Spatial and temporal patterns of serotonin release in the rat's lumbar spinal cord following electrical stimulation of the nucleus raphe magnus.

The monoamine neurotransmitter serotonin is released from spinal terminals of nucleus raphe magnus (NRM) neurons and important in sensory and motor control, but its pattern of release has remained unclear. Serotonin was measured by the high-resolution method of fast cyclic voltammetry (2 Hz) with carbon-fiber microelectrodes in lumbar segments (L3-L6) of halothane-anesthetized rats during electrical stimulation of the NRM. Because sites of serotonin release are often histologically remote from membrane transporters and receptors, rapid emergence into aggregate extracellular space was expected. Increased monoamine oxidation currents were found in 94% of trials of 50-Hz, 20-s NRM stimulation across all laminae. The estimated peak serotonin concentration averaged 37.8 nM (maximum 287 nM), and was greater in dorsal and ventral laminae (I-III and VIII-IX) than in intermediate laminae (IV-VI). When measured near NRM-evoked changes, basal monoamine levels (relative to dorsal white matter) were highest in intermediate laminae, while changes in norepinephrine level produced by locus ceruleus (LC) stimulation were lowest in laminae II/III and VII. The NRM-evoked monoamine peak was linearly proportional to stimulus frequency (10-100 Hz). The peak often occurred before the stimulus ended (mean 15.6 s at 50 Hz, range 4-35 s) regardless of frequency, suggesting that release per impulse was constant during the rise but fell later. The latency from stimulus onset to electrochemical signal detection (mean 4.2 s, range 1-23 s) was inversely correlated with peak amplitude and directly correlated with time-to-peak. Quantitative modeling suggested that shorter latencies mostly reflected the time below detection threshold (5-10 nM), so that extrasynaptic serotonin was significantly elevated well within 1 s. Longer latencies (>5 s), which were confined to intermediate laminae, appeared mainly to be due to diffusion from distant sources. In conclusion, except possibly in intermediate laminae, serotonergic volume transmission is a significant mode of spinal control by the NRM.

Spinal allografts of adrenal medulla block nociceptive facilitation in the dorsal horn.

Transplantation of chromaffin cells into the lumbar subarachnoid space has been found to produce analgesia, most conspicuously against chronic neuropathic pain. To ascertain the neurophysiological mechanism, we recorded electrical activity from wide-dynamic-range dorsal horn neurons in vivo, measuring the short-lasting homosynaptic facilitatory effect known as windup, which is induced by repetitive C-fiber input. Rats were given adrenal medulla allografts, or, as controls, striated-muscle allografts. The adrenal-transplanted rats showed analgesia 3--4 wk after transplantation, measured as a reduction in flinching reflexes 30--55 min after subcutaneous formalin injection. Recordings were made under halothane anesthesia, 3--7 days following the behavioral testing. The average C-fiber response and subsequent afterdischarge were facilitated severalfold in control rats by 1-Hz cutaneous electrical stimulation. Such facilitation was essentially absent in adrenal-transplanted animals and also in the A-fiber response of both preparations. Extirpation of transplanted tissue several hours prior to recording did not significantly affect this difference. In conclusion, the adrenal transplants block short-term spinal nociceptive facilitation, probably by stimulating some persistent cellular process that may be an important determinant, but not the only one, of their analgesic effect.

Spinal CSF from rats with painful peripheral neuropathy evokes catecholamine release from chromaffin cells in vitro.

The environment presented by host tissue may influence cellular transplants in the CNS depending on injury or disease. Here we examined whether chronic pain alters cerebrospinal fluid (CSF), thereby enhancing the analgesic effect of transplanted adrenal cells. CSF samples were taken intracisternally from rats with neuropathic pain induced by chronic constriction injury of the sciatic nerve. The samples were applied to cultured bovine chromaffin-cell clusters while catecholamine release was measured by fast cyclic voltammetry. This caused marked and sustained elevations in catecholamine levels, compared to CSF from sham-operated controls, which were reversible by the nicotinic antagonist mecamylamine. These results suggest that chronic neuropathic pain produces increased CSF levels of secretogogues for chromaffin cells, and illustrates the importance of host microenvironmental factors in determining graft function.

Interactions between brainstem and trigeminal neurons detected by cross-spectral analysis.

Cells in the nucleus raphe magnus that are inhibited by noxious skin stimuli (off-cells) have been postulated to suppress pain by continuously inhibiting spinal and trigeminal nociceptive neurons. To test this hypothesis, spontaneous activity was simultaneously recorded from off-cells (n = 15) and wide-dynamic range cells (n = 27) of the trigeminal complex (subnucleus interpolaris), in rats anesthetized with pentobarbital. Most off-cells (n = 14) had rhythmic interspike intervals, their modes averaging 106 ms. No trigeminal cell fired rhythmically. Rhythmic firing was defined quantitatively: the autospectrum's peak power had to exceed 1.75 times its asymptote. This formula was obtained by generalizing from a natural cut-off in the theoretical autospectrum for serially uncorrelated, gamma-distributed intervals, whose firing can be varied from Poissonian to highly regular by adjusting one parameter. It encompasses the qualitative judgement of autocorrelograms commonly made in neurophysiology. Cross-correlograms (n = 29) appeared noisy and otherwise featureless. However, their power spectra (cross-periodograms) sometimes showed significant peaks, compared with simulated non-interactive distributions. The latter were generated by interchanging the raphe interval sequences at one random point (as in cutting a deck of cards), thus retaining most of their serial correlation. Of 29 cross-periodograms, 21 were significant at 1 to 13 frequencies (100 points, 0.4 to 39 Hz). These frequencies were often near the peak raphe power, and sometimes near its harmonics too. Furthermore, cross-spectral phase angles at peak power were non-uniform, most falling between 0 and 180 degrees (unit vector sum 60 degrees, n = 20). To understand why the frequency domain gave better detection, cross-spectra and cross-correlations were modeled theoretically by convolving idealized input autocorrelations and synaptic response functions. This demonstrated that rhythmic firing is insufficient for better frequency-domain detection, and that serially correlated input intervals or non-additive synaptic responses are necessary. The conclusion was confirmed by stochastic simulation of a simple non-additive synapse, that required successful input spikes to fall within a specified interval of the preceding spike. Experimentally, serial correlation was found in 12 of the 15 raphe cells, and in 20 of the 27 trigeminal cells. It is proposed that the weak experimental cross-correlograms arise because many asynchronous raphe inputs converge on each trigeminal cell, possibly to optimize the resting suppression of pain. The distribution of cross-spectral phase angles at peak raphe power suggested that raphe spikes arriving at the synapses' preferred interval cause a fall in trigeminal activity. In general, cross-spectral analysis can sometimes uncover influences hidden in cross-correlograms, but the firing of one neuron must be rhythmic and non-renewal, or else certain input intervals must be favored in synaptic transmission.

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.

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.

The interpeduncular nucleus regulates nicotine's effects on free-field activity.

Partial lesions were made with kainic acid in the interpeduncular nucleus of the ventral midbrain of the rat. Compared with sham-operated controls, lesions significantly (p < 0.25) blunted the early (<60 min) free-field locomotor hypoactivity caused by nicotine (0.5 mg kg(-1), i.m.), enhanced the later (60-120 min) nicotine-induced hyperactivity, and raised spontaneous nocturnal activity. Lesions reduced the extent of immunohistological staining for choline acetyltransferase in the interpeduncular nucleus (p <0.025), but not for tyrosine hydroxylase in the surrounding catecholaminergic A10 region. We conclude that the interpeduncular nucleus mediates nicotinic depression of locomotor activity and dampens nicotinic arousal mechanisms located elsewhere in the brain.

Excitation of cells in the rostral medial medulla of the rat by the nitric oxide-cyclic guanosine monophosphate messenger system.

Analgesia has been reported to be facilitated by supraspinal nitric oxide (NO) and cyclic guanosine monophosphate (cGMP). In the rostromedial medulla, an important pain-suppressing region, iontophoretically delivered 8-bromo-cGMP excited most single recorded cells (9/10), and methylene blue (a guanylyl cyclase inhibitor) inhibited all cells (7/7). Nitrite and ferrous ions together, shown voltammetrically ex vivo to yield nitric oxide (NO), excited some cells (14/28) and inhibited others (7/28). Methylene blue blocked excitation (3/3) but not inhibition (4/4) by the putative NO. Spontaneous or glutamate-evoked firing was gradually inhibited (23/32) or unaffected by N omega-nitro-L-arginine (a NO synthase inhibitor), but was mostly inhibited by L-arginine (the NO precursor) (23/26), although a rapid onset militated against elevated NO production. These substances, excepting L-arginine, produced changes consistent with an excitatory cGMP-NO cascade contributing to analgesia.

Observations on field potentials at their point of generation.

A technique for evoking then recording field potentials through one extracellular electrode was studied in the dentate gyrus of pentobarbital-anesthetized rats. In the molecular layer (the location of granule cell dendrites), a -5 microA pulse (0.4 ms, 0.2 Hz) consistently elicited a 'focal' response the major component of which was a negative-going wave of about 1 ms latency, 10 ms duration, and -0.8 to -1.5 mV amplitude. This wave resembled, and could partially occlude, field excitatory post-synaptic potentials (EPSPs) elicited electrically from the perforant path. It fatigued during high-frequency stimulation and is suggested to consist largely of granule-cell EPSPs produced by directly activated, perforant-path terminals. Focal and perforant-path tetanic stimulation led to stable potentiation of the focal negative phase. Stimulus-response curves for the negative phase were roughly linear over most or all of the stimulus range of -1 to -5 microA, but on a finer scale were serrated and irregular. After a tetanus, different stimulus-response curves showed parallel leftward shifts or slope changes along all or part of their range, implying multiple mechanisms of potentiation that might include both threshold and amplification changes. Several uses are suggested in the paper for focal recording of compound potentials in research and diagnosis.

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)

Nicotinic activity in the interpeduncular nucleus of the midbrain prolongs recovery from halothane anesthesia.

The influence of nicotinic transmission in the interpeduncular nucleus of the ventral midbrain on recovery from general anesthesia (3% halothane in oxygen) was assessed in rats. Immediately upon withdrawal of the anesthetic, nicotine (2 microliters, 10(-5) to 10(-1) M) was injected into the interpeduncular nucleus. Larger doses of nicotine (10(-2) and 10(-1) M) significantly (P < 0.05) prolonged the recovery of righting reflexes (to 371 +/- 55 and 362 +/- 67 sec, respectively, mean +/- SE), compared with injection of saline (187 +/- 19 sec). Prior intramuscular administration of the nicotinic antagonist, mecamylamine (2 mg/kg) significantly reduced the effect of 10(-2) M nicotine (to 211 +/- 43 sec). Injection of the nicotinic antagonist, hexamethonium (10(-1) M) led to a low mean recovery time (181 +/- 21 sec), not significantly different from control. Prolongation of recovery by 10(-2) M nicotine was not found to be significant when sites more dorsal to the interpeduncular nucleus were injected. An observed tendency for injection of nicotine to slow the post-anesthesia rate of breathing was not statistically significant and not correlated anatomically with the injection site in the midbrain. Increased release of acetylcholine has been shown previously to occur in the interpeduncular nucleus during anesthesia. The present results suggest that nicotinic activation of the interpeduncular nucleus facilitates or sums with the mechanisms in the brain that produce anesthesia under halothane.

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 neuron's behavioral or physiological influences or in exploring a cell membrane's response to drugs (in terms of excitability rather than voltage and impedance).

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 midbrain's 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.

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.

Spatial and temporal variation of microstimulation thresholds for inhibiting the tail-flick reflex from the rat's rostral medial medulla.

Suppression of the tail-flick reflex by microstimulation of the rostral medial medulla in rats lightly anesthetized with barbiturates was studied with regard to spatial and temporal variations in electrical threshold. Trains of constant-current pulses with linearly descending amplitudes (called 'ramps') were passed through the extracellular brain microelectrode during noxious heating of the tail. The pulse amplitude at the time of the reflex, after allowance for conduction and reaction latencies, was taken as the threshold reading. This new method revealed a range of vertical electrode positions corresponding roughly to the nucleus raphe magnus, where the thresholds tended to be lowest (a mean of 4.1 microA for 0.4-ms pulses delivered at 50 Hz). In confirmation of the technique's validity neither the duration of the ramp nor its starting amplitude, within their useful range, significantly affected the measured threshold. Pronounced temporal fluctuation was seen in thresholds measured every 2 min. Spatial variability within the low-threshold region and differences between preparations were statistically much smaller sources of variation. The temporal fluctuation appeared to have a stationary mean for at least 20 min under constant conditions of anesthesia. In some experiments, action potentials from single neurons were recorded through the stimulating electrode, and classified into those inhibited during the tail-flick (off-cells), those excited (on-cells), and those unaffected (neutral cells). The thresholds where off-cells exhibited their maximum action potential were on average significantly lower than corresponding thresholds for on-cells. Short-range (less than 0.2 mm) spatial variations in the threshold appeared however to be uncorrelated with the distance to an individual recorded off-cell or on-cell.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

A theoretical analysis of extracellular punctate stimulation around dendrites.

The polarization of neuronal trees by external point stimulation is modelled. In one form of model, an almost spheroidal field encloses the dendritic tree. Radially projecting, electrically linear dendrites, along with extracellular medium, are considered to occupy the entire field. The spheroid is modified by a penetrating cone that can surround the stimulating microelectrode; here, and in the rest of the infinite volume outside the field, there is only extracellular medium. A second form of linear electrical model, representing sections of membrane and cytoplasm by means of lumped electrical components commonly known as compartments, is used to validate the field construct. A similar spatial distribution of induced steady-state membrane potential emerges from the two forms of model, for a given morphology and electrophysiology. Compartmental models are also used to demonstrate time-courses of membrane charging. At the soma, if the point source is nearby, charging proves to be essentially complete in less than one time-constant. The soma thresholds under steady-state polarization from different electrode distances are plotted for field models of various electrical space-constant, size and shape of spheroid, and eccentricity of the soma. Characteristic cathodal or anodal thresholds, depending on the particular cell parameters, are revealed for specific electrode trajectories. The range of threshold-distance relations obtained in previously published experiments match those given by the models, when the time-course of charging is taken into account.

Simulating stimulating bulges: the electrical meddling of neurons.

The steady-state cable equations were used to describe the response of discontinuous neuronal cylinders to extracellular electric fields. Where two unequal diameters join, there forms a strong local peak of membrane polarization. Under fields of constant gradient, this peak roughly equals the drop in external voltage over the distance given by the larger length-constant. In contrast, altering the specific membrane resistance along part of a uniformly shaped axon affects the general level of membrane potential without causing a local peak. Field-effects due to structural discontinuities, although relevant to the interpretation of experiments using electrical stimulation, are shown to be of minor functional consequence compared to chemical gating of membrane current.

Simulating stimulating bulges: the electrical meddling of neurons

El efecto de campos externos de electricida sobre cilindros neuronales fue estudiado por medio de ecuaciones de cables en estado estacionario. En el punto de unión entre dos cilindros con diámetros distintos, el potencial de membrana alcanza un valor máximo. Bajo un campo con un declive constante de voltaje, la cima es aproximadamente igual a la caída del potencial exterior a lo largo de la distancia dada por la mayor constante de espacio. Cuando hay cambios en la resistencia específica de una sección de la membrana en una axona sencilla, surge un nuevo nivel de polarización general, en vez de la cima. Estos efectos de los campos eléctricos en el funcionamiento normal del sistema nervioso son despreciables comparados con la activación química de conductancias membranales. Sin embargo, estas propiedades deben ser consideradas al interpretar los resultados de la estimulación eléctrica