotrophic lateral sclerosis 8 (ALS8). To investigate the pathogenic mechanism of P56S VAPB in ALS, we generated transgenic (Tg) mice that heterologously express human wild-type (WT) and P56S VAPB under the control of a pan-neuronal promoter Thy1.2. While WT VAPB Tg mice did not exhibit any overt motor behavioral phenotypes, P56S VAPB Tg mice developed progressive hyperactivities and other motor abnormalities. VAPB protein was accumulated as large punctate in the soma and proximal dendrites of both corticospinal motor neurons (CSMNs) and spinal motor neurons (SMNs) in P56S VAPB Tg mice. Concomitantly, a significant increase of endoplasmic reticulum stress and unfolded protein response and the resulting up-regulation of pro-apoptotic factor CCAAT/enhancer-binding protein homologous protein expression were observed in the CSMNs and SMNs of P56S VAPB Tg mice. However, only a progressive loss of CSMNs but not SMNs was found in P56S VAPB Tg mice. In SMNs, P56S VAPB promoted a rather selective translocation of VAPB protein onto the postsynaptic site of C-boutons that altered the morphology of C-boutons and impaired the spontaneous rhythmic discharges of SMNs. Therefore, these findings provide new pathophysiological mechanisms of P56S VAPB that differentially affect the function and survival of CSMNs and SMNs in ALS8.

GABAergic synaptic scaling in embryonic motoneurons is mediated by a shift in the chloride reversal potential.

Homeostatic synaptic plasticity ensures that networks maintain specific levels of activity by regulating synaptic strength in a compensatory manner. When spontaneous network activity was blocked in vivo in the embryonic spinal cord, compensatory increases in excitatory GABAergic synaptic inputs were observed. This homeostatic synaptic strengthening was observed as an increase in the amplitude of GABAergic miniature postsynaptic currents. We find that this process is mediated by an increase in chloride accumulation, which produces a depolarizing shift in the GABAergic reversal potential (E(GABA)). The findings demonstrate a previously unrecognized mechanism underlying homeostatic synaptic scaling. Similar shifts in E(GABA) have been described following various forms of neuronal injury, introducing the possibility that these shifts in E(GABA) represent a homeostatic response.

Mechanisms of excitation of spinal networks by stimulation of the ventral roots.

It has recently been demonstrated that motoneurons in neonatal rodents release an excitatory amino acid, in addition to acetylcholine, from their central terminals onto Renshaw cells. Although the function of this amino acid release is not understood, it may mediate the excitatory actions of motor axon stimulation on spinal motor networks. Stimulation of motor axons in the ventral roots or muscle nerves can activate the locomotor central pattern generator or entrain bursting in the disinhibited cord. Both of these effects persist in the presence of cholinergic antagonists and are abolished or diminished by ionotropic and metabotropic glutamate antagonists. Calcium imaging in the disinhibited cord shows that a ventral root stimulus evokes ventrolateral activity initially, which subsequently propagates to the rest of the cord. This finding suggests that excitatory interneurons excited by motoneuron recurrent collaterals are located in this region. However, motoneurons do not exhibit short latency excitatory potentials in response to ventral root stimulation indicating that the excitatory effects are mediated polysynaptically. We discuss the significance of these findings.

Excitatory actions of ventral root stimulation during network activity generated by the disinhibited neonatal mouse spinal cord.

To further understand the excitatory effects of motoneurons on spinal network function, we investigated the entrainment of disinhibited rhythms by ventral root (VR) stimulation in the neonatal mouse spinal cord. A brief train of stimuli applied to a VR triggered bursting reliably in 31/32 experiments. The same roots that entrained disinhibited bursting could also produce locomotor-like activity with a similar probability when the network was not disinhibited. The ability of VR stimulation to entrain the rhythm persisted in nicotinic and muscarinic cholinergic antagonists but was blocked by the AMPAR antagonist NBQX. Bath application of the type I mGluR1 receptor antagonist CPCCOEt reduced the ability of both dorsal root and VR stimulation to entrain the disinhibited rhythm and abolished the ability of either type of stimulation to evoke locomotor-like activity. Calcium imaging through the lateral aspect of the cord revealed that VR stimulation and spontaneously occurring bursts were accompanied by a wave of activity that originated ventrally and propagated dorsally. Imaging the cut transverse face of L(5) revealed that the earliest VR-evoked optical activity began ventrolaterally. The optical activity accompanying spontaneous bursts could originate ventrolaterally, ventromedially, or throughout the mediolateral extent of the ventral horn or very occasionally dorsally. Collectively, our data indicate that VR stimulation can entrain disinhibited spinal network activity and trigger locomotor-like activity through a mechanism dependent on activation of both ionotropic and metabotropic glutamate receptors. The effects of entrainment appear to be mediated by a ventrolaterally located network that is also active during spontaneously occurring bursts.

NKCC1 and AE3 appear to accumulate chloride in embryonic motoneurons.

During early development, gamma-aminobutyric acid (GABA) depolarizes and excites neurons, contrary to its typical function in the mature nervous system. As a result, developing networks are hyperexcitable and experience a spontaneous network activity that is important for several aspects of development. GABA is depolarizing because chloride is accumulated beyond its passive distribution in these developing cells. Identifying all of the transporters that accumulate chloride in immature neurons has been elusive and it is unknown whether chloride levels are different at synaptic and extrasynaptic locations. We have therefore assessed intracellular chloride levels specifically at synaptic locations in embryonic motoneurons by measuring the GABAergic reversal potential (EGABA) for GABAA miniature postsynaptic currents. When whole cell patch solutions contained 17-52 mM chloride, we found that synaptic EGABA was around -30 mV. Because of the low HCO3- permeability of the GABAA receptor, this value of EGABA corresponds to approximately 50 mM intracellular chloride. It is likely that synaptic chloride is maintained at levels higher than the patch solution by chloride accumulators. We show that the Na+-K+-2Cl- cotransporter, NKCC1, is clearly involved in the accumulation of chloride in motoneurons because blocking this transporter hyperpolarized EGABA and reduced nerve potentials evoked by local application of a GABAA agonist. However, chloride accumulation following NKCC1 block was still clearly present. We find physiological evidence of chloride accumulation that is dependent on HCO3- and sensitive to an anion exchanger blocker. These results suggest that the anion exchanger, AE3, is also likely to contribute to chloride accumulation in embryonic motoneurons.

Imaging the spatiotemporal organization of neural activity in the developing spinal cord.

In this review, we discuss the use of imaging to visualize the spatiotemporal organization of network activity in the developing spinal cord of the chick embryo and the neonatal mouse. We describe several different methods for loading ion- and voltage-sensitive dyes into spinal neurons and consider the advantages and limitations of each one. We review work in the chick embryo, suggesting that motoneurons play a critical role in the initiation of each cycle of spontaneous network activity and describe how imaging has been used to identify a class of spinal interneuron that appears to be the avian homolog of mammalian Renshaw cells or 1a-inhibitory interneurons. Imaging of locomotor-like activity in the neonatal mouse revealed a wave-like activation of motoneurons during each cycle of discharge. We discuss the significance of this finding and its implications for understanding how locomotor-like activity is coordinated across different segments of the cord. In the last part of the review, we discuss some of the exciting new prospects for the future.

Chloride-sensitive MEQ fluorescence in chick embryo motoneurons following manipulations of chloride and during spontaneous network activity.

Intracellular Cl(-) ([Cl(-)](in)) homeostasis is thought to be an important regulator of spontaneous activity in the spinal cord of the chick embryo. We investigated this idea by visualizing the variations of [Cl(-)](in) in motoneurons retrogradely labeled with the Cl-sensitive dye 6-methoxy-N-ethylquinolinium iodide (MEQ) applied to cut muscle nerves in the isolated E10-E12 spinal cord. This labeling procedure obviated the need for synthesizing the reduced, cell-permeable dihydro-MEQ (DiH-MEQ). The specificity of motoneuron labeling was confirmed using retrograde co-labeling with Texas Red Dextran and immunocytochemistry for choline acetyltransferase (ChAT). In MEQ-labeled motoneurons, the GABA(A) receptor agonist isoguvacine (100 muM) increased somatic and dendritic fluorescence by 7.4 and 16.7%, respectively. The time course of this fluorescence change mirrored that of the depolarization recorded from the axons of the labeled motoneurons. Blockade of the inward Na(+)/K(-)/2Cl(-) co-transporter (NKCC1) with bumetanide (20 microM) or with a low-Na(+) bath solution (12 mM), increased MEQ fluorescence by 5.3 and 11.4%, respectively, consistent with a decrease of [Cl(-)](in). After spontaneous episodes of activity, MEQ fluorescence increased and then declined to the pre-episode level during the interepisode interval. The largest fluorescence changes occurred over motoneuron dendrites (19.7%) with significantly smaller changes (5.2%) over somata. Collectively, these results show that retrogradely loaded MEQ can be used to detect [Cl(-)](in) in motoneurons, that the bumetanide-sensitive NKCC1 co-transporter is at least partially responsible for the elevated [Cl(-)](in) of developing motoneurons, and that dendritic [Cl(-)](in) decreases during spontaneous episodes and recovers during the inter-episode interval, presumably due to the action of NKCC1.

Modeling spontaneous activity in the developing spinal cord using activity-dependent variations of intracellular chloride.

We investigated how spontaneous activity is generated in developing, hyperexcitable networks. We focused our study on the embryonic chick spinal cord, a preparation that exhibits rhythmic discharge on multiple timescales: slow episodes (lasting minutes) and faster intraepisode cycling (approximately 1 Hz frequency). For this purpose, we developed a mean field model of a recurrent network with slow chloride dynamics and a fast depression variable. We showed that the model, in addition to providing a biophysical mechanism for the slow dynamics, was able to account for the experimentally observed activity. The model made predictions on how interval and duration of episodes are affected when changing chloride-mediated synaptic transmission or chloride flux across cell membrane. These predictions guided experiments, and the model results were compared with experimental data obtained with electrophysiological recordings. We found agreement when transmission was affected through changes in synaptic conductance and good qualitative agreement when chloride flux was varied through changes in external chloride concentration or in the rate of the Na+-K+-2Cl- cotransporter. Furthermore, the model made predictions about the time course of intracellular chloride concentration and chloride reversal potential and how these are affected by changes in synaptic conductance. Based on the comparison between modeling and experimental results, we propose that chloride dynamics could be an important mechanism in rhythm generation in the developing chick spinal cord.