Long-term amphetamine treatment decreases brain serotonin metabolism: implications for theories of schizophrenia.

Long-term amphetamine administration to cats (a mean of 8.75 milligrams per kilogram twice daily for 10 days) produced large decreases (40 to 67 percent in serotonin and its major metabolite, 5-hydroxyindoleacetic acid, in all brain regions examined. This treatment also produced several behaviors that are dependent on depressed central serotonergic neurotransmission, and which normally are elicited exclusively by hallucinogenic drugs. Short-term amphetamine administration (15 mg/kg) did not produce these behaviors and resulted in small decreases in brain serotonin and no change in 5-hydroxyindoleacetic acid. These data are discussed in the context of monoamine theories of schizophrenia.

Dissociations between the effects of LSD on behavior and raphe unit activity in freely moving cats.

The hypothesis that the action of hallucinogenic drugs is mediated by a depression of the activity of brain serotonergic (raphe) neurons was tested by examining the behavioral effects of d-lysergic acid diethylamide (LSD) while studying the activity of raphe neurons in freely moving cats. Although the results provide general support for the hypothesis, there were several important dissociations. (i) Low doses of LSD produced only small decreases in raphe unit activity but significant behavoiral changes; (ii) LSD-induced behavioral changes outlasted the depression of raphe unit activity; and (iii) raphe neurons were at least as responsive to LSD during tolerance as they were in the nontolerant condition.

An animal behavior model for studying the actions of LSD and related hallucinogens.

Cats injected with LSD (d-lysergic acid diethylamide) exhibit a group of behaviors that appear to be specific to hallucinogenic drugs. Two of these behaviors, limb flick and abortive grooming, have an extremely low frequency of occurrence in normal cats, but often dominate the behavior of LSD-treated cats. The frequency of occurrence of this group of behaviors is related to the dose of LSD. The behavioral changes are long-lasting following a single injection of LSD, and exhibit tolerance following the repeated administration of LSD. They are not elicited by a variety of control drugs, but are elicited by other indole nucleus hallucinogens. Because the behavioral effects are specific, reliable, easy to score, and quantifiable, they represent an animal model that can be used in studies of the effects of LSD and related hallucinogens.

Activity of dopamine-containing substantia nigra neurons in freely moving cats.

The present series of studies examined the activity of presumed dopamine-containing neurons in the substantia nigra of freely moving cats. These neurons were found to have a slow (1-9 spikes/sec) discharge rate, unusually long duration action potentials (2-4 msec) and frequently fired in bursts with progressive decreases in the amplitude of the action potential within the burst. These neurons showed no significant change in their activity across the sleep-waking cycle, and showed no changes in activity with phasic movement. Most units were unresponsive to olfactory, noxious, tactile, auditory and visual stimulation, when unit activity was integrated over several seconds following stimulus presentation. However, phasic auditory and visual stimuli produced a period of excitation lasting approximately 120 msec after a delay of about 80 msec. The period of excitation was followed by a period of inhibition lasting approximately 60 msec. Presumed dopamine-containing substantia nigra units showed no significant circadian changes in activity. The firing rates of these units were inhibited by dopamine agonists, including the direct-acting agonist, apomorphine, the dopamine precursor, L-dihydroxyphenylalanine, a dopamine releasing agent, d-amphetamine, and a dopamine reuptake blocker, bupropion, and were excited by a dopamine receptor blocker, haloperidol. Thus, these neurons show many similarities to dopamine units recorded in anesthetized rats; however, they showed several notable differences as well. Recording the activity of these units in behaving animals allows one to examine behavioral correlates of unit activity. Furthermore, the data (sensory stimulation, pharmacological, etc.) obtained in the unanesthetized preparation are far more relevant to the physiological and pharmacological effects that may occur in humans.

Effects of chronic methamphetamine administration on tryptophan hydroxylase activity, [3H]serotonin synaptosomal uptake, and serotonin metabolism in rat brain following systemic tryptophan loading.

Chronic administration of methamphetamine (20 mg/kg, i.p., every 12 hr for 6 days) produced significant decreases in the Vmax of brainstem (-32.8%) and forebrain (-31.5%) tryptophan hydroxylase when measured 12 hr after the final injection. Serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and [3H]5-HT synaptosomal uptake were decreased by a similar magnitude following chronic drug treatment. Administration of fluoxetine prior to each methamphetamine injection prevented these neurochemical changes. Neither acute nor chronic methamphetamine treatment produced any significant changes in the Km of tryptophan hydroxylase for either substrate or cofactor. Systemic tryptophan loading (50 mg/kg, i.p.) one hour prior to sacrifice in chronic methamphetamine treated rats restored brain 5-HT and 5-HIAA levels to normal. These data suggest that chronic methamphetamine treatment decreases central serotonergic neurotransmission by reducing the activity of the rate-limiting enzyme in 5-HT biosynthesis, possibly by disrupting serotonergic nerve terminals.

Buspirone decreases the activity of serotonin-containing neurons in the dorsal raphe in freely-moving cats.

Buspirone, a non-benzodiazepine anxiolytic agent, produced a dose-dependent decrease in the activity of serotonin-containing neurons in the dorsal raphe nucleus of freely-moving cats. The response ranged from no significant change at doses of 0.05 mg/kg to a nearly total suppression of activity at 1 mg/kg. These data suggest that the anxiolytic properties of buspirone may be mediated, at least part, by an action on neurons in the dorsal raphe.

Effects of chronic administration of D-amphetamine on PGO wave activity in the cat.

The effects of depletion of serotonin by the chronic administration of amphetamine (twice daily, 7.5 mg/kg per injection on days 1-6; 15 mg/kg per injection on the remaining days, i.p.) for 10-14 days on ponto-geniculo-occipital (PGO) waves was examined in cats. While the regimen of pretreatment with amphetamine produced comparable decreases in the content of central serotonin (i.e. 40-70%) to those observed in other studies (i.e. with reserpine, p-chlorophenylalanine, methiothepin) of ponto-geniculo-occipital wave activity, no ponto-geniculo-occipital waves of the rapid-eye-movement sleep type were observed during waking after chronic treatment with amphetamine. These results are discussed within the context of the "serotonin gating" hypothesis of the generation of ponto-geniculo-occipital waves, an hypothesis which states that ponto-geniculo-occipital waves emerge into waking when a critical level of the depletion of serotonin is reached, thus opening the "gate" for ponto-geniculo-occipital waves to enter waking states. The present authors suggest that a gradual depletion of serotonin, such as occurs following the chronic administration of amphetamine, will not elicit ponto-geniculo-occipital waves into waking, whereas a rapid depletion of serotonin, such as occurs following treatment with reserpine, results in the emergence of ponto-geniculo-occipital waves into walking.

Unit activity in the dorsal raphe in freely-moving cats. Effect of monoamine oxidase inhibitors.

Two commonly used monoamine oxidase inhibitors, tranylcypromine and pargyline, produced dose-dependent decreases in the activity of serotonin-containing neurons in the dorsal raphe in awake, freely-moving cats. The onset of the suppression of unit activity occurred within 15-20 min after administration of drug and persisted for 6-16 hr, depending upon dose. Parallel neurochemical studies revealed that serotonin in the brain was significantly increased following inhibition of monoamine oxidase, and that concentrations of serotonin were still significantly elevated after unit activity in the raphe had returned to baseline levels. These data suggest that autoreceptors on neurons of the dorsal raphe may become tolerant following prolonged exposure to large concentrations of serotonin.

Raphe unit activity in freely moving cats: effects of quipazine.

Quipazine produced a dose-dependent decrease in the discharge rate of serotonin-containing neurons in the dorsal raphe nucleus of freely-moving cats. This ranged from a 10% decrease at 0.5 mg/kg, (i.p.), to a virtually complete depression of activity at 5.0 mg/kg. The effects of quipazine on raphe units occurred with a short latency (5--10 min) and its duration of action was dose-dependent and lasted from 1 to 6 hr. The degree of depression of raphe unit activity was directly related to the frequency of occurrence of a number of behaviors such as limb flicking and abortive grooming. There was a close temporal correlation between the depression of raphe unit activity and the occurrence of these behaviors. These data reveal that quipazine produces behavioral and raphe unit changes similar to those observed after administration of hallucinogens with an indole nucleus.

Raphe unit activity in freely moving cats: effects of benzodiazepines.

Benzodiazepines (chlordiazepoxide and diazepam) produced a dose-dependent decrease in the discharge rate of serotonin-containing neurons in the dorsal raphe nucleus of freely moving cats. This ranged from no significant change at doses of 0.5 and 1.0 mg/kg (i.p.), to greater than 90% reductions in unit activity at 10 mg/kg. The effects of benzodiazepines on raphe units occurred within 15-30 min of injection and the duration of action was dose-dependent and lasted from 1 to more than 6 hr. Doses of benzodiazepines that significantly decreased raphe unit activity (i.e. 2.5-10 mg/kg) also produced ataxia and decreased EMG activity. These data suggest that benzodiazepine-induced suppression of raphe unit activity is closely related to general motor behavior. Raphe unit activity remained suppressed during phasic increases in EMG activity during eating, grooming, or predatory behavior, suggesting that benzodiazepines also have a direct inhibitory action on raphe cells. The present results are discussed in the context of the serotonergic hypothesis of anxiety.

Electrophysiological properties of mouse dopamine neurons: in vivo and in vitro studies.

The present experiments were conducted to determine the electrophysiological and pharmacological properties of substantia nigra neurons in the mouse. These cells were studied using extracellular single unit recording and microiontophoretic techniques in both chloral hydrate anesthetized mice and in vitro mouse slices. In the in vivo preparation the substantia nigra zona compacta neurons had long duration action potentials (greater than 4 ms), fired from 1 to 7 impulses/s, and the cells discharged with either a decremental burst pattern or with a regular pattern. The dopamine agonists apomorphine and d-amphetamine, given systemically, decreased the firing rate of these neurons and the dopamine receptor blocker, haloperidol, reversed these effects. The zona compacta neurons were inhibited by the micro-iontophoretic application of dopamine and gamma-aminobutyric acid, and systemic haloperidol selectively attenuated the effects of dopamine. In vitro recordings from substantia nigra zona compacta and zona reticulata neurons were generally similar to those found in vivo, both in terms of the electrophysiological and pharmacological properties. However, the zona compacta cells fired faster in vitro than in vivo, and the firing pattern in vitro tended to be pacemaker-like, especially when recordings were made in an incubation medium which blocks synaptic transmission (e.g. low Ca2+/high Mg2+). Our data indicate that: (a) in vivo mouse zona compacta neurons exhibit the same electrophysiological and pharmacological properties as rat dopamine-containing neurons; (b) in vitro mouse dopaminergic neurons fire with pacemaker regularity when in a low Ca2+/high Mg2+ environment; and (c) in vitro studies offer an approach to examine the basic properties of dopaminergic neurons exclusive of feedback pathways and other afferent inputs.

Effects of diazepam on behavior and dopamine-containing substantia nigra units in freely moving cats.

Administration of diazepam at doses that produced ataxia (5-10 mg/kg IP) significantly decreased the discharge rate of substantia-nigral dopamine-containing neurons by 28.1% in freely moving cats. Diazepam also altered the characteristic decremental bursting pattern of these neurons, producing a steady rhythmic discharge pattern. Similar results were obtained with another centrally acting muscle-relaxant drug, mephenesin. However, the peripherally acting muscle relaxant dantrolene did not produce these effects. These data suggest that diazepam and other centrally acting muscle relaxants may produce their effects on motoric behavior in part by altering the dopaminergic input to the striatum.

Activity of a non-hallucinogenic ergoline derivative, lisuride, in an animal behavior model for hallucinogens.

The behavioral effects of IP administration of lisuride, a non-hallucinogenic iso-lysergic acid amide analog structurally related to d-lysergic acid diethylamide (LSD), were examined in 15 cats. Ten animals were given saline or 6.25, 12.5, 25, 50, or 100 micrograms/kg of lisuride and observed for 1 h by a rater blind to dose. There was a statistically significant effect of lisuride dose on the frequency of occurrence of the behaviors limb flicking, grooming, and abortive grooming. A time-course study with five cats at the most effective lisuride dose, 50 micrograms/kg, revealed that the frequencies of occurrence of these behaviors reached a maximum during the first 2 h post dose, and were comparable to frequencies after saline by 6 h post dose. An acute tolerance study with four cats scored for 90 min post dose revealed no significant tolerance to a 50 micrograms/kg lisuride test dose administered 6, 24, or 72 h after an initial 50 micrograms/kg dose. Acute cross tolerance studies with four cats scored for 90 min after an initial dose of 50 micrograms/kg of LSD or of lisuride, followed 24 h later by 50 micrograms/kg of lisuride or LSD, revealed no significant cross tolerance. The potency of lisuride relative to LSD was evaluated in six cats that were scored for 60 min following 25 and 50 micrograms/kg of LSD and of lisuride. On a molar basis, scores after lisuride were 51% and 67% those after LSD for limb flicking and grooming. These results indicate that lisuride, a non-hallucinogenic iso-lysergic acid derivative, is a false positive in the animal behavior model for hallucinogens.

Raphe unit activity in freely moving cats: correlation with level of behavioral arousal.

Dorsal raphe unit activity in freely moving cats showed a slow, rhythmic discharge rate during quiet waking (X=2.82 +/- 0.17 spikes/sec), and displayed a strong positive correlation with level of behavioral arousal. Presentation of an auditory stimulus during quiet waking resulted in significant increases in unit activity of 112% and 39% during the first sec and first 10 sec after the stimulus, respectively. This effect rapidly habituated with repeated stimulus presentations. During active waking, unit activity was significantly increased by 22% as compared to quiet waking, but there was no correlation between unit activity and gross body movements. Raphe unit activity showed a significant decrease of 17% during drowsiness (first appearance of EEG synchronization) as compared to quiet waking, and then progressive decreases during the early (--34%), middle (--52%) and late (--68%) phases of slow wave sleep. During all phases of slow wave sleep, the occurrence of sleep spindles was frequently associated with a transitory decrease in unit activity. The discharge rate would typically decrease during the few seconds immediately preceding the spindle, remains at this low level during the occurrence of the spindle, and then increase immediately after the spindle. Raphe unit activity showed decreases of 81% during pre-REM (the 60 sec immediately before REM onset) and 98% during REM, as compared to quiet waking. Unit activity reappeared 3.2 sec before the end of REM, with significant increases in unit activity of 83% and 17% during the first sec and first 10 sec of unit activity, respectively, as compared to quiet waking. The results of these studies are discussed in relation to the hypothesis that serotonin may play a modulatory, rather than mediative, role in behavioral and physiological processes.

Usefulness of an animal behavioral model in studying the duration of action of LSD and the onset and duration of tolerance to LSD in the cat.

LSD elicits a number of emergent behaviors in the cat, including limb flicking, abortive grooming, investigatory and hallucinatory-like behaviors, which we have proposed as an animal behavior model for studying the actions of LSD and related hallucinogens. These emergent behaviors were used in the present study to investigate the duration of action of LSD, as well as the onset and duration of tolerance. A dose of 10 microgram/kg of LSD produced significant behavioral changes for up to 4h, while a dose or 50 microgram/kg produced changes lasting for at least 8 h. Tolerance to a test dose of 50 microgram/kg of LSD is virtually complete one day after a single 50 microgram/kg dose, and lasts for approximately 5 days. Tolerance to a test dose of 50 microgram/kh of LSD one day after a single dose of 10 microgram/kg is quite marked, and lasts for approximately 3 days. A significant tolerance to a test dose of 50 microgram/kg of LSD occurs within 2 h after a single injection of 10 microgram/kg. The limb flick was found to be the most sensitive index in all tests: it showed the longest time-course, as well as the most rapid and longest-lasting tolerance. These studies demonstrate that the LSD-induced behavioral syndrome in the cat parallels important parameters of the action of LSD in humans, and thus enhances the usefulness of the model.

Activity of nucleus raphe pallidus neurons across the sleep-waking cycle in freely moving cats.

The activity of serotonin-containing nucleus raphe pallidus (RPA) units was recorded by means of movable 32 or 62 microns diameter insulated nichrome wires in freely moving cats. RPA units displayed a slow, rhythmic discharge rate during waking (mean = 5.3 spikes/s) and showed no significant change in activity during slow-wave sleep. However, these neurons showed a large decrease in activity during REM sleep (mean = 1.2 spikes/s). In contrast with results from studies on serotonin-containing nucleus raphe dorsalis units, RPA neurons showed no relationship to the occurrence of sleep spindles, and were not inhibited by low doses of LSD.

Dissociations between the effects of hallucinogenic drugs on behavior and raphe unit activity in freely moving cats.

The hypothesis that the action of hallucinogenic drugs is mediated by a depression of the activity of brain serotonergic (raphe) neurons was tested by examining the behavioral effects of several hallucinogenic drugs while concurrently monitoring the activity of raphe neurons in freely moving cats. LSD produced a dose-dependent decrease in raphe unit activity and a dose-dependent increase in certain behaviors (e.g. limb flick and abortive groom), and the peak of the behavioral and unit changes were temporally correlated. However, there were three important dissociations between the behavioral and electrophysiological effects of LSD. Firstly, low doses of LSD produced only small decreases in raphe unit activity but significant behavioral changes. Secondly, the duration of LSD-induced behavioral changes significantly outlasted the depression of raphe unit activity. And thirdly, raphe neurons were at least as responsive to LSD during tolerance as they were in the nontolerant condition. Psilocin produced a dose-dependent decrease in raphe unit activity, while the behavioral changes were not dose-related. However, the peak behavioral changes corresponded to the maximal depression of raphe unit activity. The phenylethylamine hallucinogens, DOM and mescaline, both produced large behavioral changes but no overall effect on raphe neurons. Following administration of DOM or mescaline, some raphe units showed a significant increase, while some showed a significant decrease, and others showed no change in activity. Therefore, the phenylethylamine hallucinogens may exert a depressant effect upon a subset of serotonin-containing neurons, and an amphetamine-like excitatory effect upon another subset of these neurons. Consistent with previous studies, all hallucinogens produced a high concentration of slow waves in the cortical EEG. Following administration of LSD or psilocin, the appearance of slow waves in the EEG was often associated with a transitory decrease in unit activity, while this was not observed for the phenylethylamine hallucinogens. The present data, in conjunction with recent data from other laboratories, suggest that the serotonin hypothesis of hallucinogenic drug action should be re-evaluated.

Raphe unit activity in freely moving cats: effects of phasic auditory and visual stimuli.

The effects of phasic auditory or visual stimuli upon the single unit activity of serotonergic neurons within the dorsal raphe nucleus (DRN) were studied in freely moving cats. The predominant response to auditory stimulation (86% of the cells) was excitation, with a mean latency of 40 +/- 3 ms (S.E.M.) and a mean duration of 64 +/- 4 ms. This was typically followed by a longer period (206 +/- 32 ms) with unit activity below the baseline level. This did not appear to be a stimulus-induced inhibition of unit activity, however, since its duration closely corresponded to the normal interspike interval for that particular neuron. The response to repetitive auditory stimulation showed no evidence of habituation and was even present during sleep. A similar response, although generally of lesser magnitude, was evoked by a phasic visual stimulation in 64% of the cells tested. The mean latency for the response to visual stimulation was 53 +/- 4 ms, the mean duration of excitation was 76 +/- 7 ms, and the mean duration of the subsequent suppressed period was 239 +/- 37 ms. The response to the visual stimulus also showed no evidence of habituation. These data indicate that serotonergic neurons of the DRN are driven, with similar temporal characteristics, by stimuli in two different sensory modalities. We hypothesize that these similar effects are attributable to a common excitatory input.

Raphe unit activity during REM sleep in normal cats and in pontine lesioned cats displaying REM sleep without atonia.

Previous studies have shown that the activity of serotonin-containing raphe neurons in cats is almost completely suppressed during rapid eye movement (REM) sleep. However, since raphe unit activity is known to be grossly correlated with the level of behavioral arousal or tonic motor activity, this decrease in activity during REM sleep may be simply due to the fact that tonic EMG activity or motoric output is at a minimum. On the other hand, raphe unit activity may be related to the state (i.e. REM sleep) of the organism. To test these competing hypotheses, in the present study we compared raphe unit activity in normal cats with that in cats that display REM sleep without atonia (produced by bilateral lesions of the pontine tegmentum). These lesioned cats manifest episodes which, by all criteria, appear to be REM sleep except that they display overt behavior, presumably because the mechanism normally responsible for producing atonia has been disrupted. Although the activity of raphe neurons in lesioned cats during REM sleep without atonia was significantly below that seen in these cats during waking, the level of activity was often impressive. This is especially true when those animals that displayed the greatest degree of tonic motor activity during REM sleep (group IV animals) are considered separately. In these cats, the depression was only 40.5% below their quiet waking level, whereas in lesioned cats displaying less tonic motor activity (Group II animals), raphe discharge rate was 65.6% below their quiet waking level. The discharge rate of raphe neurons during REM sleep in lesioned cats was more than 6-fold greater than that seen in normal animals. These data, in conjunction with other recent results from our laboratory, suggest that the decrease in raphe unit activity during REM sleep is largely a concomitant of the atonia which characterizes that state. These data are discussed within the general context of the relationship between raphe unit discharge and the activity of central motor systems.

A metabolic profile of the rat caudate microvasculature: a histochemical study.

Arterioles of the rat caudate nucleus were examined histochemically to determine their metabolic profile. These microvessels appear capable of aerobic and anaerobic metabolism with a potential for nucleic acid and protein synthesis. Little intramural lipid storage occurs and any fatty acids utilized are provided via the blood supply. Likewise, glycogen is not seen in the arteriolar wall and may be rapidly turned over as a substrate for anaerobic metabolism.