A serotonergic, midbrain-raphe model of tonic immobility

Biobehavioral Reviews, Vol. 1, pp. 35-43, 1977. Copyright © ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.

A Serotonergic, Midbrain-Raphe Model of Tonic Immobility L A R R Y B. WALLNAU AND GORDON G. GALLUP, JR. l

Department o f Psychology, State University o f New York at Albany (Received 19 June 1976)

WALLNAU, L. B. AND G. G. GALLUP, JR. A serotonergic, midbrain-raphe model of tonic immobility. BIOBEHAV. REV. 1(1) 35-43, 1977. - Research involving the effects of serotonergic and other pharmacological manipulations on tonic immobility is reviewed. An attempt is made to show how these findings parallel independent research involving precursor availability and drug-induced changes in the electrical activity of raphe neurons. Central serotonergic neuronal mechanisms appear to play a crucial role in modulating tonic immobility. Tryptophan Serotonin Sleep Acetylcholine Catecholamines Tonic immobility Animal hypnosis Efferent inhibition

Raphe nuclei

Catatonia

RATIONALE

TONIC IMMOBILITY (TI), also known as animal hypnosis, is reliably elicited by a brief period of physical restraint. Under conditions of manual restraint, which is the typical laboratory procedure, one simply holds the animal down on a flat surface. The animal reacts by struggling and trying to escape, but after a few seconds these frantic reactions subside and it assumes a motionless, catatonic-like posture which now persists in the absence of further restraint (see Fig. 1). The response is characterized by inhibition of movement, intermittent eye closure, hypertonicity of skeletal muscles, tremors of the extremities, waxy flexibility, mydriasis, and decreased vocalization. Physiological changes occurring during TI are varied and include altered EEG patterns, changes in respiration, heart rate, and core temperature (e.g., [ 50,65 ] ). Tonic immobility has been demonstrated in animals from such diverse groups as insects, fish, amphibians, reptiles, avians, and mammals [36]. Domestic fowl are often studied because they display a pronounced immobility response that is readily induced and easily quantifiable. Response durations in naive chickens may last from a few seconds to over several hours. Tonic immobility has proven especially sensitive to manipulations designed to affect fear, and has been conceptualized as the terminal defensive reaction in a sequential series of distant-dependent predator defenses [36,69]. Tonic immobility has also been proposed as a laboratory model of catatonic schizophrenia [37].

The published literature on TI dates back to 1636, and since that time at least 800 reports have appeared in print [57]. Over the years TI even attracted the attention of such notable figures as Darwin and Pavlov. Among several theories [36], a recurring interpretation has been that it may represent a sleep state [19,78]. To view TI as a form of sleep is consistent with the obvious absence of movement. Eye closure during TI also often gives the impression of sleep, and the amount of eye closure is positively correlated with the duration of the immobility episode [40]. In terms of psychopharmacology it has been known for some time that the putative neurotransmitter and indolamine, serotonin (5-HT, 5-hydroxytryptamine) is related to sleep [49], and our attention was drawn to serotonin as a possible neurochemical mediator of TI for a number of reasons. Drugs thought to affect central serotonergic systems lead to profound changes in tonic immobility (e.g., [39, 42, 45, 58]). In a broader sense, a number of investigators have suggested that 5-HT may participate in behavioral inhibition. Margules and Margules [55] proposed that serotonergic mechanisms cause a general inhibition of operant behavior, as, for example, freezing in a fear provoking situation. Serotonergic neurons have also been implicated in the development of passive avoidance responses which are prompted by punishment [76,85]. Evidence for serotoner-

1We thank E. P. Riley for comments on an earlier version of this paper. Send reprint requests to G. G. Gallup, Jr., Department of Psychology, State University of New York at Albany, Albany, NY 12222. 35

36

WALLNAU AND GALLUP

FIG. l. A three and one-half week old Production Red chicken exhibiting the tonic immobility reaction.

gic inhibition of general locomotor activity is provided by studies that demonstrate an elevation in activity following the depletion of brain serotonin (e.g, [30,54]), and a reduction in activity following injections of its immediate precursor 5-hydroxytryptophan (5-HTP). Similarly, Hollister et al. [46] have shown that tryptophan and 5-HTP will block the amphetamine-induced hyperactivity associated with food deprivation. The present paper reviews the recent neurochemical research on TI, and attempts to parallel many of these findings to research involving altered plasma amino acid levels and pharmacological effects on raphe electrical activity in the mammalian brainstem.

feedback to the raphe cells, producing a firing decrement [3,5] and presumably a correlated reduction in 5-HT release [ 1 ]. A number of drugs which affect brain 5-HT and also alter raphe firing have been shown to affect TI durations in chickens [45, 56, 58]. Drugs which are known to inhibit raphe firing tend to increase T! duration, and vice versa. That this area may play a role in TI is also implicated by brain transection data which show that the integrity of the medullary-reticular formation is essential for tonic immobility [59]. Some of these drugs and their influence on both TI and raphe electrical activity are depicted in Table 1.

Psychotomimetic Drugs TONIC IMMOBILITY AND RAPHE ELECTRICAL ACTIVITY Most of the cell bodies of serotonergic fibers are located in raphe nuclei; structures that are found in the upper brainstem and lower midbrain area [20]. Aghajanian and co-workers [ 1,3-8] have studied the effect of a variety of drugs on the firing of raphe neurons in rats. Their work implicates the existence of a negative feedback mechanism in which postsynaptic neurons modulate the electrical activity of raphe units. According to this hypothesis, excess 5-HT at postsynaptic receptor sites results in inhibitory

Administration of d-LSD increases TI duration [ 58] and suppresses the firing of raphe neurons [3, 4, 8]. Among several effects, d-LSD is thought to compete with 5-HT for postsynaptic receptor sites and consequently may activate the postulated negative feedback loop which inhibits raphe firing [7]. The LSD derivative BOL-148, which acts on the serotonergic system in a fashion similar to d-LSD but without hallucinagenic effects, was also observed to inhibit raphe electrical activity [4] and enhance TI duration [58]. However, the inactive isomer, 1-LSD had no effect [58].

SEROTONIN AND TONIC IMMOBILITY

37 TABLE 1

DRUG EFFECTS ON TONIC IMMOBILITY AND RAPHE FIRING Drug

d-LSD BOL-148 Pargyline Iproniazid 5-HT (central) Tryptophan 5-HTP PCPA 5-HT (peripheral) d-Amphetamine Imipramine Norepinephrine

Duration of Tonic Immobility [13, 21, 39, 42, 56, 58] + + + + + + 0 0 _a, +b 0a

Raphe Electrical Activity [1, 3-8, 14, 34, 63, 74, 82, 84] 0, 0 + + _a 0a, +b

Note: Adapted from Maser et al. [58]. + = increase, - = decrease, 0 = no change. aPeripheral administration. bCentral administration. S e r o t o n i n a n d its Precursors

Tryptophan, the ultimate precursor to 5-HT, has been shown to depress the activity of raphe units [1]. Intraperitoneal injections of tryptophan also cause increases in histochemical fluorescence of raphe neurons [2]. As evidence for an inverse relationship between raphe electrical activity and duration of TI, tryptophan has been found to potentiate tonic immobility [ 39]. An earlier study showed no effect of the immediate 5-HT precursor, 5-HTP on raphe activity [6]. More recently, however, Gallager and Aghajanian [35] found that 5-HTP suppressed raphe firing when administered in a higher dose (150 mg/kg) along with peripheral decarboxylase inhibition. Trulson and Jacobs [82] report similar findings. While Maser e t a l . [58] employed peripheral decarboxylase inhibition ( R O 4 - 4 6 0 2 ) , they found no evidence of an effect of 5-HTP on TI when birds were tested 20 rain after injection. However, it is possible that the dose of 5-HTP was insufficient (69 mg/kg) and that the injection-test interval was too short. Gallager and Aghajanian report the onset of raphe inhibition 40 rain after 5-HTP administration. As would be expected, serotonin administered microiontophoretically suppresses raphe electrical activity [7]. When injected intraventricularly, 5-HT also produces an increment in the duration of tonic immobility [42]. Maser et al. [58], however, found that intravenous injections of 5-HT caused a decrease rather than an increase in TI duration. While seemingly at odds with a serotonergic, midbrain-raphe model, the effects of intravenous 5-HT injections on TI correspond nicely with the finding that peripherally administered 5-HT causes an increase instead of a suppression of raphe firing [63]. Serotonin Depletion

Since p-chlorophenylalanine (PCPA) is a widely used depletor of brain 5-HT due to its inhibitory action on tryptophan hydroxylase [48,51], it would seem to follow

that PCPA should cause an increase in raphe firing and a corresponding decrease in tonic immobility. However, Aghajanian e t a l . [6] found that PCPA had no effect on raphe electrical activity, and several subsequent studies have failed to show an effect of PCPA on tonic immobility (e.g., [21,58]). On the other hand, PCPA pretreatment blocks the potentiating effect of morphine [45], and the attenuating effect of amphetamine [13] on tonic immobility. Similarly, PCPA has been found to block the effect of MAO inhibitors on raphe firing [6]. It is not known why PCPA alone fails to affect raphe firing or TI, however, the absence of an effect is consistent with the apparent covariation between TI duration and raphe firing. (Recent reports have questioned the selectivity of 5-HT depletion via PCPA, noting that PCPA also depletes norepinephrine (e.g., [67] ). However, Koe and Weissman [40] in their original article report the time course effects of a single injection of PCPA (316 mg/kg) on brain catecholamines and serotonin. Three days following injection, brain catecholamines were only slightly lowered, while brain 5-HT levels were depleted by as much as 90 percent. We employed similar time and dose parameters in all our PCPA studies.)

M o n o a m i n e Oxidase I n h i b i t o r s

Since MAO inhibitors allow 5-HT to accumulate by preventing oxidation in the synaptic cleft (e.g., [16] ), this consequent accumulation should act on postsynaptic receptor sites and lead to inhibition of raphe neurons. Moreover, if there is an inverse relation between raphe activity and TI, one would expect a corresponding increase in response duration following administration of MAO inhibitors. As predicted, iproniazid and pargyline were found to inhibit raphe neurons in rats [6], and increase immobility duration in domestic fowl [58]. Recent work in our laboratory has shown that tranylcypromine, a potent MAO inhibitor which inhibits raphe firing [6], also potentiates tonic immobility (unpublished data).

38 lmipramine Tricyclic antidepressants such as imipramine HCI inhibit raphe firing following peripheral injections [ 14,74]. Among several pharmacological effects, imipramine blocks the uptake of 5-HT by the presynaptic neuron [70], and should trigger the inhibitory feedback mechanism by the action of excess 5-HT on postsynaptic receptor sftes. Further support for the role of raphe activity in modulating TI is provided by the fact that imipramine caused increases in TI following intraventricular administration [42]. This recent finding, however, contrasts with an earlier one in which intramuscular injections of imipramine caused a reduction in TI duration [56]. d-A mphetamine The inverse relationship between TI and raphe activity is symmetrical as evidenced by the fact that d-amphetamine potentiates raphe firing rate [34], but suppresses TI duration [13,80]. Selective depletion of 5-HT by PCPA blocks the amphetamine effect on tonic immobility [13]. Similarly, Schrold and Squires [73] found that PCPA blocked other more general behavioral effects of damphetamine in young chicks. It has been suggested by several investigators [15,46] that the serotonergic system may play a role in mediating the effects of d-amphetamine. Since d-amphetamine is a norepinephrine-releasing agent [75,89], it could be argued, in spite of these data, that the effect of amphetamine on Tl reflects some noradrenergic involvement. Contrary to such a contention, Thompson etal. [81] failed to find an effect of norepinephrine on tonic immobility. It is also interesting to note that intravenous administration of norepinephrine has no effect on raphe electrical activity [34]. While microiontophoretic application of norepinephrine does affect the firing rate of raphe units, the effect appears to be indirect and variable {77/. Temperature and Illumination During TI core temperature decreases, while a distinct hyperthermic reaction is observed following response termination [65]. Weiss and Aghajanian [84] have found that changes in raphe firing are positively correlated with induced changes in core temperature. Specifically, increased core temperature is associated with increases in raphe firing. It is possible that the hypothermic reaction during TI and the hyperthermia following the response may reflect corresponding activity changes of central serotonergic neurons. If the hyperthermia following TI termination is in fact related to an increase in raphe firing, then it is interesting that d-amphetamine, which increases raphe firing [34], causes hyperthermia in chickens [9] and abbreviates TI duration [ 13,80]. Rovee and co-workers [71,72] have reported circadian periodicity of TI, with the shortest reactions occurring during light periods and the longest during darkness. Mosko and Jacobs [64] found that midbrain raphe neurons tend to respond at higher rates under conditions of normal room illumination than in darkness. If raphe firing rate changes as a function of the light-dark cycle, then a serotonergic, midbrain-raphe mechanism might also be involved in the apparent circadian fluctuations in TI duration.

WALLNAU AND GALLUP PLASMA TRYPTOPHAN MANIPULATIONS Fernstrom and Wurtman [24] found increases in brain tryptophan and brain 5-HT concentrations in rats when the level of plasma tryptophan was elevated by a single injection of this essential, dietary amino acid precursor. They concluded that the level of brain tryptophan and consequently brain 5-HT was dependent on the total amount of tryptophan present in the blood. Using similar time and dose parameters, Gallup etal. [39] found that tryptophan injections significantly increased the duration of TI in chickens, with an optimal effect at 55 mg/kg. While brain 5-HT increases are asymptotic one hour after injection in rats [24], the potentiation of TI in chickens following tryptophan preloading was maximal after 30 min. This disparity may reflect differences between avian and rodent metabolic and/or blood-brain transport rates. On the other hand, when Fernstrom and Wurtman maintained rats on a high-protein diet, plasma tryptophan was greatly increased, but there was no corresponding change in either brain tryptophan or brain serotonin [27]. Apparently tryptophan competes with other plasma neutral amino acids (tyrosine, phenylalanine, leucine, isoleucine, and valine) for the same mechanism which provides transport across the blood-brain barrier [10]. Thus, the level of brain tryptophan and brain 5-HT seems to depend more on the ratio of plasma tryptophan to competing neutral amino acids than it does on the absolute level of plasma tryptophan [271. In support of this view, the ratio of plasma tryptophan to its competing amino acids, rather than its absolute level, is a better predictor of brain tryptophan and brain 5-HT concentrations [27]. Fernstrom, Wurtman and co-workers have also demonstrated that levels of brain 5-HT can be manipulated by regulating dietary intake of tryptophan (for reviews see [28,29]). Since tryptophan is an essential amino acid in mammals, its only natural source is dietary. It has also been suggested that tryptophan hydroxylase is a low-affinity enzyme, and thus the amount of tryptophan normally present in the brain is not enough to saturate this enzyme [29,87]. As long as tryptophan hydroxylase is not saturated by its substrate, it will not synthesize 5-HT at its maximum rate. If this is the case then the availability of tryptophan could be a rate-limiting factor in 5-HT synthesis, rather than the amount of tryptophan hydroxylase [24, 28, 29, 87]. Given this assumption along with the fact that the only source of tryptophan is dietary, Fernstrom and Wurtman predicted that dietary manipulations of tryptophan should have profound effects on the level of brain tryptophan, and consequently on the rate of 5-HT synthesis (e.g., [ 2 5 - 2 7 ] ). It is now well established that rats maintained on a chronic corn diet (corn is low in tryptophan) exhibit reduced levels of plasma tryptophan, brain tryptophan, brain 5-HT, and in some cases the 5-HT metabolite, 5-hydroxyindolacetic acid [23, 26, 53]. The depletion of these substances can either be prevented by the addition of tryptophan to the diet [26] or reversed by a single injection of tryptophan [23,53]. It should be noted that the underlying principle influencing the availability of tryptophan under conditions of dietary depletion is the same as for dietary augmentation. That is, dietary depletion of tryptophan should shift the plasma ratio in favor of the other amino acids, giving them a competitive advantage for the transport mechanism.

SEROTONIN AND TONIC IMMOBILITY Consistent with these findings, chickens maintained on a corn diet for a period of 11 days displayed greatly attenuated durations of immobility, with a mean TI duration of only 47.5 sec as compared to 359.3 sec for control animals [39]. When additional birds were placed on a diet completely free of tryptophan, the effect was even more pronounced, with a mean response duration of 9.5 sec compared to 507.4 sec for those that received a balanced diet [39]. In order to circumvent the possible confounding influence of niacin depletion (an alternate end-product of tryptophan), this vitamin was added to the specially prepared tryptophan-free diet. It is also interesting to note that tryptamine, another metabolic product of tryptophan, has no effect on TI duration [39]. Thus, dietary manipulations that have been shown to decrease plasma tryptophan, brain tryptophan, and brain 5-HT also produce corresponding decreases in the duration of tonic immobility. Such effects are reversible since following depletion of tryptophan, birds that were given a normal diet showed a partial reinstatement of the immobility response, and did not differ significantly from control animals [39]. Taken together, the effects of tryptophan injections, dietary depletion, and dietary replenishment strongly support the possibility of serotonergic involvement in tonic immobility. Behavioral effects of tryptophan depletion are not limited to tonic immobility. Lytle e t al. [53] reported that rats which were maintained on a corn diet showed increased sensitivity to electric shock. Flinch and jump thresholds to electric shock decreased as early as two weeks following confinement to a corn diet. They also showed that dietary rehabilitation and tryptophan injections served to reinstate the thresholds to control levels. Contrary to these findings, however, we have found that when the presence or absence of electric shock is factorially combined with the presence or absence of a tryptophan-free diet, chickens show no evidence of hyperalgesia. Exposure to electric shock immediately prior to induction reliably prolonged TI in both normal and tryptophan deprived birds [83]. Moreover, when we used PCPA in a similar paradigm to deplete brain 5-HT, rather than the tryptophan-free diet, only a main effect of shock was obtained. PCPA did not block the shock effect nor did it interact with shock. The apparent absence of hyperalgesia in these data might bear on the problem of separating shock sensitivity (i.e., detection thresholds) from shock reactivity. An alternative explanation for the data reported by Lytle e t al. is that the rats showed increased reactivity to shock, but not sensitivity. That is, serotonin depletion may remove behavioral inhibition, and the reaction to electric shock might therefore reflect an efferent rather than an afferent effect. In other words, shock sensitivity and shock reactivity may not be isomorphic. This interpretation is supported by the finding that detection thresholds for electric shock are unchanged, whereas locomotor activity in response to suprathreshold shock is increased following administration of PCPA to rats [33]. The effect of 5-HT depletion may be to simply alter the animal's response bias. This possibility is bolstered by the finding that rats depleted of 5-HT by raphe lesions demonstrate an increase in spontaneous crossings in a shuttle box avoidance task [52]. The sensitivity-reactivity problem could be further resolved by signal detection methods (e.g., [41 ] ). CATECHOLAMINES

One must be cautious about attributing the behavioral

39 effects of tryptophan to increases in brain serotonin. Tryptophan is also the precursor to several other endproducts, including tryptamine and niacin. Engel and Modigh [22] have argued that tryptophan-induced behavioral effects may not be mediated, as one might expect, by an increase in brain 5-HT, or even by other tryptophan metabolites. Their assertion is supported by the observation that a tryptophan-induced decrease of locomotor activity in mice was not reversed when the various metabolic routes of tryptophan (formation of 5-HT, tryptamine, and formylkynurenine) were blocked [62]. Instead, Engel and Modigh propose that catecholamines (CA) participate in the behavioral effects of tryptophan preloading. For example, they found that a tryptophan-induced suppression of avoidance behavior in rats could be blocked by injections of L-DOPA, the immediate precursor of dopamine. Engel and Modigh suggest that tryptophan may in some way impair CA synthesis which in turn can be reversed by administration of L-DOPA. Since plasma tryptophan competes with tyrosine and phenylalanine, amino acid precursors of the catecholamines, for blood-brain transport, an impairment of CA synthesis following tryptophan injections could occur. Modigh (unpublished data, cited in [22]) found that 800 mg/kg of tryptophan caused a 50 percent reduction in CA synthesis. Thus, since tryptophan increases TI duration, its effect on TI might be mediated by a reduction in CA synthesis rather than an increase in brain serotonin. While the effects of L-DOPA on TI are unknown, several facts discount the tenability of the CA-impairment hypothesis. A single injection of 55 mg/kg of tryptophan constitutes the maximally effective dose for potentiating TI duration. Engel and Modigh [22], however, did not observe behavioral effects with tryptophan doses of less than 600 mg/kg. Likewise, Modigh (cited in [22]) found CA synthesis impairment with a dose of 800 mg/kg, but lower doses were not investigated. Wurtman e t a l . [88] found that a 50 mg/kg dose of tryptophan caused a 32 percent reduction of DOPA accumulation in rat brains. However, it cannot readily be inferred that tryptophan impaired CA synthesis in this instance, since in the Wurtman e t al. study DOPA accumulation was made possible by pretreatment with the decarboxylase inhibitor R 0 4 - 4 6 0 2 , and the rate of CA synthesis is controlled by end-product inhibition. Also discounting CA involvement are the findings that norepinephrine, and two dopamine-~-hydroxylase inhibitors (U 14, 624 and Sch 10595) have negligible effects on tonic immobility [81]. In addition, recent work in our laboratory has failed to show an effect of dopamine on TI in chickens (unpublished data). The problem of seemingly inevitable confounding of CA and 5-HT effects following precursor loading (e.g., injections of tryptophan, tyrosine, 5-HTP, or L-DOPA) is further circumvented by the finding that direct intraventricular administration of 5-HT produces an increase in TI duration [42]. CHOLINERGIC EFFECTS Another neurochemical view of response inhibition is that it is mediated by a central cholinergic mechanism (e.g., [17,18] ). Support for this position is provided by studies which demonstrate increased behavioral arousal following treatment with anticholinergic agents [61,66], a decrease in locomotor activity following treatment with cholinomimetics [31,32], and a disruption of DRL responding by

40

WALLNAU AND GALLUP

scopolamine [60]. Thompson etal. [79] suggest that a similar system mediates tonic immobility. Scopolamine, an anticholinergic agent, impairs immobility duration in chickens and physostigmine, a drug which prevents the metabolic breakdown of acetylcholine, causes an increase in tonic immobility [79]. While these data clearly provide evidence which implicates cholinergic involvement, other findings caution against assuming a direct cholinergic influence on tonic immobility. Atropine, a specific anticholinergic agent, has no effect on tonic immobility [44,58]. Moreover, scopolamine has been shown to have fear-reducing properties [68]. Since TI is potentiated by manipulations which increase fear [36], it is possible that the effect of scopolamine on TI reflects its fear-reducing properties, rather than a direct mediating action of the cholinergic system. It is also interesting to note that anticholinergics such as scopolamine and atropine have no effect on raphe firing [4]. Further complicating any simple interpretation is the possibility of a curious mammalian-avian reversal in the effects of cholinergic compounds. Scopolamine, for example, potentiates TI duration in rabbits [43] and guinea pigs [86], while physostigmine abbreviates the response in both of these animals. These effects are just the opposite of those reported for chickens [79] and ducks [86]. CURRENT STATUS Although TI resembles sleep in a number of ways, recent evidence shows that during the immobility episode, animals, much like schizophrenic patients in catatonic stupors [37], remain acutely aware of what is going on around them and continue to monitor events in the environment. Immobile animals remain capable of processing information, retrieving previously learned associations, and even forming new associations during this trance-like state [36,40]. Tonic immobility also differs from REM sleep in a number of other important ways

[501. While TI is affected by a variety of drugs thought to produce changes in brain 5-HT levels, drug-induced changes in the electrical activity of midbrain raphe neurons seem to provide the most powerful predictive framework for anticipating drug effects on T1 duration. Brain transection studies and electrical recording [50,59], show that the integrity of an area in the medullary reticular formation is essential to the immobility reaction. It may be more than merely coincidental that many raphe neurons are found in roughly the same area of the brain. Changes in core temperature during TI also appear to parallel temperatureinduced changes in raphe activity, and a raphe model may even subsume circadian rhythm effects on TI as a result of light-induced changes in raphe firing. Thus, there may be a functional relationship between raphe neurons and TI, such that raphe activity antagonizes TI, while both physiological and neurochemical events which prolong TI do so through the inhibition of raphe units. Tonic immobility shows a striking resemblance to a drug-induced form of catatonia recently reported in rats [11,47]. The specific substance, called ~-endorphin, is one of several endogenous morphinomimetic brain peptides thought to have analgesic-like properties. Following central administration of ~-endorphin, rats become rigidly immobile, showing signs of waxy flexibility, mydriasis,

decreased rectal temperature, exopthalamus, and loss of the righting reflex. Such effects last up to one hour, while recovery is abrupt with no apparent aftereffects. Sudden changes in stimulation cause subjects to resume normal postures. All of these symptoms are also characteristic of animals in tonic immobility [37]. In support of the possibility that these may represent homologous behaviors, morphine has been shown to be an exceptionally potent immobility agonist [45], and endorphin-induced catatonia is completely reversed by the opiate antagonist naloxone [ 11,47 ]. In light of this parallel it is curious to note that painful electric shock serves to potentiate tonic immobility (e.g., [36,38]). While it might seem paradoxical that a powerful analgesic like morphine has the same effect on TI as pain, pain may trigger the release of naturally occuring morphinelike compounds in the brain, such as /3-endorphin. Thus, morphine may simulate the release of such substances and thereby provide for response enhancement. The presence of this parallel is not inconsistent with the focus of this paper, since the neurochemistry of these opiate-like compounds may be related to serotonin. As noted previously, PCPA completely eliminates the effect of morphine on tonic immobility [45]. CONCLUSION Despite some ambiguities and inconsistencies between pharmacological effects and research on the serotonergic system, the majority of data provide a strong inferential base for suggesting that neuronal mechanisms related to 5-HT participate in modulating tonic immobility. The work of Fernstrom and Wurtman (e.g., [24, 28, 29]) has provided a means by which brain tryptophan and brain 5-HT levels can be readily manipulated via systemic alterations of tryptophan and diet. Comparable manipulations provide convergent validation for the importance of central serotonergic mechanisms in tonic immobility. The evidence may be summarized as follows. (A) Injections of tryptophan increase brain tryptophan and 5-HT levels, presumably by increasing the plasma amino acid ratio in favor of tryptophan. Likewise, injections of tryptophan increase TI duration in chickens by more than five times relative to water-injected controls. (B) Diets low in tryptophan (e.g., corn) which decrease the ratio of plasma tryptophan to other neutral amino acids, cause decreases in brain tryptophan, and brain 5-HT in the rat. When chickens are placed on a corn diet or a specially prepared tryptophan-free diet, TI durations are greatly reduced. Niacin supplements and the absence of a tryptamine effect on TI argue against other end-product alternative interpretations. (C) The behavioral changes that accompany brain 5-HT depletion due to long-term consumption of food which is low in tryptophan can be reversed when rats are given diets supplemented with tryptophan. Similarly, the attenuation of TI duration following dietary restriction of tryptophan is reversed by dietary rehabilitation. Additional evidence for central serotonergic involvement is provided by the striking parallel (see Table 1) between drug effects on TI and central serotonergic neurons. Putative serotonergic manipulations known to alter the firing of raphe neurons, have just the opposite directional effects on the duration of TI in domestic fowl. At the present time the best predictor of the effect of a serotonergic-acting drug on TI is its effect on raphe

SEROTONIN AND TONIC IMMOBILITY

41

electrical activity, rather than changes in brain levels of serotonin per se. This point is highlighted by the effects of PCPA. While PCPA is a p o w e r f u l depletor of brain 5-HT, it has n o effect on either TI or raphe firing when administered alone. However, PCPA can be used to block drug effects on b o t h raphe electrical activity and the duration of tonic immobility. The role, if any, of catecholamines in tonic i m m o b i l i t y seems to be negligible since d o p a m i n e , norepinephrine, and dopamine-~-hydroxylase inhibitors have no effect on the response. A l t h o u g h capable of producing robust effects, the cholinergic influence on TI appears to be indirect. Atropine, an anticholinergic, has no effect on tonic i m m o b i l i t y . The effect of scopolamine, a n o t h e r anticholinergic agent, may be due to fear reduction, and neither atropine or scopolamine affect raphe electrical activity. Finally, scopolamine and physostigmine affect TI duration in rabbits and guinea pigs in a m a n n e r exactly opposite to their effects on chickens and ducks. Even t h o u g h m a n y of the underlying neural mechanisms of TI and o t h e r forms of behavioral suppression are likely to be different, in a very general sense the serotonergic studies of TI provide more collaborative evidence that central 5-HT mechanisms may be involved in behavioral inhibition. It is i m p o r t a n t to n o t e that since m a n y drug manipulations affect m o r e than one n e u r o t r a n s m i t t e r , it is not possible, nor do we claim to base our conclusion on a single manipulation. Rather, it is the data provided by a variety of drug effects which converge to implicate the possible role of a central serotonergic system in tonic immobility. ADDENDUM Since this paper was submitted for publication, there has been a report which questions the existence of a negative feedback loop b e t w e e n raphe units and postsynaptic neurons. Mosko and Jacobs (Brain Res. 119: 2 9 1 - 3 0 3 , 1.977) found that brain transections rostral to the midbrain

raphe in rats did not disrupt the inhibition of raphe neurons caused by drugs which increase synaptic c o n c e n t r a t i o n s of serotonin. Since midbrain raphe efferents are primarily ascending, Mosko and Jacobs argue that negative feedback from postsynaptic neurons should have been eliminated. As a possible alternative interpretation, they suggest that such drugs may inhibit raphe electrical activity by their action at serotonergic synapses intrinsic to the midbrain raphe. There have also been a n u m b e r of recent developments which bear on opiate effects and tonic i m m o b i l i t y . We have found n a l o x o n e in doses as high as 2 mg/kg to have no effect on TI in chickens, and we have also d e t e r m i n e d that n a l o x o n e fails to block the m o r p h i n e e n h a n c e m e n t of tonic i m m o b i l i t y (unpublished data). Consistent with these findings, H. J. Haigler (personal c o m m u n i c a t i o n ) has found m o r p h i n e to inhibit the firing rate of midbrain raphe neurons, an effect which n a l o x o n e also failed to block. Thus, c o n t r a r y to the possible involvement of #-endorphin, the effects of m o r p h i n e on midbrain raphe neurons and on TI does not appear to be a specific narcotic effect. On the o t h e r hand, Carli (e.g., Psychol. Rec. 27: 1 2 3 - 1 4 3 , 1977) reports that TI duration in rabbits is reduced by naloxone. However, such effects required unusually high doses of from 15 to 25 m g / k g of naloxone. A l t h o u g h previous a t t e m p t s to d e m o n s t r a t e an effect of atropine have failed [ 4 4 , 5 8 ] , we recently found that high doses of atropine (75 mg/kg) attenuate TI duration in chickens. However, the existence of a mammalian-avian reversal in cholinergic effects on TI still complicates a clear interpretation. In support of the generality of the m o d e l developed in the present paper, parallel effects of such c o m p o u n d s as m o r p h i n e and d - a m p h e t a m i n e on TI have been obtained in b o t h rabbits (W. M. Davis, Arch. int. Pharmacodyn. 142: 3 4 9 - 3 6 0 , 1963) and chickens [ 13,45]. Finally, the effect of L-DOPA on TI has recently been e x a m i n e d in our laboratory. With birds tested 30 minutes following intraperitoneal injection, TI was unaffected by doses as high as 100 mg/kg.

REFERENCES 1. Aghajanian, G. K. Influence of drugs on the firing of serotonin-containing neurons in brain. Fedn Proc. 31: 91-96, 1972. 2. Aghajanian, G. K. and I. M. Asher. Histochemical fluorescence of raphe neurons: Selective enhancement by tryptophan. Science 172: 1159-1161, 1971. 3. Aghajanian, G. K., W. E. Foote and M. H. Sheard. Lysergic acid diethylamide: Sensitive neuronal units in the midbrain raphe. Science 161: 706-708, 1968. 4. Aghajanian, G. K., W. E. Foote and M. H. Sheard. Action of psychotogenic drugs on single midbrain raphe neurons. J. Pharmac. exp. Ther. 171: 178-187, 1970. 5. Aghajanian, G. K. and D. K. Freedman. Biochemical and morphological aspects of LSD pharmacology. In: Psychopharmacology: A Review o f Progress, edited by E. H. Efron. Washington, D. C.: U.S. Govt. Printing Office, 1968. 6. Aghajanian, G. K., A. W. Graham and M. H. Sheard. Serotonincontaining neurons in brain: Depression of firing by monoamine oxidase inhibitors. Science 169: 1100-1102, 1970. 7. Aghajanian, G. K. and H. J. Haigler. Direct and indirect actions of LSD, serotonin and related compounds on serotonincontaining neurons. In: Serotonin and Behavior, edited by J. Barchas and E. Usdino New York: Academic Press, 1973.

8. Aghajanian, G. K., H. J. Haigler and F. E. Bloom, Lysergic acid diethylamide and serotonin: Direct actions on serotonincontaining neurons. Life Sei. 11: 615-622, 1972. 9. Allen, D. J. and E. Marley. Effect of sympathomimetic and allied amines on temperature and oxygen consumption. Br. J. Pharmac. Chemother. 31: 290-312, 1967. 10. Blasberg, R. and A. Lajtha. Substrate specificity of steady-state amino acid transport in mouse brain slices. Archs Biochem. Biophys. 122: 361-377, 1965. 11. Bloom, F., D. Segal, N. Ling and R. Guillemin. Endorphins: Profound behavioral effects in rats suggest new etiological factors in mental illness. Science 194: 630-632, 1976. 12. Bolton, T. B. The physiology of the nervous system. In: Physiology and Biochemistry of the Domestic Fowl, Vol. 2, edited by D. J. Bell and B. M. Freeman. New York: Academic Press, 1971. 13. Boren, J. and G. G. Gallup, Jr. Amphetamine attenuation of tonic immobility. Physiol. Psychol. 1976, in press. 14. Bramwell, G. J. Effect of imipramine on unit activity in the midbrain raphe of rats. Br. J. Pharmac. Chemother. 44: 345-346, 1972. 15. Breese, G. R., B. R. Cooper and R. A. Mueller. Evidence for involvement of 5-hydroxytryptamine in the actions of amphetamine. Br. J. Pharmac. Chemother. 52: 307-314, 1974.

42

16. Brodie, B. B., S. Spector and P. A. Shore. Interaction of monoamine oxidase inhibition with physiological and biochemical mechanism in brain. Ann. N. Y. Acad. Sci. 80: 6 0 9 - 6 1 4 , 1959. 17. Carlton, P. L. Cholinergic mechanisms in the control of behavior by the brain. Psychol. Rev. 7 0 : 1 9 - 3 9 , 1963. 18. Carlton, P. L. Brain-acetylcholine and inhibition. In: Reinforcement and Behavior, edited by J. L. Tapp. New York: Academic Press, 1969. 19. Chertok, L. Animal hypnosis. In: Abnormal Behavior in Animals, edited by M. W. Fox. Philadelphia: Saunders, 1968. 20. Cooper, J. R., F. E. Bloom and R. H. Roth. The Biochemical Basis o f Neuropharmacology. New York: Oxford University Press, 1974. 21. Edson, P. H. Chemical correlates of tonic immobility in chickens. Unpublished doctoral dissertation, Tulane University, 1973. 22. Engel, J. and K. Modigh. Tryptophan-induced suppression of conditioned avoidance behavior in rats. In: Advances in Biochemical Psychopharmaeology, Vol. 11, edited by E. Costa and P. Greengard. New York: Raven Press, 1974. 23. Fernstrom, J. D. and M. J. Hirsch. Rapid repletion of brain serotonin in malnourished corn-fed rats following Ltryptophan injection. Life Sci. 17: 4 5 5 - 4 6 4 , 1975. 24. Fernstrom, J. D. and R. J. Wurtman. Brain serotonin content: Physiological dependence on plasma tryptophan levels. Science 173: 149-152, 1971. 25. Fernstrom, J. D. and R. J. Wurtman. Brain serotonin content: Increase following ingestion of carbohydrate diet. Science 174: 1023-1025, 1971. 26. Fernstrom, J. D. and R. J. Wurtman. Effect of chronic corn consumption on serotonin content of rat brain. Nat. New Biol. 234: 6 2 - 6 4 , 1971. 27. l:ernstrom, J. D. and R. J. Wurtman. Brain serotonin content: Physiological regulation by plasma neutral amino acids. Science 178:414 416, 1972. 28. Fernstrom, J. D. and R. J. Wurtman. Control of brain serotonin levels by the diet. In: Advances in Biochemical Psychopharrnacology, Vol. 11, edited by E. Costa and P. Greengard. New York: Raven Press, 1974. 29. Fernstrom, J. D. and R. J. Wurtman. Nutrition and the brain. Scient. Am. 2 3 0 : 8 4 91, 1974. 30. Fibiger, H. C. and B. A. Campbell. The effect of parachlorophenylalanine on spontaneous locomotor activity in tile rat. Neuropharmacology 10: 2 5 - 32, 1971. 31. Fibiger, H. C., G. S. Lynch and H. P. Cooper, A biphasic action of central cholinergic stimulation on behavioral arousal in the rat. Psychopharnlacologia 2 0 : 3 6 6 382, 1971. 32. Fibiger, H. C., L. D. Lytle and B. A. Campbell. Cholinergic nrodulation of adrenergic arousal in the developing rat. J. comp. physiol. Psychol. 72: 3 8 4 - 3 8 9 , 1970. 33. I:ibiger, H. C., P. H. Mertz and B. A. Campbell. The effect of para-chlorophenylalanine on aversion thresholds and reactivity to foot shock. Physiol. Behav. 8: 2 5 9 - 2 6 3 , 1972. 34. Foote, W. F., M. H. Sheard and G. K. Aghajanian. Comparison of effects of LSD and amphetamine on midbrain raphe units. Nature 2 2 2 : 5 6 7 569, 1969. 35. Gallager, D. W. and G. K. Aghajanian. Inhibition of firing of raphe neurones by tryptophan and 5-hydroxytryptophan: Blockade by inhibiting serotonin synthesis with R 0 - 4 - 4 6 0 2 . Neuropharrnacology 15:149 156, 1976. 36. Gallup, G. G., Jr. Animal hypnosis: Factual status of a fictional concept. Psychol. Bull. 8 1 : 8 3 6 853, 1974. 37. Gallnp, G. G., Jr. and J. D. Maser. Tonic immobility: Evolutionary underpinnings of human catalepsy and catatonia. In: Psychopathology: Experimental Models, edited by J. D. Maser and M. E. P. Seligman. San Francisco: Freeman, in press. 38. Gallup, G. G., Jr., R. 1.'. Nash, R. J. Potter and N. H. Donegan. Effect of varying conditions of fear on immobility reactions in domestic chickens (Gallus gallus). J. comp physiol. Psychol. 7 3 : 4 4 2 445, 197(I.

WALLNAU AND GALLUP

39. Gallup, G. G., Jr., L. B. Wallnau, J. L. Boren, G. J. Gagliardi, J. D. Maser and P. H. Edson. Tryptophan and tonic immobility in chickens: Effects of dietary and systemic manipulations. J. comp. physiol. Psyehol. 1977, in press. 40. Gallup, G. G., Jr., L. B. Wallnau and G. J. Gagliardi. Evidence for the integrity of central processing during tonic immobility. Paper presented at the meeting of the Psychonomic Society, St. Louis, November 1976. 41. Green, D. M. and J. A. Swets. Signal Detection Theory and Psychophysics. New York: Wiley, 1966. 42. Harston, C. T., D. H. Sibley, G. G. Gallup, Jr. and L. B. Wallnau. Effects of intraventricular injections of imipramine and 5-hydroxytryptamine on tonic immobility in chickens. Bull. Psychon. Soc. 8: 4 0 3 - 4 0 5 , 1976. 43. Hatton, D. C., M. L. Woodruff and M. E. Meyer. Cholinergic modulation of tonic immobility in the rabbit {Oryctolagus cuniculus). J. cornp, physiol. Psychol. 80: 1053-1060, 1975. 44. Hicks, L. E. Cholinergic underpinnings of habituation in domestic chickens. Unpublished doctoral dissertation. Tulane University, 1975. 45. Hicks, L. E., J. D. Maser, G. G. Gallup, Jr. and P. H. Edson. Possible serotonergic mediation of tonic immobility: Effects of morphine and serotonin blockade. Psychopharmacologia 42: 51 56, 1975. 46. Hollister, A. S., G. R. Breese, C. M. Kuhn, B. R. Cooper and S. M. Schanberg. An inhibitory role for brain serotonincontaining systems in the locomotor effects of d-amphetamine. J. Pharrnac. exp. Ther. 198: 1 2 - 2 2 , 1976. 47. Jacquet, Y. F. and N. Marks. The C-fragment of B-lipotropin: An endogenous neuroleptic or antipsychotogen? Science 194: 6 3 2 - 6 3 5 , 1976. 48. Jequier, E., W. Lovenberg and A. Sjoerdsma. Tryptophan hydroxylase inhibition: The mechanism by which pchlorophenylalamine depletes rat brain serotonin. Molec. Pharrnac. 3: 2 7 4 - 2 7 8 , 1967. 49. Jouvet, M. and J. F. Pujol. Effects of central alterations of serotonergic neurons upon the sleep-waking cycle. In: Advances in Biochemical Psyehopharmacology, Vol. 11, edited by E. Costa and P. Greengard. New York: Raven Press, 1974. 50. Klemm, W. R. Neurophysiologic studies of the immobility reflex ("animal hypnosis"). In: Neuroseience Research, Vol. 4, edited by S. Ehrenpreis and O. C. Solnitzky. New York: Academic Press, 1971. 51. Koe, B. K. and A. Weissman. p-Chlorophenylalanine: A specific depictor of brain serotonin. 3". Pharrnac. exp. Ther. 154: 4 9 9 - 5 1 6 , 1966. 52. Lorens, S. A., J. P. Sorensen and L. M. Yunger. Behavioral and neurochemical effects of lesions in the raphe system of the rat. J. cornp, physiol. Psychol. 77: 4 8 - 5 2 , 1971. 53. Lytle, L., R. B. Messing, L. Fisher and L. Phebus. Effects of long-term corn consumption on brain serotonin and the response to electric shock. Science 190: 6 9 2 - 6 9 4 , 1975. 54. Mabry, P. D. and B. A. Campbell. Ontogeny of serotonergic inhibition of behavioral arousal in the rat. J. comp. physiol. Psychol. 8 6 : 1 9 3 201, 1974. 55. Margules, D. L. and A. S. Margules. The development of operant responses by noradrenergic activation and cholinergic suppression of movements. In: Efferent Organization and the Integration o f Behavior, edited by J. D. Maser. New York: Academic Press, 1973. 56. Maser, J. D. and G. G. Gallup, Jr. Tonic immobility in chickens: Catalepsy potentiation by uncontrollable shock and alleviation by imipramine. Psychosom. Med. 36: 199-205, 1974. 57. Maser, J. D. and G. G. Gallup, Jr. Tonic immobility and related phenomena: A partially annotated, tricentenial bibliography, 1636-1976. Psychol. Rec. 27: 177-217, 1977. 58. Maser, J. D., G. G. Gallup, Jr. and L. E. Hicks. Tonic immobility in chickens: Possible involvement of monoamines. J. cornp, physiol. Psychol. 8 9 : 3 1 9 328, 1975.

S E R O T O N I N AND TONIC I M M O B I L I T Y

59. McBride, R. L. and W. R. Klemm. Mechanisms of the immobility reflex ("animal hypnosis"): I. Influences of repetition of induction, restriction of auditory-visual input, and destruction of brain areas. Communs behav. Biol. 3: 33-41, 1969. 60. Meyer, M. E., G. A. Severson and R. W. Thompson. Scopolamine, methylscopolamine, and response conditioned inhibition in rats. Physiol. Psychol. 4: 4 3 - 4 4 , 1976. 61. Meyers, B., K. H. Roberts, R. H. Riciputi and E. F. Domino. Some effects of muscarinic cholinergic blocking drugs on behavior and the electrocorticogram. Psychopharmacologia 5: 289-300, 1964. 62. Modigh, K. Effects of L-tryptophan on motor activity in mice. Psychopharmacologia 3 0 : 1 2 3 - 1 3 4 , 1973. 63. Mosko, S. and B. L. Jacobs. Effect of peripherally administered serotonin on the neuronal activity of midbrain raphe neurons. Brain Res. 79: 315-320, 1974. 64. Mosko, S. S. and B. L. Jacobs. Midbrain raphe neurons: Spontaneous activity and response to light. Physiol. Behav. 13: 589-593, 1974. 65. Nash, R. F., G. G. Gallup, Jr. and D. A. Czech. Psychophysiological correlates of tonic immobility in the domestic chicken (Gallus gallus). Physiol. Behav. 1 7 : 4 1 3 - 4 1 8 , 1976. 66. Payne, R. and D. C. Anderson. Scopolamine-produced changes in activity and in the startle response: Implications for behavioral activation. Psychopharmacologia 12: 83-90, 1967. 67. Peters, D. A. V., M. Filczewski and I. M. KazurkiewiczKwilecki. Effect of para-chlorophenylalanine on catecholamine synthesis in rat brain, heart and adrenals. Biochem. Pharmac. 21: 2282-2284, 1972. 68. Plotnik, R., S. Mollenauer and E. Snyder. Fear reduction in the rat following central cholinergic blockade. J. comp. physiol. Psychol. 8 6 : 1 0 7 4 - 1 0 8 2 , 1974. 69. Ratner, S. C. Comparative aspects of hypnosis. In: Handbook o f Clinical and Experimental llypnosis, edited by J. E. Gordon New York: Macmillan, 1967. 70. Ross, S. B. and A. L. Renyi. Inhibition of the uptake of tritiated 5-hyroxytryptamine in brain tissue. Eur. J. Pharmac. 7: 270-277, 1969. 71. Rovee, C. K., W. J. Chiapparelli and L. W. Kaufman. The influence of altered lighting regimes on the periodicity of death feigning. Physiol. Behav. 1976, in press. 72. Rovee, C. K., L. W. Kaufman and S. Lelek. Arousal-induced changes in the amplitude of death-feigning periodicity. Paper presented at the meeting of the Psychonomic Society, St. Louis, November 1976. 73. Schrold, J. and R. F. Squires. Behavioral effects of damphetamine in young chicks treated with p-Cl-phenylalanine. Psychopharmacologia 20: 85-90, 1971. 74. Sheard, M. H., A. Zolovick and G. K. Aghajanian. Raphe neurons: Effect of tricyclic antidepressant drugs. Brain Res. 43: 690-694, 1972.

43 75. Stein, L. and C. D. Wise. Release of norepinephrine from hypothalamus and amygdala by rewarding medial forebrain bundle stimulation and amphetamine. J. comp. physiol. Psychol. 67: 189-198, 1969. 76. Stein, L. and C. D. Wise. Serotonin and behavioral inhibition. In: Advances in Biochemical Psychopharmacology, Vol. 11, edited by E. Costa and P. Greengard. New York: Raven Press, 1974. 77. Svensson, T. H., B. S. Bunney and G. K. Aghajanian. Inhibition of both noradrenergic and serotonergic neurons in brain by the a-adrenergic agonist clonidine. Brain Res. 92: 291-306, 1975. 78. Svorad, D. "Animal hypnosis" (Totstell reflex) as experimental model for psychiatry. Archs Neurol. Psychiat. 77: 533-539, 1957. 79. Thompson, R. W., J. Piroch, D. Fallen and D. Hatton. A central cholinergic inhibitory system as a basis for tonic immobility ("animal hypnosis") in chickens. J. comp. physiol. Psychol. 8 7 : 5 0 7 - 5 1 2 , 1974. 80. Thompson, R. W., J. Piroch and D. Hatton. The effect of sympatholytic and sympathomimetic drugs on the duration of tonic immobility ("animal hypnosis") in chickens. Paper presented at the meeting of the Midwestern Psychological Association, Chicago, May 1973. 81. Thompson, R. W., R. Scuderi and J. Boren. The effects of the catecholamines on tonic immobility ("animal hypnosis") in chickens. Paper presented at the meeting of the Rocky Mountain Psychological Association, Denver, May 1974. 82. Trulson, M. E. and B. L. Jacobs. Raphe neurons: Depression of activity by L-5-hydroxytryptophan. Brain Res. 97: 350-355, 1975. 83. Wallnau, L. B. and G. G. Gallup, Jr. In preparation, 1976. 84. Weiss, B. L. and G. K. Aghajanian. Activation of brain serotonin metabolism by heat: Role of midbrain raphe neurons. Brain Res. 26: 37-48, 1971. 85. Wise, C. D., B. D. Berger and L. Stein. Brain serotonin and conditioned fear. Proc. 78th Ann. Cony. APA. 821-822, 1970. 86. Woodruff, M. L., D. C. Hatton, M. B. Frankl and M. E. Meyer. Effects of scopolamine and physostigmine on tonic immobility in ducks and guinea pigs. PhysioL Psychol. 4: 198-200, 1976. 87. Wurtman, R. J. and J. D. Fernstrom. L-tryptophan, L-tyrosine, and the control of brain monoamine biosynthesis. In: Perspec-

tives in Neuropharmacology: A Tribute to Julius Axelrod, edited by S. H. Snyder. New York: Oxford University Press, 1972. 88. Wurtman, R. J., F. Larin, S. Mostafapour and J. D. Fernstrom. Brain catechol synthesis: Control by brain tyrosine concentration. Science 185: 183-184, 1974. 89. Ziance, R. J., A. J. Azzaro and C. O. Rutledge. Characteristics of amphetamine-induced release of norepinephrine from rat cerebral cortex. J. Pharmac. exp. Ther. 182: 284-294, 1972.