Rob Bakels

Effects of histone deacetylation inhibition on neuronal differentiation of embryonic mouse neural stem cells

Neural stem cells (NSCs) are multipotent cells that have the capacity for self-renewal and for differentiation into the major cell types of the nervous system, i.e. neurons, astrocytes and oligodendrocytes. The molecular mechanisms regulating gene transcription resulting in NSC differentiation and cell lineage specification are slowly being unraveled. An important mechanism in transcriptional regulation is modulation of chromatin by histone acetylation and deacetylation, allowing or blocking the access of transcriptional factors to DNA sequences. The precise involvement of histone acetyltransferases and histone deacetylases (HDACs) in the differentiation of NSCs into mature functional neurons is still to be revealed. In this in vitro study we have investigated the effects of the HDAC inhibitor trichostatin A (TSA) on the differentiation pattern of embryonic mouse NSCs during culture in a minimal, serum-free medium, lacking any induction or growth factor. We demonstrated that under these basic conditions TSA treatment increased neuronal differentiation of the NSCs and decreased astrocyte differentiation. Most strikingly, electrophysiological recordings revealed that in our minimal culture system only TSA-treated NSC-derived neurons developed normal electrophysiological membrane properties characteristic for functional, i.e. excitable and firing, neurons. Furthermore, TSA-treated NSC-derived neurons were characterized by an increased elongation and arborization of the dendrites. Our study shows that chromatin structure modulation by HDACs plays an important role in the transcriptional regulation of the neuronal differentiation of embryonic NSCs particularly as far as the development of functional properties are concerned. Manipulation of HDAC activity may be an important tool to generate specific neuronal populations from NSCs for transplantation purposes.

Functionally deficient neuronal differentiation of mouse embryonic neural stem cells in vitro.

Embryonic mouse neural stem cells (NSCs) were isolated from E14 mice, multiplied in medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and plated in laminin-coated wells in basic serum-free neurobasal medium. After 7 days in vitro, approximately 20% of the embryonic mouse NSCs developed into morphologically and biochemically fully maturated neurons, with extensive dendrites and multiple synaptic contacts. However, even after 22 days of culture, none of these neurons developed voltage-dependent sodium-channels characteristic for a functional neuron. Apparently, the morphological differentiation and the electrophysiological maturation of an embryonic mouse NSC into a neuron are independently regulated.

Human spinal cord injury: motor unit properties and behaviour

Spinal cord injury (SCI) results in widespread variation in muscle function. Review of motor unit data shows that changes in the amount and balance of excitatory and inhibitory inputs after SCI alter management of motoneurons. Not only are units recruited up to higher than usual relative forces when SCI leaves few units under voluntary control, the force contribution from recruitment increases due to elevation of twitch/tetanic force ratios. Force gradation and precision are also coarser with reduced unit numbers. Maximal unit firing rates are low in hand muscles, limiting voluntary strength, but are low, normal or high in limb muscles. Unit firing rates during spasms can exceed voluntary rates, emphasizing that deficits in descending drive limit force production. SCI also changes muscle properties. Motor unit weakness and fatigability seem universal across muscles and species, increasing the muscle weakness that arises from paralysis of units, motoneuron death and sensory impairment. Motor axon conduction velocity decreases after human SCI. Muscle contractile speed is also reduced, which lowers the stimulation frequencies needed to grade force when paralysed muscles are activated with patterned electrical stimulation. This slowing does not necessarily occur in hind limb muscles after cord transection in cats and rats. The nature, duration and level of SCI underlie some of these species differences, as do variations in muscle function, daily usage, tract control and fibre‐type composition. Exploring this diversity is important to promote recovery of the hand, bowel, bladder and locomotor function most wanted by people with SCI.

Measures of ''fastness'': Force profiles of twitches and partly fused contractions in rat medial gastrocnemius and tibialis anterior muscle units

Recordings of isometric force were obtained for twitches and (sub)maximal tetani of gastrocnemius medialis (MG) and tibialis anterior (TA) muscle units in female Wistar rats. We assessed the relationships between unit properties that have all been associated with “speed”: (1) the relative degree of peak force attained during repetitive activation at 40 Hz (P40/Pmax), (2) the relative degree of final twitch fusion during the same test burst (Fus-end), and (3) various measures of the time-course of single twitches, including twitch time-to-peak and a parameter referred to as “initial fusion ratio” (Fus-in; relative decline from peak force at 25 ms from twitch onset). The various measures of twitch time-course were significantly correlated to each other with correlation coefficients varying over a fairly wide range (0.35–0.64 for MG; 0.50–0.80 for TA). Twitch time-course was also significantly correlated with Fus-end during the 40-Hz repetitive activation; the highest correlation coefficient (0.69 for MG, 0.80 for TA) was obtained for Fus-in, which was also numerically similar to Fus-end. Thus, the degree of fusion indeed seemed to be largely dependent upon aspects of twitch time-course. However, the relative degree of force mobilization obtained in the same contractions elicited by stimulation at 40 Hz was not consistently better correlated with Fus-end than with measures of single twitch time-course. Furthermore, in fast-twitch units having the same twitch time-to-peak, the force mobilization elicited by stimulation at 40 Hz (P40Pmax) was the same for MG and TA, while the degree of fusion was significantly smaller for TA than for MG units. The results demonstrate the complexity of the concept of isometric “speed” and underline the need for using several speed indicators in parallel in studies concerning the differentiation of muscle (unit) properties.

Inadvertent Contralateral Activity during a Sustained Unilateral Contraction Reflects the Direction of Target Movement

Strong unilateral contractions are accompanied by excitatory effects to the ipsilateral cortex. This activity can even result in overt contractions of muscles in the contralateral limb. We used this inadvertent, associated activity to study whether the cortical presentation of movements is organized in a directional-related or a muscle-related reference frame. We assessed the contralateral activation for the left index finger during a sustained maximal abduction of the right index finger. In the first experiment, both hands were held vertically in a symmetrical orientation, and in the second experiment the hands were in an asymmetrical orientation (left hand, palm downward; right hand, vertical). In both experiments, the direction of the contralateral associated contraction was upward, i.e., in the symmetrical hand orientation the contralateral force increased mainly in abduction direction, whereas in the asymmetrical hand orientation the contralateral force increased in the extension direction. Thus, the contralateral contractions reflected the direction of the target movement rather than simply the activity of the muscles activated on the target side. These observations provide strong evidence that motor commands are organized in an extrinsic, direction-related reference frame, as opposed to an internal muscle-related reference frame.

Do Additional Inputs Change Maximal Voluntary Motor Unit Firing Rates After Spinal Cord Injury

Background. Motor unit firing frequencies are low during maximal voluntary contractions (MVCs) of human thenar muscles impaired by cervical spinal cord injury (SCI). Objective. This study aimed to examine whether thenar motor unit firing frequencies increase when driven by both maximal voluntary drive and other concurrent inputs compared with an MVC alone. Methods. Motor unit firing rates, force, and surface electromyographic activity (EMG) were compared across 2 contractions: (a) MVC alone and (b) MVC combined with another input (combination contraction). Other inputs (conditions) included vibration, heat, or cold applied to the anterior surface of the forearm, electrical stimulation delivered to the anterior surface of the middle finger, a muscle spasm, or a voluntary contraction of the contralateral thenar muscles against resistance. Results. The maximal firing frequency (n = 68 units), force, and electromyographic activity (n = 92 contraction pairs) were all significantly higher during the combined contractions compared with MVCs alone. There was a 3-way interaction between contraction, condition, and subject for maximal motor unit firing rates, force, and EMG. Thus, combined contraction responses were different for conditions across subjects. Some conditions (eg, a muscle spasm) resulted in more effective and more frequent responses (increases in unit firing frequency, force, EMG in >50% contractions) than others. Recruitment of new units also occurred in combined contractions. Conclusions. Motoneurons are still responsive to additional afferent inputs from various sources when rate modulation from voluntary drive is limited by SCI. Individuals with SCI may be able to combine inputs to control functional tasks they cannot perform with voluntary drive alone.

Corticotropin-releasing factor and urocortin regulate spine and synapse formation: structural basis for stress-induced neuronal remodeling and pathology

Dendritic spines are important sites of excitatory neurotransmission in the brain with their function determined by their structure and molecular content. Alterations in spine number, morphology and receptor content are a hallmark of many psychiatric disorders, most notably those because of stress. We investigated the role of corticotropin-releasing factor (CRF) stress peptides on the plasticity of spines in the cerebellum, a structure implicated in a host of mental illnesses, particularly of a developmental origin. We used organotypic slice cultures of the cerebellum and restraint stress in behaving animals to determine whether CRF in vitro and stress in vivo affects Purkinje cell (PC) spine density. Application of CRF and urocortin (UCN) to cerebellar slice cultures increased the density of spines on PC signaling via CRF receptors (CRF-Rs) 1 and 2 and RhoA downregulation, although the structural phenotypes of the induced spines varied, suggesting that CRF-Rs differentially induce the outgrowth of functionally distinct populations of spines. Furthermore, CRF and UCN exert a trophic effect on the surface contact between synaptic elements by increasing active zones and postsynaptic densities and facilitating the alignment of pre- and post-synaptic membranes of synapses on PCs. In addition, 1 h of restraint stress significantly increased PC spine density compared with those animals that were only handled. This study provides unprecedented resolution of CRF pathways that regulate the structural machinery essential for synaptic transmission and provides a basis for understanding stress-induced mental illnesses.

Hypothyroidism in the rat results in decreased soleus motoneurone soma size

Adult female rats were thyroidectomized. After an average of 17 weeks, horseradish peroxidase (HRP) was injected into the right side soleus muscle. Two days later, left side soleus muscle properties were recorded and muscles and spinal cord were removed for further histological measurements. Soleus muscles from hypothyroid rats no longer contained type IIA fibers. Contraction and half-relaxation times of twitches had increased significantly compared to control rats. The average cross-sectional surface areas of HRP-labeled soleus motoneurones from hypothyroid rats were slightly but significantly smaller than those of control rats. A similar decrease in size was found for other (presumed moto-) neurones lying ventrolaterally to the soleus motor nucleus. It is concluded that changes in the soleus muscle fiber composition, as caused by lowered levels of thyroid hormone, are paralleled by corresponding changes in the size of its motoneurones and also of other spinal (moto)neurones.

Motor unit firing rates during spasms in thenar muscles of spinal cord injured subjects.

Involuntary contractions of paralyzed muscles (spasms) commonly disrupt daily activities and rehabilitation after human spinal cord injury (SCI). Our aim was to examine the recruitment, firing rate modulation, and derecruitment of motor units that underlie spasms of thenar muscles after cervical SCI. Intramuscular electromyographic activity (EMG), surface EMG, and force were recorded during thenar muscle spasms that occurred spontaneously or that were triggered by movement of a shoulder or leg. Most spasms were submaximal (mean: 39%, SD: 33 of the force evoked by median nerve stimulation at 50 Hz) with strong relationships between EMG and force (R2 > 0.69). Unit recruitment occurred over a wide force range (0.2–103% of 50 Hz force). Significant unit rate modulation occurred during spasms (frequency at 25% maximal force: 8.8 Hz, 3.3 SD; at maximal force: 16.1 Hz, 4.1 SD). Mean recruitment frequency (7.1 Hz, 3.2 SD) was significantly higher than derecruitment frequency (5.4 Hz, 2.4 SD). Coactive unit pairs that fired for more than 4 s showed high (R2 > 0.7, n = 4) or low (R2:0.3–0.7, n = 12) rate-rate correlations, and derecruitment reversals (21 pairs, 29%). Later recruited units had higher or lower maximal firing rates than lower threshold units. These discrepant data show that coactive motoneurons are drive both by common inputs and by synaptic inputs from different sources during muscle spasms. Further, thenar motoneurons can still fire at high rates in response to various peripheral inputs after SCI, supporting the idea that low maximal voluntary firing rates and forces in thenar muscles result from reduced descending drive.