Localization of serotonin receptors in the rat thalamus by electrophysiology and the action of 5-HTP-DP-hex.

This study was designed to pinpoint the site at which N-hexanoyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP-hex) exerts its previously reported effect on thalamic neurons in rats. The animals were prepared under chloralose-urethane anesthesia for a stereotaxic approach to either the nucleus ventralis posterolateralis (nVPL) or the centrum medianum-parafascicularis complex (CM-Pf) of the thalamus. Individual neurons in these nuclei were separately activated by single-pulse stimulation of the sciatic nerve or the thalamic fibers that form reciprocal connections between the CM-Pf and the second somatosensory (SII) region of the nVPL. Poststimulus time histograms were constructed from computer readouts of the stimulus-evoked responses of a neuron during a 500-ms period accumulated in a digital computer 100X. In addition, the number of spikes accumulated in each histogram was compared to the number of spikes accumulated under identical conditions on the same neuron after intracarotid infusion of 5-HTP-DP-hex. The effect of the drug was reversed by the infusion of 5-HTP. Statistical evaluation of the accumulated spike counts indicated that 5-HTP-DP-hex suppressed only the excitation of CM-Pf neurons from the SII of the nVPL; the input of the sciatic nerve into the CM-Pf remained unaltered. Furthermore, no effect was exerted by this dipeptide on the afferent excitation of neurons in the SII of the nVPL.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of a serotonin receptor antagonist, 5-HTP-DP-hex, on spinal and thalamic nociceptive neurons in rats.

The antinociceptive properties of a new synthetic dipeptide (N-hexanoyl-5-hydroxytryptophyl-5-hydroxytryptophan amide, or 5-HTP-DP-hex) were studied in rats by an electrophysiological method. After an i.p. injection of alpha-chloralose and urethane, the animals were prepared for stereotaxic approach to the nucleus ventralis posterolateralis of the thalamus. With tungsten microelectrodes, individual nociceptive neurons in the nucleus were identified by the sequence of spikes emitted in response to single-pulse stimulation of the sciatic nerve. In addition to the usual short-latency spikes, a nociceptive neuron fired late spikes at regular intervals within 500 ms following each stimulus. When the spikes were accumulated in poststimulus time histograms, the short-latency spikes compiled an intensity-related (I) peak. The late spikes formed modality-related (M) peaks with spacing characteristic of nociception. Intracarotid infusion of 5-HTP-DP-hex (1 mg/kg) elevated the delayed portion of the I peak and the first M peak. This effect was followed in 25 min by suppression of all M peaks. The control record could be reinstated at any time by 5-hydroxytryptophan (3.5 mg/kg), or by natural recovery in 2.5 h. Responses evoked from a thalamic nociceptive neuron by single-pulse stimulation of the spinothalamic tract were modified by 5-HTP-DP-hex in a similar manner, except that no elevation of the activity peaks was observed. As shown previously, elevation of the delayed I peak and M1 indicated an increased input of A-delta and C fibers, respectively. The increased input lowers the response threshold and may represent hyperalgesia. Suppression of the M peaks may result from altered function of the positive feedback loop in the nociceptive system at the thalamic level, and may represent analgesia. Naloxone, methysergide, as well as ketanserin had no significant effect on the response histograms. These findings suggested that 5-HTP-DP-hex, a known serotonin receptor antagonist, targeted its action on very specific receptors, and thus interfered with particular synaptic activity within the spinal cord and on the thalamic level.

Stimulation of the periaqueductal gray subdues sensitized pain in morphine- and meperidine-dependent rats.

Because increased tolerance of narcotics is marked by progressive deactivation of the descending antinociceptive system, a question was raised whether stimulation of the periaqueductal gray matter (PAG) would have any electroanalgetic effect in animals adapted to increasing doses of narcotics. The daily dose of morphine (10 mg/kg) administered to rats was increased on alternate days by 10 mg/kg to 100 mg/kg/day. To another group, the daily dose of meperidine was increased from 15 mg/kg by 15 mg/kg to 150 mg/kg/day. Electrophysiological experiments were conducted under chloralose and urethane anesthesia 16 h after the last injection of morphine or meperidine. Spike potentials evoked from individual neurons of the nucleus ventralis posterolateralis by single-pulse stimulation of the sciatic nerve were accumulated in poststimulus time histograms. For nociceptive neurons the histograms were characterized by a short-latency activity peak and at least two late (270 and 420 ms) peaks. For non-nociceptive neurons the histograms had no late activity peaks. In control rats, stimulation of the PAG (400 ms at 70/s) prior to each sciatic nerve pulse reorganized the late activity peaks of the nociceptive neurons: a single late peak occurred during the 280 to 400 ms poststimulus interval, indicating suppression of pain by electroanalgesia. In rats adapted to morphine or meperidine, intracarotid infusion of naloxone lowered the nociceptive threshold. Stimulation of the PAG reorganized the late peaks but only if the sciatic nerve stimulation was not increased. At the voltage used to stimulate the sciatic nerve in control animals, two separate late peaks appeared, which were subdued by PAG stimulation after intracarotid infusion of 5-hydroxytryptophan (5-HTP). These results affirmed previous findings that electroanalgesia is induced by activity in an ascending and a descending pathway, both originating from the PAG. Since the function of the descending pathway is impaired by repeated administration of narcotics, only the pathway ascending to the somesthetic thalamus can be activated to mask pain, unless 5-HTP is injected. The latter renews the functional capacity of the descending pathway and thus reinstates the full capacity of electroanalgesia.

Functional changes in the descending antinociceptive system of morphine-dependent rats.

Functional changes of the descending antinociceptive system were studied in morphine-dependent rats by two types of experiments: (a) by recording the effects produced on nociceptive responses of the somesthetic thalamus by stimulation of the nucleus raphe magnus (NRM), and (b) by analyzing changes in the tonic activity of neurons in the periaqueductal gray matter (PAG). Poststimulus time histograms indicated that the input of A-alpha, A-delta, and C fibers into the spinal cord was conveyed via the spinothalamic tract (STT) to particular neurons of the nucleus ventralis posterolateralis (nVPL). Spikes of their responses were grouped in a short-latency burst followed by late spikes that occurred at the appropriate intervals for the arrival of the excitations originally initiated in the A-delta and C fibers. Only the histograms with late peaks were evaluated for the influence of the antinociceptive system. Stimulation of the NRM prior to stimulation of the sciatic nerve promptly suppressed the late A-delta and C-fiber) peaks in morphine-naive animals. In morphine-dependent rats, NRM stimulation had little or no effect on the histograms. 5-Hydroxytryptophan (5-HTP) had no effect on the nVPL neurons in morphine-naive rats, but in the morphine-dependent rats it renewed the ability of NRM stimulation to suppress the late activity. The tonic activity of the PAG neurons was significantly higher in morphine-dependent compared with that of the morphine-naive animals. Naloxone had a differential effect on the activity of the PAG neurons with regard to the two types of animals: it decreased the PAG activity drastically in the morphine-dependent rats. Apparently, the high tonic activity induced in the PAG by repeated administration of morphine acts on the descending antinociceptive fibers of the NRM and exhausts the synaptic transmitter substance, serotonin, in the NRM terminals, thus decreasing the ability of these terminals to block A-delta and C-fiber excitation of the STT. By assisting the synthesis of serotonin, 5-HTP renews the capacity of the NRM fibers to counteract STT excitation, and thus reinstates the normal function of the antinociceptive system.

Changes in thalamic nociception resulting from morphine- and meperidine-dependence in rats.

Rats were injected with progressively increasing doses of morphine or meperidine during a period of 3 to 40 days. From this colony of animals individual rats were used at 3- to 4-day intervals for electrophysiologic experiments to analyze the activity of nociceptive neurons in the somesthetic thalamus. After an i.p. injection of chloralose-urethane and the appropriate preparation for a stereotaxic microelectrode penetration of the thalamus, a nociceptive neuron was identified in the nucleus ventralis posterolateralis by its unique spacing of spike potentials emitted in response to pricking the foot with a pin. In addition to the short-latency response that formed a high activity peak on poststimulus time histograms, spikes following the stimulus up to 500 ms also formed activity peaks. Single-pulse stimulation of the sciatic nerve evoked the same response as pinpricks, but innocuous stimuli (pin shielded with a piece of cork) evoked a response without the late activity peaks. Only neurons that exhibited this differential response were regarded as nociceptive. Their response and spontaneous activity were accumulated separately on a digital computer. Following this, naloxone was infused i.v. and the computer accumulations were repeated. It was found that during naloxone-precipitated narcotic withdrawal, innocuous stimuli evoked responses indicative of pain; the nociceptive system was sensitized. Furthermore, a small dose or morphine or meperidine heightened the sensitization. This action of the narcotic agents was reversed by 5-hydroxytryptophan, which assisted the narcotics in suppressing pain in morphine- or meperidine-dependent rats but had no demonstrable effect in control animals. The spontaneous tonic activity of the nociceptive neurons of the somesthetic thalamus was high in rats exhibiting narcotic dependence. Naloxone decreased the count, but not to the value of the control animals. The sensitization of nociception can be explained by a decreased action of a neural pathway that descends from the periaqueductal gray matter via the nucleus raphe magnus to the spinal cord and there blocks the excitation of the spinothalamic tract cells by A-delta and C fibers. The mechanisms that increase the spontaneous activity of the thalamic nociceptive neurons remain unclear.

Interaction of neural systems which control nutritional balance.

The functional relationships of 5 nuclear masses of the cat brain were analyzed under conditions of general anesthesia by a combination of neurophysiological methods. The nuclear masses are known as (1) the area lateralis hypothalami (ALH), (2) the nucleus entopeduncularis (nEp), (3) the nucleus semilunaris accessorius (nSA), (4) the nucleus ventromedialis hypothalami (nVmH), and (5) the posterolateral portion of the zona incerta (ZI). Neurons of the ALH, nEp, and nVmH were tested for their responsiveness to electrical stimulation of the thalamic taste nucleus (nSA) and to intracarotid infusions of glucose and insulin. Following this, lesions were placed in some of the nuclei, and the responsiveness of the same neurons to glucose and insulin was re-evaluated. Additional experiments were performed to determine the influence of certain peripheral receptors on the 5 nuclear masses. Results indicated the following. (1) ALH-Ep neurons which are influenced by the nSA stimulation receive information about changes in plasma glucose from two sources: taste receptors and receptors located in the splanchnic area. The former are excited during the application of glucose to the tongue or with an increase in the plasma glucose concentration (intravascular taste); the latter, with a lowering of the plasma glucose level. (2) Destruction of the nSA leaves the ALH-Ep neurons unresponsive to glucose and insulin. (3) A negative feedback loop interrelates certain neurons of the ALH-Ep with the nVmH. (4) Interactions of the neurons of the 5 nuclear masses can account for the major events of the feeding cycle and for the specific hungers for some substances. Moreoever, the interactions can resolve several seemingly discordant experimental findings which have presented obstacles in previous attempts to link the control of nutrition with the availability of glucose to tissues.

Thalamic mechanisms that process a temporal pulse code for pain.

The sequential ordering of spikes emitted by single thalamic neurons which respond to noxious stimulation was studied using rats anesthetized with a mixture of alpha-chloralose and urethane. An electrical stimulus applied to the sciatic nerve contralateral to the thalamic recording site fired single thalamic SII neurons with a short latency spike burst and with long latency spikes which occurred at relatively fixed intervals. On repetition of stimulation, the short latency spike burst formed a high amplitude peak on sequential spike density histograms (I of Fig. 2B); the long latency spikes formed peaks of relatively low amplitude (M1, M2, M3 of Fig. 2B). Histograms of touch and light pressure relay neurons found within the thalamic SII differed conspicuously from that of Fig. 2B. Further experiments revealed that the I peak contained frequency coded information about the stimulus intensity, whereas the M peaks with their temporal relationship to the I peak coded information pertaining to a particular sensory modality. The M peaks are formed by timed firing in a positive feedback loop found between the thalamic SII and the nucleus centrum medianum-nucleus parafascicularis (CM-Pf) neurons. Consequently, the M peaks can be abolished without losing the I peak by a lesion placed in a portion of the CM-Pf complex or by the administration or morphine which is able to disorganize the timing mechanism of the feedback loop. Therefore, it is reasonably certain that the modality coded by the M peaks is pain.