Spinal motoneurones are intrinsically more responsive in the adult G93A SOD1 mouse model of amyotrophic lateral sclerosis.

KEY POINTS: Although in vitro recordings using neonatal preparations from mouse models of amyotrophic lateral sclerosis (ALS) suggest increased motoneurone excitability, in vivo recordings in adult ALS mouse models have been conflicting. In adult G93A SOD1 models, spinal motoneurones have previously been shown to have deficits in repetitive firing, in contrast to the G127X SOD1 mouse model. Our in vivo intracellular recordings in barbiturate-anaesthetized adult male G93A SOD1 mice reveal that the incidence of failure to fire with current injection was equally low in control and ALS mice (∼2%). We show that failure to fire repetitively can be a consequence of experimental protocol and should not be used alone to classify otherwise normal motoneurones as hypo-excitable. Motoneurones in the G93A SOD1 mice showed an increased response to inputs, with lower rheobase, higher input-output gains and increased activation of persistent inward currents. ABSTRACT: In vitro studies from transgenic amyotrophic lateral sclerosis models have suggested an increased excitability of spinal motoneurones. However, in vivo intracellular recordings from adult amyotrophic lateral sclerosis mice models have produced conflicting findings. Previous investigations using barbiturate anaesthetized G93A SOD1 mice have suggested that some motoneurones are hypo-excitable, defined by deficits in repetitive firing. Our own previous recordings in G127X SOD1 mice using different anaesthesia, however, showed no repetitive firing deficits and increased persistent inward currents at symptom onset. These discrepancies may be a result of differences between models, symptomatic stage, anaesthesia or technical differences. To investigate this, we repeated our original experiments, but in adult male G93A SOD1 mice, at both presymptomatic and symptomatic stages, under barbiturate anaesthesia. In vivo intracellular recordings from antidromically identified spinal motoneurones revealed that the incidence of failure to fire with current injection was equally low in control and G93A SOD1 mice (∼2%). Motoneurones in G93A SOD1 mice fired significantly more spontaneous action potentials. Rheobase was significantly lower and the input resistance and input-output gain were significantly higher in both presymptomatic and symptomatic G93A SOD1 mice. This was despite a significant increase in the duration of the post-spike after-hyperpolarization in both presymptomatic and symptomatic G93A SOD1 mice. Finally, evidence of increased activation of persistent inward currents was seen in both presymptomatic and symptomatic G93A SOD1 mice. Our results do not confirm previous reports of hypo-excitability of spinal motoneurones in the G93A SOD1 mouse and demonstrate that the motoneurones show an increased response to inputs.

Rhythmic activity of feline dorsal and ventral spinocerebellar tract neurons during fictive motor actions.

Neurons of the dorsal spinocerebellar tracts (DSCT) have been described to be rhythmically active during walking on a treadmill in decerebrate cats, but this activity ceased following deafferentation of the hindlimb. This observation supported the hypothesis that DSCT neurons primarily relay the activity of hindlimb afferents during locomotion, but lack input from the spinal central pattern generator. The ventral spinocerebellar tract (VSCT) neurons, on the other hand, were found to be active during actual locomotion (on a treadmill) even after deafferentation, as well as during fictive locomotion (without phasic afferent feedback). In this study, we compared the activity of DSCT and VSCT neurons during fictive rhythmic motor behaviors. We used decerebrate cat preparations in which fictive motor tasks can be evoked while the animal is paralyzed and there is no rhythmic sensory input from hindlimb nerves. Spinocerebellar tract cells with cell bodies located in the lumbar segments were identified by electrophysiological techniques and examined by extra- and intracellular microelectrode recordings. During fictive locomotion, 57/81 DSCT and 30/30 VSCT neurons showed phasic, cycle-related activity. During fictive scratch, 19/29 DSCT neurons showed activity related to the scratch cycle. We provide evidence for the first time that locomotor and scratch drive potentials are present not only in VSCT, but also in the majority of DSCT neurons. These results demonstrate that both spinocerebellar tracts receive input from the central pattern generator circuitry, often sufficient to elicit firing in the absence of sensory input.

Dendrite-derived supernumerary axons on adult axotomized motor neurons possess proteins that are essential for the initiation and propagation of action potentials and synaptic vesicle release.

Axotomy can trigger profound alterations in the neuronal polarity of adult neurons in vivo. This can manifest itself in the development of new axon-like processes emanating from the tips of distal dendrites. Previously, these processes have been defined as axonal based on their axonal morphology. This study extends this definition to determine whether, more importantly, these processes possess the prerequisite molecular machinery to function as axons. Using a combination of intracellular labeling and immunohistochemistry, we demonstrate that the distribution of voltage-gated sodium channels on these processes matches the arrangement of these channels that is necessary for the initiation and conduction of action potentials. At terminal bouton-like structures they possess key proteins necessary for the release of synaptic vesicles (SV2 and synaptophysin). Thus, axon-like processes emanating from the tips of distal dendrites represent a rearrangement of neuronal polarity whereby axotomized neurons can develop additional functional axons in vivo.

Reciprocal Ia inhibition contributes to motoneuronal hyperpolarisation during the inactive phase of locomotion and scratching in the cat.

Despite decades of research, the classical idea that 'reciprocal inhibition' is involved in the hyperpolarisation of motoneurones in their inactive phase during rhythmic activity is still under debate. Here, we investigated the contribution of reciprocal Ia inhibition to the hyperpolarisation of motoneurones during fictive locomotion (evoked either by electrical stimulation of the brainstem or by l-DOPA administration following a spinal transection at the cervical level) and fictive scratching (evoked by stimulation of the pinna) in decerebrate cats. Simultaneous extracellular recordings of Ia inhibitory interneurones and intracellular recordings of lumbar motoneurones revealed the interneurones to be most active when their target motoneurones were hyperpolarised (i.e. in the inactive phase of the target motoneurones). To date, these results are the most direct evidence that Ia inhibitory interneurones contribute to the hyperpolarisation of motoneurones during rhythmic behaviours. We also estimated the amount of Ia inhibition as the amplitude of Ia IPSC in voltage-clamp mode. In both flexor and extensor motoneurones, Ia IPSCs were always larger in the inactive phase than in the active phase during locomotion (n = 14) and during scratch (n = 11). Results obtained from spinalised animals demonstrate that the spinal rhythm-generating network simultaneously drives the motoneurones of one muscle group and the Ia interneurones projecting to motoneurones of the antagonist muscles in parallel. Our results thus support the classical view of reciprocal inhibition as a basis for relaxation of antagonist muscles during flexion-extension movements.

A high-affinity, bivalent PDZ domain inhibitor complexes PICK1 to alleviate neuropathic pain.

Maladaptive plasticity involving increased expression of AMPA-type glutamate receptors is involved in several pathologies, including neuropathic pain, but direct inhibition of AMPARs is associated with side effects. As an alternative, we developed a cell-permeable, high-affinity (~2 nM) peptide inhibitor, Tat-P4 -(C5)2 , of the PDZ domain protein PICK1 to interfere with increased AMPAR expression. The affinity is obtained partly from the Tat peptide and partly from the bivalency of the PDZ motif, engaging PDZ domains from two separate PICK1 dimers to form a tetrameric complex. Bivalent Tat-P4 -(C5)2 disrupts PICK1 interaction with membrane proteins on supported cell membrane sheets and reduce the interaction of AMPARs with PICK1 and AMPA-receptor surface expression in vivo. Moreover, Tat-P4 -(C5)2 administration reduces spinal cord transmission and alleviates mechanical hyperalgesia in the spared nerve injury model of neuropathic pain. Taken together, our data reveal Tat-P4 -(C5)2 as a novel promising lead for neuropathic pain treatment and expand the therapeutic potential of bivalent inhibitors to non-tandem protein-protein interaction domains.

Axonal regeneration and development of de novo axons from distal dendrites of adult feline commissural interneurons after a proximal axotomy.

Following proximal axotomy, several types of neurons sprout de novo axons from distal dendrites. These processes may represent a means of forming new circuits following spinal cord injury. However, it is not know whether mammalian spinal interneurons, axotomized as a result of a spinal cord injury, develop de novo axons. Our goal was to determine whether spinal commissural interneurons (CINs), axotomized by 3-4-mm midsagittal transection at C3, form de novo axons from distal dendrites. All experiments were performed on adult cats. CINs in C3 were stained with extracellular injections of Neurobiotin at 4-5 weeks post injury. The somata of axotomized CINs were identified by the presence of immunoreactivity for the axonal growth-associated protein-43 (GAP-43). Nearly half of the CINs had de novo axons that emerged from distal dendrites. These axons lacked immunoreactivity for the dendritic protein, microtubule-associated protein2a/b (MAP2a/b); some had GAP-43-immunoreactive terminals; and nearly all had morphological features typical of axons. Dendrites of other CINs did not give rise to de novo axons. These CINs did, however, each have a long axon-like process (L-ALP) that projected directly from the soma or a very proximal dendrite. L-ALPs were devoid of MAP2a/b immunoreactivity. Some of these L-ALPs projected through the lesion and formed bouton-like swellings. These results suggest that proximally axotomized spinal interneurons have the potential to form new connections via de novo axons that emerge from distal dendrites. Others may be capable of regeneration of their original axon.

Decerebrate mouse model for studies of the spinal cord circuits.

The adult decerebrate mouse model (a mouse with the cerebrum removed) enables the study of sensory-motor integration and motor output from the spinal cord for several hours without compromising these functions with anesthesia. For example, the decerebrate mouse is ideal for examining locomotor behavior using intracellular recording approaches, which would not be possible using current anesthetized preparations. This protocol describes the steps required to achieve a low-blood-loss decerebration in the mouse and approaches for recording signals from spinal cord neurons with a focus on motoneurons. The protocol also describes an example application for the protocol: the evocation of spontaneous and actively driven stepping, including optimization of these behaviors in decerebrate mice. The time taken to prepare the animal and perform a decerebration takes ∼2 h, and the mice are viable for up to 3-8 h, which is ample time to perform most short-term procedures. These protocols can be modified for those interested in cardiovascular or respiratory function in addition to motor function and can be performed by trainees with some previous experience in animal surgery.

Rostrocaudal distribution of motoneurones and variation in ventral horn area within a segment of the feline thoracic spinal cord.

Retrograde transport of horseradish peroxidase, applied to cut peripheral nerves, was used to determine the rostrocaudal distribution of motoneurones supplying different branches of the ventral ramus for a single mid- or caudal thoracic segment in the cat. The motoneurones occupied a length of spinal cord equal to the segmental length but displaced rostrally from the segment as defined by the dorsal roots, with the number of motoneurones per unit length of cord higher in the rostral part of a segment (close to the entry of the most rostral dorsal root) than in the caudal part. The cross-sectional area of the ventral horn showed a rostrocaudal variation that closely paralleled the motoneurone distribution. The ratio between the number of motoneurones per unit length in the caudal and rostral regions of a segment (0.70) was similar to the ratio previously reported for the strength of functional projections of expiratory bulbospinal neurones (0.63). This is consistent with the motoneurones being the main targets of the bulbospinal neurones.

Control and role of plateau potential properties in the spinal cord.

In this review we will first give a historical account of how the discovery of persistent inward currents (PICs) and plateau potentials changed the understanding of the operation and function of the "final common path", i.e. the motoneurons themselves. A major function of voltage-dependent PICs is to serve as an adjustable amplifier of classical synaptic inputs. The complex control of this, and other intrinsic properties, certainly adjusts the performance of the motoneurons to the needs of the behavioral settings. It has emerged that supraspinal facilitation, mainly by monoaminergic projections, is a prerequisite for the normal function of the PIC channels. When these pathways are interrupted following a spinal lesion the "gain" of the transmission across the motoneurons is reduced and this is likely to be an important explanation for the spinal shock. However, after a few weeks the "plateau properties" of the motoneurons return - now without descending monoaminergic control. This plasticity after spinal lesion is likely to contribute to the hyperreflexia (spasticity) seen after spinal lesions. We then review the current knowledge on PICs in other spinal (inter-)neurons. The monoaminergic systems seem to play a pivotal role in activating the spinal network generating the rhythm and basic motor pattern of locomotion and scratch - the spinal "central pattern generators" (CPGs). We give a short historical background of this research with a special emphasis on the importance of the descending monoaminergic systems.