Ian D. Hentall

Serotonergic, cholinergic and nociceptive inhibition or excitation of raphe magnus neurons in barbiturate-anesthetized rats

Neurons in the nucleus raphe magnus were recorded extracellularly from barbiturate-anesthetized rats, and were classified by their responses to noxious mechanical stimulation as either pinch-excited, pinch-inhibited or biphasic (inhibited then excited). They were then subjected to iontophoresis of serotonin, some serotonergic agonists and antagonists, acetylcholine, and gamma-amino-n-butyric acid. Serotonin reduced the spontaneous firing of most pinch-inhibited cells (79%). Significantly fewer (P < 0.05) pinch-excited and biphasic cells were inhibited by serotonin (40% and 45%, respectively); in these two cell classes, the observed response was often excitation (30% and 14%), or inhibition for 10-30s followed by excitation for the next 1-2 min (25% and 36%). Acetylcholine showed a similar, statistically significant distribution of effects (P < 0.05), inhibiting all pinch-inhibited neurons (n = 10) but fewer pinch-excited (53%, n = 17) and biphasic neurons (20%, n = 10). Excitation, or excitation then inhibition, was again found frequently among the remaining pinch-excited and biphasic cells. The effect of gamma-amino-n-butyric acid was only inhibitory. In all three nociceptive classes, the serotonin-1A agonist buspirone (n = 15) was inhibitory (87%) and the serotonin-1C/2 antagonist ketanserin (n = 20) was excitatory (35%). The mixed serotonin-1/2 antagonist methysergide (n = 10) was inhibitory (50%) or excitatory (40%). 8-Hydroxy-dipropylaminotetralin (n = 3) was found to increase spontaneous activity (possibly because of partial serotonin-1A agonsim), and +/- propranolol (n = 4) to reduce it (possibly through beta-adrenoceptor antagonism, not serotonin-1A antagonism).(ABSTRACT TRUNCATED AT 250 WORDS)

The alleviation of pain by cell transplantation.

Publisher Summary The mushrooming of the field of cellular transplantation in recent years has given rise to a new way of supplying analgesic molecules. In animal models, cell-transplant analgesia has proven successful both with cells from primary tissue (mostly in studies with adrenal medullary chromaffin cells) and with immortalized, bioengineered cell lines that secrete pain-reducing neuroactive substances. The choice of donor material for cell-transplant analgesia presents some interesting contrasts with other neural transplant therapies. Homotypic tissue is a realistic choice as replacement material for repair of deficits, such as those seen in Parkinsons disease or spinal trauma. However, heterotypic tissue shows an apparently coincidental but useful concordance with the function of the local host tissue. This is most remarkable in the case of the adrenal medulla for spinal analgesia. Preclinical pain models of several types have by now confirmed that adrenal medullaty chromaffin cells can be analgesic when transplanted into the rats spinal subarachnoid space.

Correlations between serotonin level and single-cell firing in the rat's nucleus raphe magnus.

The relation between serotonin release and electrical activity was examined in the nucleus raphe magnus of rats anesthetized with pentobarbital. Serotonin levels were monitored through a carbon-fiber microelectrode by fast cyclic voltammetry (usually at 1 Hz). Single-cell firing was recorded through the same microelectrode, except during the voltammetry waveform and associated electrical artifact (totaling about 30 ms). Multi-barrel micropipettes incorporating the voltammetry electrode were used for iontophoresis of drugs. Cells were inhibited, excited or unaffected by noxious mechanical skin stimulation. These were respectively designated as off(M) cells, on(M) cells and neutral(M) cells, M denoting mechanical. During 3 min of pinching, serotonin slowly rose near seven of 14 on(M) cells and 26 of 46 off(M) cells; it fell near two off(M) cells; it was unchanged near all other cells, including six neutral(M) cells. On a finer spatiotemporal scale, near four of seven on(M) cells, 10 of 14 off(M) cells and 0 of four neutral(M) cells, average serotonin levels fell significantly within +/- 100 ms of spontaneous spikes. Lower serotonin may have caused the higher spike probability; the converse is theoretically unlikely, since delays between release and detection are estimated to exceed 100 ms. Increased serotonin and decreased firing were always seen following iontophoresis or intravenous injection (1 mg/kg) of the serotonin re-uptake inhibitor clomipramine (n = 7). Iontophoresis of +/- propranolol, whose serotonergic actions include antagonism and partial agonism at 5-HT1 receptors, also increased serotonin and decreased firing (n=4). Methiothepin (intravenous, 1 mg/kg), whose serotonergic actions include 5-HT1 and 5-HT2 antagonism, typically raised serotonin levels (four of five cells) and always blocked inhibition by clomipramine (n = 3). Iontophoresis of glutamate always lowered serotonin and increased firing (n = 4). Since serotonin levels and firing were usually inversely correlated, except near on(M) cells during pinch, we propose that serotonin is released from terminals of incoming nociceptive afferents. Prior neuroanatomical knowledge favors a midbrain origin for these afferents, while some of the drug findings suggest that their terminals possess inhibitory serotonergic autoreceptors, possibly of 5-HT1b subtype. The released serotonin could contribute to the inhibition of off(M) cells and excitation of on(M) cells by noxious stimulation, since inhibitory 5-HT1a receptors and excitatory 5-HT2 receptors, respectively, have previously been shown to dominate their serotonergic responses.

Acetylcholine release from the midbrain interpeduncular nucleus during anesthesia.

An exceptional local rise in metabolism during general anesthesia has been noted previously in the ventral midbrains highly cholinoceptive interpeduncular nucleus (IPN). We report here a functional correlate. Increased interstitial acetylcholine (ACh) was measured in the IPN of rats through chronically implanted microdialysis probes upon anesthesia by inhalation of 3% halothane (mean 1425% of pre-anesthesia baseline at 30 min, n = 5) and by i.p. injection of 100 mg kg-1 ketamine (mean 387%, n = 6). With 50 mg kg-1 i.p. pentobarbital (n = 8), ACh either climbed or fell repeatably in each animal; a positive correlation (p less than 0.05) emerged between the baseline preanesthetized level and the percentage change after 60 min. Mapping of the brainstem under ketamine (n = 2) or pentobarbital (n = 3) anesthesia showed the ACh source to lie in the IPN. We conclude that physiological responses to the chemically and pharmacologically diverse anesthetics halothane and ketamine, and probably also to pentobarbital, converge to enhance the output of the IPN.

The interpeduncular nucleus excites the on-cells and inhibits the off-cells of the nucleus raphe magnus.

The interpeduncular nucleus (IPN) was stimulated electrically while single-cell activity was recorded in the nucleus raphe magnus (NRM) of rats under pentobarbital anesthesia. Two classes of NRM cell were examined, those inhibited (off-cells) and those excited (on-cells) by noxious mechanical skin stimulation. Off-cells (92%) were found to be inhibited by IPN stimulation, whereas on-cells (50%) were excited. Based on previously suggested roles for the NRM neurons, we argue that both effects are hyperalgetic.

Coincident recording and stimulation of single and multiple neuronal activity with one extracellular microelectrode

This paper describes how an extracellular microelectrode may be used to stimulate neurons with brief, rectangular pulses and afterwards directly record the resultant activity. Two obstacles are the stimulus artifact lingering in the electrical circuitry and transient tip potentials (TTPs) arising from ion depletion at the electrode-tissue interface. Electronic switching between the stimulus source and the recording amplifier eliminates direct stimulus artifact from the electrical circuitry, although high but acceptable switching artifact remains. TTPs revert with time constants that are prominent in the desired recording (0.1-1 ms) and can reach 50 mV when more than 1 microA passes through a typical electrolyte-filled micropipette (for example 2-4 M omega, filled with 3 M NaCl, and placed in 0.1 M NaCl). They are always negative when cations flow into the tip, they are accompanied by a rise in microelectrode impedance, and they increase as a function of the resting electrode impedance, the duration and amplitude of applied current, and the dilution of the external electrolyte. TTPs were substracted by differential recording and stimulation through matched micropipettes (one in the brain and one in contiguous electrolyte) and in addition were reduced by pressure ejection of electrolyte. Directly elicited spikes (single or multiple) were detected about 0.5 ms after delivery of a rectangular stimulus pulse in the cerebellar cortex of pentobarbital-anesthetized rats. Typically, 3-4 units could be excited by less than 3 microA cathodal currents at any recording site. All-or-nothing properties, thresholds, and refractoriness to a second pulse within 2-4 ms verified the neuronal nature of the recorded signals. Complex wave forms, probably generated synaptically, were also seen. The technique of coincident extracellular recording and stimulation can be used as a universal search stimulus during microelectrode penetrations through the brain and in determining threshold-distance relations for extracellular stimulation. Where cell penetrations are unstable, it might be usefully substituted for intracellular technique in testing a neurons behavioral or physiological influences or in exploring a cell membranes response to drugs (in terms of excitability rather than voltage and impedance).

Responses of neurons in the ventromedial midbrain to noxious mechanical stimuli.

Neurons of the ventromedial midbrain in pentobarbital-anesthetized rats were examined by extracellular recording for responses to mechanical stimulation of the skin. Responses were absent from neurons clearly located in the interpeduncular nucleus (IPN) (n = 20), and from 92% of linear raphe (LR) neurons (n = 26). However, 37% of neurons in the ventral tegmental area of Tsai (VTA) (n = 38) and 63% of neurons in the small interfascicular nucleus (IF) (n = 9) were inhibited, often recovering with a delay of 1-2 min. A few cells (n = 4) were weakly excited in these 4 nuclei; none responded to innocuous mechanical stimulation of the skin. It is concluded that noxious cutaneous stimuli will not modify (by feedback) any influence of the IPN on pain perception, but could dampen behavior-reinforcing effects of the VTA and IF.

Evidence for rhythmic firing being caused by feedback inhibition in pinch-inhibited raphe magnus neurons

Raphe magnus cells that are inhibited by skin pinching fire spontaneously with strongly preferred interspike intervals (mean cycle 85 ms, n = 33). In pentobarbital-anesthetized rats, mid-cycle cathodal activation (0.3 ms) or end-cycle anodal black (30-60 ms) at approximately 1 Hz through the extracellular recording microelectrode delayed expected spikes; respective post-stimulus latencies peaked on average at 1.17 (n = 14) and 0.40 (n = 6) cycles. Feedback inhibition following random excitation, but not free-running intrinsic or afferent oscillations, may therefore cause the rhythm.