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Modulation of synaptic transmission, studies on spinal cord slices in vitro
Mrózková, Petra ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee)
Modulation of a synaptic transmission in the spinal cord dorsal horn plays a key role in nociceptive signalling, especially in states of pathological pain. The goal of this study was to develop a method for calcium imaging in spinal cord slices in vitro. This method allowed us to record changes of intracellular free calcium ions concentration (iCa2+ ), that are a major mediator of neuronal plasticity. In this work, we have focused on application of this method in a conventional fluorescence microscope and on the role of different neuromodulators of synaptic activity. Changes of iCa2+ induced by dorsal root electrical stimulation were recorded altogether in 744 dorsal horn (lamina I and II) neurons. In the first series of experiments, stimulation protocols activating preferentially A and A + C dorsal root fibers were used and long-term stability of the calcium responses was verified. The dorsal root stimulation induced in the neurons fast and delayed type of calcium response. Application of AMPA and NMDA receptors antagonists, CNQX (50μM) and MK801 (45μM), reduced the calcium response amplitude and confirmed the importance of glutamate receptors in synaptic activation. In several experiments the effect of capsaicin a TRPV1 receptors agonist, application was tested. Application of even low...
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Pathological pain states, the role of synaptic modulation at spinal cord level
Nerandžič, Vladimír ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee)
(English) Modulation of synaptic transmission in dorsal horn of spinal cord plays a key role in nociceptive signalling. Recent studies have indicated a great importance of presynaptic TRPV1 receptors (transient receptor potential vanilloid) in spinal cord. These receptors act as molecular integrator of nociceptive stimulation on periphery. The way of their activation and the effect on modulation of the synaptic transmission are not clarified yet. Previous studies demonstrated the influence of many inflammatory mediators and cytokins on TRPV1 receptors. The aim of our research was to show changes in activation of presynaptic TRPV1 receptors in the spinal cord following the application of endogenous agonist N-oleoyl dopamine (OLDA) in a model of peripheral neuropathy, after incubation with cytokine TNFα and to show the effect of precursor of anandamide N-acylphosphatidylethanolamine (NAPE). In our experiments, we have recorded miniature excitatory postsynaptic currents (mEPSC) from neurons of acute spinal cord slices by the patch-clamp method. The first series of experiments tested sensitivity to application of the endogenous agonist OLDA 5 days after evoking peripheral neuropathy. The frequency of mEPSC increased significantly - to 250 % of base level after applying a low concentration of OLDA (0,2...
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Structural and Functional Study on Transient Receptor Potential Vanilloid 1 (TRPV1) and Ankyrin Receptor (TRPA1) Channels
SAMAD, Abdul
Investigations of structural and functional relationships of rat transient receptor potential cation channel, subfamily V, member 1 (TRPV1), also known as the capsaicin receptor, and human transient receptor potential cation channel, subfamily A, member 1, also known as TRPA1, are presented. Capsaicin induced Ca2+ -dependent desensitization of rat TRPV1 channel is studied and lead to the identification of key amino acid residues in the C- terminal domain of TRPV1 interacting with the membrane phospholipid PIP2 and an intradomain interaction that controls the open and desensitized state of the TRPV1 channel. Further the molecular basis of agonist AITC- and voltage-dependent gating on TRPA1 is explained. Hereby, residue P949 located near the center of the sixth transmembrane spanning helix (S6) is structurally required for normal functioning of the receptor and the distal bi-glycine G958XXXG962 motif controls its activation/deactivation properties. Furthermore, the gating region is extended towards the cytoplasmic part of the channel, putatively located near the inner mouth of the channel pore. A following series of experiments lead to the identification of a limited number of residues that appear important for allosteric regulation of the channel by chemical and voltage stimuli (K969, R975, K989, K1009, K1046, K1071, K1092 and K1099). In addition, three charge-neutralizing `gain-of- function{\crq} mutants (R975A, K988A, and K989A) which exhibited higher sensitivity to depolarizing voltages were characterized, indicating that these residues are directly involved in voltage-dependent modulation of TRPA1.
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