Neurotransmitters and receptors in the dorsal horn of the spinal cord

Modulation of the sensory input can occur within the dorsal horn of the spinal cord where the primary afferent fibers synapse with neurons that transmit to the higher centers. The transmission of the sensory information begins with activation of the peripheral receptors of primary afferent neurons whose cell bodies lie within the dorsal root ganglia and whose central terminals project to secondary neurons in the dorsal horn of the spinal cord. Several neurotransmitters and a large variety of receptors have been found in the superficial laminae of the dorsal horn. The present work reviews the major classes of transmitters and receptors that have been implicated in the transmission and modulation of spinal afferent and pain processing. The role of excitatory and inhibitory amino acids, tachykinin and opioid peptides, calcitonin gene-related peptide (CGRP), nociceptin and nocistatin, biogenic amines, acetylcholine, ATP, nitric oxide as well as capsaicin and vanilloid receptors will be discussed along with the most recent developments in the field. It seems probable that transmission of the somatosensory information from the primary afferent fibers to the secondary dorsal horn neurons depends on the balance between the excitatory effects of excitatory amino acids and the inhibitory actions of several other transmitter systems

Ultralow-noise headstage and main amplifiers for extracellular spike recording

This methodological paper provides a detailed description of a novel ultralow-noise pre- and main-amplifier system designed for the extracellular recording of voltage spikes produced by neuronal action potentials. The main difficulties with extracellular recording are the unwanted electrical signals that lower the recording quality. They include destructive interferences by alternating electric or magnetic fields, in addition to thermal and other random noises resulting from the intrinsic properties of the substances from which the electrode and the electrical circuit are made. The preamplifier is placed in a specially built metal headstage probe. It is designed so that microelectrodes can be plugged directly into the probe, keeping the electrode and preamplifier in the closest possible proximity. Unique electrode holder adaptors at the same time make the probe for the electrode holder with the added benefit of extended electrical shielding. The main amplifier contains tuned circuit band pass filters optimized for metal and carbon fiber microelectrodes. Electromagnetic interference pickup is reduced by the enclosing tin-plated iron box and a built-in 50/60 Hz reject filter. In test experiments, an excellent signal-to-noise ratio and low-noise baseline recording were achieved. When carbon fiber microelectrodes were applied in the medulla of anesthetized rats, the total peak-to-peak noise level of the system was about 25 µV, i.e., about 6 µV RMS, which is only a few µV higher than the theoretical random noise. It is concluded that, in combination with carbon fiber electrodes, the present amplifiers do not contribute significantly to the overall noise

Differential inhibition by trifluoperazine of responses of hippocampal CA1 pyramidal cells to NMDA and AMPA in vivo

The effects of trifuoperazine (TFP), a phenothiazine neuroleptic drug having potent anticalmodulin activity, were studied on the responses of hippocampal CA1 pyramidal cells to N-methyl-D-aspartic acid (NMDA) and (RS)-A-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in vivo. Single-unit activity was recorded using multibarrel carbon fiber containing microelectrodes whilst all drugs were delivered by microiontophoresis. NMDA and AMPA were iontophoresed alternately so that they evoked comparable responses in terms of peak heights as peristimulus time histograms were recorded. We observed that changing the stimulation intensity of one classes of receptors (e.g. NMDA) greatly influenced the responsiveness of the other (e.g. AMPA) and vice versa. In the presence of iontophoretically applied TFP responses to both NMDA and AMPA were significantly decreased. More interestingly, NMDA-evoked responses were significantly more inhibited by TFP than responses to AMPA under the same experimental conditions. In our conclusions, these results are due to the inhibition by TFP of the second messenger cascade events leading from NMDA receptors via Ca2+/calmodulin to AMPA receptors and, in consequence, for the blocking of phosphorylation of AMPA receptors and their sensitization. It is also likely that the function of NMDA receptors by itself is, at least in part, dependent on the Ca2+/calmodulin-mediated events

Electrochemical responses of carbon fiber microelectrodes to dopamine in vitro and in vivo

Cylindrical, 7 μm in diameter carbon fiber (CF) microelectrodes were constructed and tested for their responses to dopamine in constant potential amperometry or fast scan cyclic voltammetry (FSCV). Amperometry was carried out in a miniature perfusion chamber whereas background subtracted FSCV was performed both in vitro and in vivo. For calibration performance of the microelectrodes, peak oxidation currents were determined using unmodified carbon tips of varying lengths and plotted against the tip lengths. A very close linear correlation (r = 0.997) was found between the two variables for tip lengths ranging from 25 to 300 μm. Also, a very close linear correlation was found between the oxidation current at a given carbon tip length in response to increasing dopamine concentrations measured by either amperometry or FSCV. In vivo experiments were carried out in the visual cortex of the anaesthetized rat to detect dopamine release in response to visual stimulation. Indeed, background subtracted cyclic voltammograms showed an increase in the current at 0.65 V which is the typical dopamine oxidation potential

Novel carbon fiber microeletrodes for extracellular electrophysiology

Single- and multibarrel carbon fiber microelectrode blanks were constructed and pulled to electrodes to be used for extracellular recording and microiontophoresis. A unique spark etching method was developed to produce a sharp-pointed, conical carbon tip protruding 15-20 µm from the glass pipette(s). The shape and size of the carbon fiber tip were examined by scanning electron microscopy. In test experiments, extracellular recordings were made from spinal dorsal horn neurons of the spinal cord in anesthetized rats. The sharp carbon tip allowed these electrodes to penetrate the arachnoid membrane over the spinal cord with ease. The electrodes picked up extracellular spikes with an excellent signal-to-noise ratio. Under the given experimental conditions, the peak-to-peak noise level was about 20 µV. To test the performance of the iontophoresis barrels, neurons were stimulated by iontophoretic application of N-methyl-D-aspartate (NMDA) or kainic acid or by noxious heat delivered to the cutaneous receptive fields in the tail. After the iontophoretic ejection of naloxone, the responses to iontophoresed kainic acid and noxious heat were significantly increased. Spikes from dorsal horn neurons were counted and peristimulus time histograms were displayed online by means of a LabView-based system. These carbon fiber microelectrodes are excellent for extracellular spike recording and microiontophoresis and may additionally be suitable for electrochemical measurements and for the development of enzyme- or antibody-based microbiosensor

Polymer insulation of ultramicro carbon fiber electrodes for electrophysiological, electrochemical and biosensor applications

There is an obvious need for electrodes with extremely small electroactive areas and structural dimensions that offer great promise for electrochemical microscopy used for neuronal analysis in ultrasmall environments. In the present study we have developed ultramicro carbon fiber (CF) electrodes with combined thin layers of poly(oxyphenylene) and epoxy resin coatings for electrical insulation. The thickness of the borosilicate glass housing and insulating layer of our standard CF microelectrodes is about 1 |im and the carbon tip protrudes by about 20 |im from the glass assembly. Functionalization requires longer sections of the exposed, uninsulated carbon fiber where submicron tips are shaped and chemical modifications are made. Electrodeposition of poly(oxyphenylene) was carried out using anodic currents at 2V against an Ag/AgCI half-cell. After finishing the polymerization at 150°C for 2 hours, electrical impedances of the electrodes were 17.1 ±2.8MQ (mean ± SD, n= 22). An additional epoxy coating was formed by dipping the CF electrodes in diluted epoxy resins followed by dipping in a mixture of diluted curing agents. The epoxy layer significantly increased the effectiveness of the insulation as the impedance for each of the 38 prepared CF electrodes was higher than the upper range limit (200 MQ) of our impedance meter. The thickness of the combined insulating layer was less than 1 |im as estimated by electron microscopic studies. Removal of the insulation from the very tip was carried out using high voltage spark or electrochemical etching. These submicron CF electrodes are suitable for extracellular spike recording, electrochemical and biosensor applications

Anti-calmodulins and Tricyclic Adjuvants in Pain Therapy Block the TRPV1 Channel

Ca2+-loaded calmodulin normally inhibits multiple Ca2+-channels upon dangerous elevation of intracellular Ca2+ and protects cells from Ca2+-cytotoxicity, so blocking of calmodulin should theoretically lead to uncontrolled elevation of intracellular Ca2+. Paradoxically, classical anti-psychotic, anti-calmodulin drugs were noted here to inhibit Ca2+-uptake via the vanilloid inducible Ca2+-channel/inflamatory pain receptor 1 (TRPV1), which suggests that calmodulin inhibitors may block pore formation and Ca2+ entry. Functional assays on TRPV1 expressing cells support direct, dose-dependent inhibition of vanilloid-induced 45Ca2+-uptake at µM concentrations: calmidazolium (broad range)≥trifluoperazine (narrow range)>chlorpromazine/amitriptyline>fluphenazine>>W-7 and W-13 (only partially). Most likely a short acidic domain at the pore loop of the channel orifice functions as binding site either for Ca2+ or anti-calmodulin drugs. Camstatin, a selective peptide blocker of calmodulin, inhibits vanilloid-induced Ca2+-uptake in intact TRPV1+ cells, and suggests an extracellular site of inhibition. TRPV1+, inflammatory pain-conferring nociceptive neurons from sensory ganglia, were blocked by various anti-psychotic and anti-calmodulin drugs. Among them, calmidazolium, the most effective calmodulin agonist, blocked Ca2+-entry by a non-competitive kinetics, affecting the TRPV1 at a different site than the vanilloid binding pocket. Data suggest that various calmodulin antagonists dock to an extracellular site, not found in other Ca2+-channels. Calmodulin antagonist-evoked inhibition of TRPV1 and NMDA receptors/Ca2+-channels was validated by microiontophoresis of calmidazolium to laminectomised rat monitored with extracellular single unit recordings in vivo. These unexpected findings may explain empirically noted efficacy of clinical pain adjuvant therapy that justify efforts to develop hits into painkillers, selective to sensory Ca2+-channels but not affecting motoneurons