1. Cellular and molecular mechanisms of pain transmission and modulation at spinal cord level.

Pathological pain states characterized by pain to innocuous stimuli (allodynia), hyperlagesia and spontaneous chronic pain often become debilitating diseases recalcitrant to the best treatment efforts, as the cellular and physiological mechanisms underlying the development of chronic pain and associated phenomena are still not fully understood. The aim of our projects is to further identify the mechanisms of synaptic modulation at the spinal cord level that can underlie different pathological pain states. The focus is to study the role of transient receptor potential vanilloid-1 (TRPV1) receptors and cytokines in nociceptive signaling and modulation at spinal cord level, under control and pathological conditions. Behavioral, electrophysiological and immuno-histochemical methods are used. Elucidating these mechanisms could help to identify new potential targets for pain therapy.

Main results

The role of the TRPV1 endogenous agonist N-Oleoyldopamine in modulation of nociceptive signaling at the spinal cord level.

Transient receptor potential vanilloid (TRPV1) receptors are abundant in a subpopulation of primary sensory neurons that convey nociceptive information from the periphery to the spinal cord dorsal horn. The TRPV1 receptors are expressed on both the peripheral and central branches of these dorsal root ganglion (DRG) neurons and can be activated by capsaicin, heat, low pH, and also by recently described endogenous lipids. Using patch-clamp recordings from superficial dorsal horn (DH) neurons in acute spinal cord slices, the effect of application of the endogenous TRPV1 agonist N-oleoyldopamine (OLDA) on the frequency of miniature excitatory postsynaptic currents (mEPSCs) was evaluated. A high concentration OLDA (10 microM) solution was needed to increase the mEPSC frequency, whereas low concentration OLDA (0.2 microM) did not evoke any change under control conditions. The increase was blocked by the TRPV1 antagonists SB366791 or BCTC. Application of a low concentration of OLDA evoked an increase in mEPSC frequency after activation of protein kinase C by phorbol ester (PMA) and bradykinin or in slices from animals with peripheral inflammation. Increasing the bath temperature from 24 to 34 degrees C enhanced the basal mEPSC frequency, but the magnitude of changes in the mEPSC frequency induced by OLDA administration was similar at both temperatures. Our results suggest that presumed endogenous agonists of TRPV1 receptors, like OLDA, could have a considerable impact on synaptic transmission in the spinal cord, especially when TRPV1 receptors are sensitized. Spinal TRPV1 receptors could play a pivotal role in modulation of nociceptive signaling in inflammatory pain.

Spicarova D, Palecek J., J Neurophysiol. 2009 Jul;102(1):234-43. Epub 2009 Apr 15.

Modulation of AMPA excitatory postsynaptic currents in the spinal cord dorsal horn neurons by insulin.

Glutamate AMPA (α-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid) receptors are critical for sensory transmission at the spinal cord dorsal horn. Plasma membrane AMPA receptor endocytosis that can be induced by insulin may underlie long term modulation of synaptic transmission. Insulin receptors (IRs) are known to be expressed on spinal cord dorsal horn (DH) neurons, but their possible role in sensory transmission has not been studied. In this work the effect of insulin application on fast excitatory postsynaptic currents (EPSCs) mediated by AMPA receptors evoked in DH neurons was evaluated. Acute spinal cord slices from 6-10 day old mice were used to record EPSCs evoked in visually identified superficial DH neurons by dorsal root primary afferent stimulation. AMPA EPSCs could be evoked in all of the tested neurons. In 75% of the neurons the size of the AMPA EPSCs was reduced to 62.1% and to 68.9% of the control values when 0.5 µM or 10 µM insulin was applied. There was no significant change in the size of the AMPA EPSCs in the remaining 25% of DH neurons. The membrane permeable protein tyrosine kinase inhibitor, lavendustin A (10 µM), prevented the insulin induced AMPA EPSC depression. Our results suggest a possible role of the insulin pathway in modulation of sensory and nociceptive synaptic transmission in the spinal cord.

Spicarova D, Palecek J., Neuroscience (2009) in press

Post-operative pain behavior in rats is reduced after single high-concentration capsaicin application.

Surgical procedures associated with tissue injury are often followed by increased sensitivity to innocuous and noxious stimuli in the vicinity of the surgical wound. The aim of this study was to evaluate the role of transient receptor potential vanilloid 1 receptor (TRPV1) containing nociceptors in this process, by their functional inactivation using a high-concentration intradermal injection of capsaicin in a rat plantar incision model. Paw withdrawal responses to mechanical stimuli (von Frey filaments 10-367 mN) and to radiant heat applied on plantar skin were tested in animals treated with capsaicin or the vehicle 6 days and 24 h before or 2 h after the incision was made. In the vehicle-treated animals, mechanical and thermal sensitivity increased significantly 1-96 h following the incision. Capsaicin applied 24 h before the surgery was most effective and significantly diminished the development of post-incisional mechanical allodynia and hyperalgesia. Thermal hypoalgesia was present in the incised paw after the capsaicin treatment. Capsaicin application 6 days before the incision induced thermal hypoalgesia before the incision but did not prevent completely the thermal hyperalgesia after the incision, while there was also a reduction of mechanical hypersensitivity. Application of the capsaicin injection after the incision showed its first effect at 2 h after the injection and at 24 h the effect was comparable with the 6 days pretreatment. Our results show an important role of TRPV1-containing nociceptors in the development of post-surgical hypersensitivity and suggest that local, high-concentration capsaicin treatment could be used to reduce it.

Pospisilova E, Palecek J., Pain. 2006 Dec 5;125(3):233-43. Epub 2006 Jun 22.

2. Effect of exo- and endogenous factors on muscle differentiation, mRNA and protein isoforms of contractile and Ca 2+ transporting proteins.

Studies of morphogenetic interactions between primary sensory axons and target tissues during development and regeneration of peripheral mechanoreceptors. The role of neural versus non-neural factors is analysed by electron or light microscopy and histo- or immunocytochemistry.

A new research field focuses on an analysis of intrinsic - myogenic, and extrinsic - mainly neurogenic and hormonal influences regulating the expression of muscle phenotype. Ultrastructure and expression of myosin heavy chain (MHC) isoforms during development and regeneration of extrafusal and intrafusal muscle fibres is studied using electron microscopic, histochemical and immunocytochemical methods and techniques of molecular biology.

Main results

1. We have confirmed that sensory innervation plays a key role in the induction of typical morphological characteristics and in the expression of specific MHC isoforms in intrafusal muscle fibres, whereas motor innervation contributes to the diversity in the expression and distribution of different MHC isoforms along the length of intrafusal fibres. We have shown that one type of intrafusal satellite cells give rise to myotubes capable, if innervated by sensory axons, to differentiate into a special intrafusal phenotype. This part of our research is covered in the invited review published in Microscop. Res. Tech. (Soukup et al. Microscop Res Tech 30: 390407, 1995).

2. The current experiments are based on our own model of heterochronous isotransplantation. We have shown that after isotransplantation, the intrafusal satellite cells retain a great plasticity as they are able to differentiate into fibres with extrafusal fast or slow phenotype, when reinnervated solely by extrafusal motor axons. Furthermore, this model enables us to compare the contribution of genetic factors, nerve impulse frequency and thyroid hormone levels to the differentiation of muscle phenotype. This topic is summarized by the review published in Physiological Research.

The first results on differentiation of muscle fibre types in hypo- eu- and hyperthyroid rats demonstrate that the grafted, genetically predetermined, slow muscle reinnervated by fast nerve differentiates as fast muscle, but the percentage of 2B, 2X/D, 2A and type 1 fibres varies depending on to the thyroid status.

Publications