Neurochemical Correlates of Behavior1

NEURQCHEMICAL CORRELATES OF BEHAVIOR1 By M. H. Aprison and J.

N.Hingtgen’

Section of Neurnbiology, The Institute of Psychiatric Research and Departments of Psychiatry and Biochemistry, Indiana University Medical Center, Indianapolis, Indiana

I. Introduction . . . . . . . . . . 11. Behavioral Depression and Increases in Brain Serotonin 111. Behavioral Depression and Decreases in Brain Serotonin IV. Behavioral Excitation and Decreases in Brain Acetylcholine V. Methodological Problems . . . . . . . VI.Summary . . . . . . . . . . . References . . . . . . . . . .

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I. Introduction

Our motivation for the development of a research program to investigate relationships between central nervous system ( CNS ) transmitters and behavior stems from a desire to learn more about the neurobiological mechanisms which underlie abnormal behavior. If the brain is the source of the events which finally govern the behavior of an organism, then impaired behavior may be the result of biophysical, biochemical andl or anatomical lesions in that part of the nervous system. When “key” neurons are affected by the lesions, their most important function, the transfer of information to other cells, is often disrupted or impaired. Since it is generally accepted that mediation of synaptic excitation and inhibition (transfer of information) within the mammalian nervous system is mainly chemical, we choose to concentrate on the study of these neurochemical mechanisms and their effect on behavior. While we have restricted our immediate goals to looking for neurochemical correlates of behavior in lower animals, it is our hope that these data will some day be useful for understanding the specific biochemical mechanisms which may cause certain types of abnormal human behavior. Our studies were initiated in 1954 when Aprison, Nathan, and Himwich found asymmetric acetylcholinesterase ( AChE ) activities in the right and left cerebral cortices and caudate nuclei from rabbits exhibiting compulsive circling after a unilateral intracarotid injection of a potent anticholinesterase drug, diisopropylfluorophosphate ( Aprison et al., 1954). Subsequent studies measuring the transmitter levels instead Supported in part by grant MH-03225 from National Institute of Mental Health,

U.S.P.H.S.

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of the enzyme activities showed that the rate of turning correlated with the asymmetric accumulation of acetylcholine ( ACh) in the cerebral cortices ( Aprison, 1958, 1965). These early studies led to the formulation of a theoretical framework which suggested possible neurobiological mechanisms operating in the emission of atypical or abnormal behavior (Fig. 1). Whatever type of ‘lesion” occurs due to general causes such as diet, stress, and drugs, the effect is invariably on the ability of various nerve cells to function normally. Thus, two main defects are produced: transmitter system defects and nontransmitter system defects. We have been interested in the former, because much evidence has accumulated which suggests that a number of specific nitrogen-containing compounds suspected of acting as neurotransmitters or modulators ( Aprison, 1962; Aprison and Takahashi, 1965)2 have major effects on the behavior of *The authors in these references as well as the present authors prefer to distinguish between transmitters and modulators. A modulator as defined by us is a compound which once released from specific nerve endings may influence the effective transmitter concentration at certain synapses by competing with it for the receptor site. It may even influence the release or uptake of the true transmitter thus also affecting the latter’s influence at that receptor. This definition allows for the possibility that a modulator can also function as a transmitter at other synapses within the same organism or within other animals.

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living organisms. We think these effects are due to the individual or grouped action of these substances after their release from the presynaptic neurons. The action is at receptor sites or sensitive portions of the postsynaptic membrane located in the synapses of various neuronal pathways being activated or utilized. Deviation from stable emission of a learned response, which we define as abnormal behavior, may therefore be due to the change in the effective concentration of the free transmitter acting at the postsynaptic receptor sites of a group of important synapses involved in key neuronal pathways utilized for the “generation” or performance of that particular behavior. If we can affect one or more of the systems labeled 1 to 5 in the last line of Fig. 1, we should be able to simulate abnormal behavior. This can be done in a number of ways including the following: ( u ) injection of known transmitter precursors; ( b ) injection of drugs known to affect components of systems involved in 1 to 5 (Fig. 1); ( c ) diet; ( d ) direct production of tissue pathology. The quantitative measurement of both the specific neurochemical changes in discrete areas and the changes in behavior of the same animal during periods of simulated abnormal behavior present the possibility of making meaningful correlations during a critical time period. Such correlations, when they occur, may help in a formulation of some general behavioral-neurochemical relationships applicable not only to animals but also to humans. We would like to describe in some detail our research program which involves the measurement of changes in levels of transmitter or modulator suspects such as ACh, 5-hydroxytryptamine (serotonin or 5-HT) and norepinephrine ( N E ) as well as their associated enzyme systems in specific brain areas, and their temporal relationship to changes in the quantitatively measured behavior of these animals. Although we d o not suggest this program serves as a model, we do feel that the general approach would be of interest to other researchers in neurobiology. II. Behavioral Depression and Increases in Brain Serotonin

The first experiments began in 1959 with the 5-HT-monoamine oxidase (MAO) system because of the keen interest that had developed in this subject in the five preceding years. The early interest in 5-HT developed when it was shown in pharmacological studies on smooth muscle that d-lysergic acid diethylamide, a compound which provokes schizophreniolike states in man, is a 5 H T antagonist at certain doses (Gaddum, 1954), but acts synergistically at lower doses (Costa, 1956). Studies with antimetabolites of 5-HT also added impetus for work in this area (Woolley and Shaw, 1954a,b). All these studies suggested to many investigators that 5 H T may have a role in brain function. The metabolic

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steps involved in the formation and destruction of 5-HT indicate that the level of 5-HT can be elevated by ( a ) injecting its precursor 5-hydroxytryptophan (5-HTP); ( b ) injecting an MA0 inhibitor such as iproniazid; or ( c ) injecting 5-HTP after iproniazid pretreatment. Once these pharmacological manipulations were performed and the increase in 5-HT measured, it became necessary to obtain equally quantitative behavioral measures for the animals in which the biochemical correlate was sought. In studies involving the correlation of changes in brain chemistry with changes in behavior, the behavioral measurements should match the neurochemical measurements for objectivity, reliability, and sensitivity. One such behavioral methodology makes use of operant conditioning techniques (Skinner, 1938; Ferster and Skinner, 1957; Honig, 19%). The operant behavior measured in our studies was emitted under either an approach or avoidance schedule of reinforcement. The approach schedule used with pigeons, maintained at 80% of free-feeding weight, consisted of a multiple fixed-ratio 50, fixed-interval 10 (Mult FR 50 FI l o ) , in which key pecking was reinforced with grain. For rats, the approach schedule was a variable ratio 40 (VR 40), in which lever pressing was reinforced with chocolate milk. The avoidance schedule, used only with rats, consisted of a response-shock interval of 40 sec and a shock-shock interval of 20 sec (RS40 SS20). In this latter schedule a lever press postponed an electric shock (1.6 mA, 0.5 sec duration) for 40 sec, but, during periods of no responding, shocks were presented at 20 sec intervals. Our initial attempt to assess the behavioral changes resulting from increased levels of brain 5-HT consisted of establishing a dose-response relationship following injections of 5 - H P into pigeons working on a Mult FR 50 FI 10 schedule of reinforcement. Intramuscular (I.M.) injections of %, 50, and 75 mg/kg doses of D L - ~ H Tlowered P the pigeons' rate of responding whereas doses below 25 mg/kg had little or no effect; since only the L isomer readily passes the blood-brain barrier, the effective dose is one half that shown (Aprison and Ferster, 1960, 1961c). The duration of the effect was usually over before the 6-hour experimental session ended. Since little 5-HT crosses the blood-brain barrier under normal physiological conditions compared to its precursor 5HTP (Costa and Aprison, 1958), a study was made to determine the behavioral effect of an I.M. injection of 5-HT on pigeons performing on a A multiple FR 50 FI 10 schedule (Aprison and Ferster, 1960, 1961~). comparable behavioral effect to 5-HTP was produced but with a much smaller dose of 5-HT; this effect was thought to be due principally to the peripheral actions of 5-HT. Since small doses of 5 H T had large behavioral effects, it might be

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supposed that the behavioral effects of 5-HTP were also due to 5-HT being formed peripherally. There is evidence that in spite of the fact that SHTP acting peripherally may affect the animal's behavior, its major influence is through the CNS. It was hypothesized that the behavioral effects were due mainly to a central mechanism involving free or physiologically effective 5-HT. Iproniazid, which is a much better central than peripheral MA0 inhibitor in vivu, enhanced the behavioral effects of injected 5-HTP. This greater behavioral disruption is best explained in terms of elevated brain 5-HT levels resulting from reduced brain M A 0 activity. When the same dose of 5-HTP was injected into iproniazid-pretreated pigeons at different intervals over a long period of time, both the behavior and brain MA0 activity returned to normal levels in about 35 days, whereas liver MA0 activity was back to normal in only 12 days; the return of the behavioral effect was readily correlated with the recovery of the brain M A 0 activity (Aprison and Ferster, 1961a,b). In studies where the dose of 5-HTP was varied and the dose of iproniazid held constant, it was found that at any level of brain MA0 activity (during the recovery period), the greatest behavioral effect was obtained at the highest 5-HTP dose injected. Even more important, these data show that at any given dose of 5-HTP, the greatest behavioral effect is obtained at the lowest brain M A 0 level during the recovery period ( Aprison and Ferster, 1961a,b), The free 5-HT in brain, which apparently is involved in the production of the behavioral effect (as a transmitter or modulator), probably comprises the 5-HT found at any instant in the synaptic cleft plus that of a labile storage pool within the presynaptic nerve endings. The latter pool can be thought of as a source of readily available 5-HT at a specific synapse and is probably in equilibrium with a second pool of 5-HT, the firmly bound storage pool. Serotonin re-entering the presynaptic nerve ending either enters the labile storage pool or is destroyed by MA0 in the mitochondria. Since an identical amount'of 5-HTP was injected at various times during the period that brain MA0 activity was returning to normal in the above mentioned experiments, the observed behavioral change should be due to action of available cerebral 5-HT formed from its precursor and released at the appropriate synapses. As the M A 0 activity increased, more 5-HT was destroyed and less 5-HT was available to produce physiological and behavioral effects. The changes seen in the birds' behavior appeared due to the action of increased free 5-HT in brain with the concentration controlled by the level of its catabolic enzyme located in the presynaptic neurons. It therefore became imperative to study the kinetic relationship between the 5-HTP induced behavioral change and brain 5-HT concentration in

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animals not given an M A 0 inhibitor. Serotonin was measured in four specific brain areas, as well as in liver, heart, lung, and blood in pigeons (Aprison et al., 1962) sacrificed at various time intervals during the period of measurable behavioral disruption, T ( Hingtgen and Aprison, 1965). Since the time course of the behavioral effect in any given animal is relatively invariant with a constant dose of 5-HTP while there is marked variation in the time course of response from animal to animal, we made use of a unique method of treating our data. The 5-HT data were plotted against the percent of the behavioral effect (in time) rather than length of time after 5-HTP administration. In this way, the variation in behavioral effect of the same dose of 5-HTP in each pigeon was weighted, thus markedly reducing the variability of the data. Only in the telencephalon and diencephalon (plus optic lobes) did the 5-HT concentrations return to normal when the behavior of the pigeons returned to normal. The time course of change in both parameters was therefore remarkably similar and confirms the hypotheses and our original explanation of the brain 5-HT-MAO-behavior correlation ( Aprison and Ferster, 196la,b; Aprison et al., 1962). It became evident that dopamine ( D A ) and NE levels in the four brain areas under investigation might also change after the injection of 5-HTP because the enzyme which synthesizes 5 H T , S-hydroxytryptophan decarboxylase, is thought to be the same as 3,4-dihydroxyphenylalanine decarboxylase, the enzyme which synthesizes DA, the precursor of NE. Consequently, another group of trained pigeons was given injections (I.M.) of 50 mg/kg 5-HTP. The average period of depressed behavior ( T ) following these injections was measured for each bird and at various percentages of T, the pigeons were sacrificed by decapitation, and the four brain parts were assayed for DA and NE (these data as well as 5 H T for three brain areas are shown in Fig. 2 ) . We found no significant changes in DA and NE concentrations in the telencephalon (Aprison and Hingtgen, 1964, 1965). Changes were found in the NE levels of the diencephalon (plus optic lobes) , pons-medulla oblongata and cerebellum, but these changes were not related to the observed behavioral changes. The NE levels of the pons-medulla oblongata in the experimental birds were depressed below the normal range both FIG.2. Serotonin, dopamine, and norepinephrine concentrations in telencephalon, diencephalon plus optic lobes, and pons plus medulla oblongata of pigeons during the complete period of atypical behavior ( T ) and after behavior had returned to normal. Each point represents a single determination from a pigeon working on a multiple FR 50 FI 10 schedule of reinforcement, injected with 50 mg/kg 5-HTP (I.M.) and killed at a percentage of its previously determined mean period of abnormal behavior. On the abscissa scale N.B. refers to normal behavior.

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during and after the period of behavioral disruption. Changes in DA occurred in diencephalon (plus optic lobes), pons-medulla oblongata, and cerebellum, but only in the first two brain parts is a temporal correlation with behavioral changes suggested. Whether this change in DA concentation contributes to the disruption of behavior, or reflects instead a fall in the NE precursor level, remains to be determined. In this regard, it should be noted that the DA concentration in the telencephalon, which contains the basal ganglia, is approximately 5 times higher than that in the midbrain and pons-medulla oblongata. The 5HT, DA, and NE concentrations in the telencephalon, diencephalon plus optic lobes, cerebellum, and pons plus medulla oblongata after 5-HTP administration suggest that the depressed approach behavior noted can best be explained by the action of free 5-HT on appropriate sites in the brain, but the role of DA in the midbrain and pons-medulla oblongata is still to be clarified. To verify these findings in another species, rats working on a VR 40 schedule of reinforcement were injected with 50 mg/kg D L - ~ H T P ( Aprison and Hingtgen, 1966a). Similar correlations over the period of disruption were found between the 5-HT changes in the telencephalon and changes in behavior. The elevated 5 H T level in the brain stem returned to normal before the behavior returned to normal. During the period of behavioral disruption, the NE concentration in the telencephalon did not vary from normal values. Further, after the behavior returned to normal, the NE concentration in the brainstem was slightly depressed. In general, these data on rats agree remarkably well with the data found in pigeons. Few studies have contained both behavioral and biochemical data on the same experimental animal, and these usually involve data from a single behavioral schedule. Experiments including measurements of two or more neurohumoral agents in specific brain areas from experimental animals working in two or more behavioral situations known to be differentially disrupted by either the injection of a transmitter precursor or drugs are even fewer in number. With such data, correlations between neurochemical and behavioral changes should occur just as in the single behavioral situation. Furthermore, these data could provide a basis for determining whether a specific biochemical system (i.e., serotonergic, noradrenergic, cholinergic) is involved in the specific production of the behavioral response (i.e., approach, avoidance). It was therefore of extreme interest to us to determine whether rats were equally affected by 5-HTP when working on either an approach or on an avoidance schedule of reinforcement. No behavioral effect followed an injection of 50 mg/kg DL-S-HTP

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into rats working on a RS40 SS20 schedule, although there was a comparable increase in brain 5-HT in these rats to that found in the approach rats ( Aprison and Hingtgen, 1966a). Furthermore, the avoidance rats still had elevated 5-HT levels beyond the time the levels in the approach rats returned to normal. These data suggest that avoidance behavior may not be as sensitive to brain 5-HT changes as is approach behavior, but that avoidance schedules may impose certain stresses on the rats to prolong the period of 5-HT elevation following 5-HTP administration. Ill. Behavioral Depression and Decreases in Brain Serotonin

Inasmuch as incremes in brain 5-HT are followed by disruptions in approach behavior, the question naturally arises: What behavioral changes would follow decreases in brain 5-HT? One method of reducing brain 5HT uses clrugs which deplete bound stores in nerve endings. Although many drugs, such as reserpine, tetrabenazine, a-methyl-m-tyrosine ( aMMT), cause a fall in more than one transmitter, a differential fall in any one compound over a reasonably long time period can be used to advantage by the investigator. For instance, after the injection of a-MMT in various species including the pigeon, brain 5-HT, NE, and DA levels are differentially depleted. Taking advantage of this differential depletion, we investigated the relationships between quantitatively measured behavioral changes after the injection of a-MMT (100 mglkg) into pigeons (Hingtgen and Aprison, 1963) and the brain levels of NE, DA, 5-HT, and a-mei-hyl-m-tyramineplus aramine, the decarboxylation products of a-MMT (Aprison and Hingtgen, 196613). The data indicate that both the change in 5-HT concentration and the change in mean behavioral response rates, including the return to normal levels of both parameters, followed the same time course (Fig. 3). This relationship was not true in the case of the other neurohumoral agents measured nor the decarboxylation products of a-MMT. In addition it was found that the a-methyl-rn-tyramines return to normal levels in pigeon brain at approximately 2-4 days after an injection of a-MMT. However, during the 8-hour period of decreased behavioral response rates following a-MMT, the almost stoichiometric displacement of DA and NE by their methyl analogs suggested that DA and NE changes are not directly responsible for the behavioral change since the a-methyl-rn-tyramines may function at the Dii and NE receptor sites (Carlsson, 1964). Although the suggestion had been made that an anorexic side effect was responsible for the behavioral results with pigeons (Carlton and Furgiuele, 1965), this explanation was shown to be inadequate since pigeons working on a multiple FR 50 FI 10 schedule of reinforcement as well as naive pigeons

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TIME A F T E R 1 0 0 m g / k g N - M M T (IM)

FIG.3. Temporal changes in behavioral response rates and brain monoamines in pigeons injected with 100 mg/kg cu-methyl-m-tyrosine (I.M. ). The behavioral data were obtained from pigeons working on a multiple FR 50 FI 10 schedule of reinforcement. [Reprinted with permission from Life Sciences 5, 1971 ( 1966).]

(both groups being maintained at 80%of free-feeding weight) consumed equivalent amounts of food in control sessions and sessions following the injection of 100 mg/kg a-MMT (Hingtgen and Aprison, 1966). At the present time we are not sure whether the behavioral disruption is due to SHT, dopamine, both of these compounds, or some unknown transmitter. However, the first lead that must be tested further is the one which indicates that a temporal relationship exists between the fall in both the total 5-HT levels and the behavioral responding. We have shown that increased total (free plus bound) brain 5-HT following an injection of 5-HTP produced a period of depressed responding in the pigeon working on a Mult FR 50 FI 10 schedule, whereas after a-MMT administration, decreased total brain 5-HT is also followed by a period of depressed responding on the same behavioral schedule. One explanation for the two opposite biochemical situations which result in virtually the same behavioral changes is found in the concept that a neurohumoral agent or neurochemical modulator must be free (to

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diffuse to the proper receptor site) rather than bound (in synaptic vesicles) in order to produce its physiological effect. Although the time course of the behavioral effects due to an injection of SHTP and a-MMT are distinctly different, one can offer the hypothesis that in both these cases more free 5-HT is released than in the normal state. Therefore, a physiological situation can exist in the brains of these pigeons where greater amounts of the free form are available at synapses to produce its effect. If one assumes that free and total transmitter levels follow the same time course, this relationship could be tentatively explained in terms of the corresponding changes in the free component during the behavioral disruption. The possibility must be supported by additional data. Another drug, tetrabenazine ( TBZ ) , has reserpine-like depressive effects on brain monoamines (Quinn et al., 1959) and on both approach and avoidance operant behavior ( Aprison and Hingtgen, 1966a ) . The 5-HT and NE data from the TBZtreated ( 2 mg/kg S.C.) rats working on a VR 40 approach schedule indicate that this drug caused a depletion of both 5-HT and NE from the telencephalon and brain stem of all the rats at 30 and 80%T. By 100%T (return to normal behavior), the 5-HT concentration in these brain areas returned to normal levels as did the NE concentration in the brain stem. Although disruptions in approach behavior appear to be correlated with 5-HT changes (and possibly brain stern NE) following TBZ, the disruptions in avoidance behavior did not correlate with any of the transmitters measured ( Aprison and Hingtgen, 1966a). These data suggest that another transmitter system, possibly the cholinergic system, may be involved in disruptions of certain types of avoidance behavior. IV. Behavioral Excitation and Decreases in Brain Acetylcholine

By administering specific drugs alone or in combination to animals it is possible to induce different behavioral patterns of normal, depressed, or enhanced responding. Using a RS40 SS20 avoidance schedule, we injected 2 mg/kg TBZ in rats and found decreased response rates (depression). When rats were preinjected with 50 mg/kg iproniazid before the same dose of TBZ was given, the response rates increased for a finite length of time (excitation). Since numerous studies show that the ACh concentration varies inversely with the degree of functional activity of the brain, ACh is a logical compound to measure in the brain of animals exhibiting enhanced or depressed behavioral response rates. Therefore we repeated the psychopharmacological experiments and killed trained rats by freezing during each behavioral state as well as in several control conditions. ACh concentrations were measured and found

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to be lowered in the telencephalon, midbrain and ponsrmedulla oblongata of rats exhibiting increased avoidance response rates, whereas, in depressed rats, only the midbrain ACh concentration increased (Tom et al., 1966). In a later study we extended these measures to include the complete period of excitation and have measured the changes in ACh plus 5-HT and NE concentrations in all three brain areas as well as in changes in increased response rates in the same rats (Aprison et al., 1968). Acetylcholine concentration in all three brain areas decreased and returned to normal levels at different times (Fig. 4). The time course of increased response rates correlated best with the changing ACh levels in the telencephalon. Both the 5-HT and NE concentrations remained elevated similar to the iproniazid control values during the period of behavioral excitation. However, the NE concentration in the midbrain showed a continuous decreasing trend toward naive control levels, the latter being approximately 61%of the iproniazid controls. These data suggested that changes in a cholinergic system in the telencephalon and a noradrenergic system in the midbrain probably operate together in the maintenance of the behavioral excitation ( Aprison et al., 1%8), with the former appearing to be more important. Since the ACh concentration changes follow the behavioral excitation so closely during most of the time period, a possible reason is suggested for the lack of correlation between disruptions of avoidance behavior and the changes in 5-HT, NE, and DA reported in the earlier studies. These data open a new dimension to our understanding of excitation produced under the above-mentioned circumstances because most studies in the past referred only to NE as the transmitter involved in the behavior described above ( Aprison et al., 1%8). The duration of behavioral excitation following iproniazid plus TBZ administration varies from animal to animal. It was noted that a small number of rats in our experiments did not exhibit excitation following the usual sequence of administration of these drugs. When nonexcited rats were killed at times comparable to the times when excited rats were killed, the ACh levels in the three brain areas did not vary significantly from control values ( Aprison and Hingtgen, 1969). These data support the suggestion that excitatory behavior and lowered ACh levels in brain ( especially telencephalon) are related. The question naturally arises: Why were some of these animals not excited? One explanation comes from the assumption that the cholinergic, serotonergic, and noradrenergic systems are interrelated in some way. The fall in brain ACh is probably due to the increased release of ACh (free) and its subsequent destruction by AChE in the postsynaptic membrane. Possibly the release of ACh is due to the biochemical changes

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in one or both of the other systems. The NE and 5-HT levels in the brains of the nonexcited rats may not have been elevated sufficiently to release ACh following one injection of iproniazid and TBZ. If ACh was not released in increased quantities at cholinergic synapses, its destruction by AChE would remain normal, and the levels of this neurotransmitter in brain tissue would not decrease. Therefore, a second

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injection of iproniazid was given to rats which did not show the typical elevated avoidance response rates ( excitation) following the usual iproniazid and TBZ injection sequence. The second iproniazid dose reversed the TBZ-induced depression of avoidance behavior and the animals response rates increased to above normal levels. In our laboratories, successive injections of 50 mg/kg iproniazid into rats at 12-hour intervals do not produce excitation until after the third injection. Therefore, a second dose of iproniazid could not account for the excitation now seen. However, in combination with the TBZ injection, the excitation of the previously nonexcited rats may be due to a change in the critical levels of 5-HT and NE in one or more brain areas which in turn cause a release of ACh, or ACh changes may produce a release of more NE. Our most recent studies indicate that the period of excitation can be prolonged or curtailed depending on the dose of atropine injected and the time sequence of the injections. Low doses (0.1 mg/kg) given 60 to 120 minutes before TBZ but after iproniazid caused an increase in the duration of excitation. Higher doses (0.8 mglkg) of atropine decreased the excitation period. These data with atropine support the involvement of the cholinergic system in behavioral excitation since low doses enhance the effect whereas high doses block it (Hingtgen and Aprison, 1970).

V.

Methodological Problems

Although the introduction of quantitative behavioral measures and the development of specific transmitter or modulator assays have facilitated the study of neurochemical-behavioral relationships, many methodological problems still remain. The ultimate experimental design for the determination of neurochemical correlates of behavior would involve the measurement of the neurochemical and behavioral parameters in the same animal at the same time. For example, in a study in which relationships between behavioral excitation and transmitter levels were being correlated, a minute by minute record of the excitatory behavior would be compared to minute by minute analyses of ACh, 5-HT, NE, etc., concentrations m discrete areas of the brain. Although our behavioral measures are kinetic, we are not yet able to assay CNS tissue in the live animal. The method of second choice, which we have described in various studies above, is to kill animals during various periods of behavioral responding, “freezing” the chemical changes at this point, and then determining levels following subsequent dissection and neurochemical assay. Another methodological shortcoming is that in our studies using concomitant measures of behavioral and neurochemical transmitter concentration changes, the best correlations should be obtained when the

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free or physiologically effective pool of the transmitter is measured. Unfortunately, neurochemists have accurately measured only totd neurohumoral concentrations in excised brain parts or metabolic turnover rates of a specific transmitter. Examination of such turnover rates of transmitter concentrations might provide additional insights into neurochemical-behavioral relationships. On the other hand, if turnover rate measurements reflmt the turnover of the total metabolic and functional pools rather than the turnover of only the free transmitter pool, these measures may not provide any better correlations with behavioral changes than were noted in our studies. It is assumed that an important factor in the stable emission of a specific learned response is the change in concentrations of the transmitters acting at the postsynaptic membrane of a group of important synapses involved in key neuronal pathways utilized for the performance of that particular behavior. If one is measuring the “correct” transmitter under conditions where specific drugs, metabolites, or enzyme inhibitors are injected, and the change in the effective concentration of its free pool follows the same time course as the change in the total transmitter level, then explanations as we have suggested become meaningful. Maximal efforts in our current work are being directed to overcoming the problem of measuring the change in the free transmitter pool in behavioral experiments where the response rates are continuously being measured. We hope that our studies reviewed here will stimulate many more collaborative efforts in this frontier area of neurochemicalbehavioral research.

VI.

Summary

A research strategy has been developed which continues to serve as a basis of designing new experiments in the field of neurochemical correlates of behavior. This strategy is based on the fact that evidence has accumulated to suggest that a number of nitrogen-containing compounds called neurotransmitters or modulators have major effects on the behavior of living organisms by their individual or grouped action on the central nervous system. It therefore becomes important to study how abnormal levels of such compounds have major effects on behavior and also to attempt to correlate temporal changes in their levels in different structures of the brain with concomitant changes in the behavior of the organism. This has been done by employing techniques which permit the investigator to measure quantitatively both the levels of the specific neurochemical agent in the brain and the behavior in the same experimental animal. Rats and pigeons were trained to emit stable response rates under various approach and avoidance schedules of reinforcement. Following the

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injection of a number of different biogenic amine precursors and drugs known to affect neurotransmitter or modulator levels, animals were killed at specific times, brains removed and dissected into specific areas and then assayed for one or more of the following compounds: acetylcholine, norepinephrine, serotonin, etc. Several correlations between biochemical (brain) and behavioral parameters have been found during the period of atypical or abnormal responding. Injections of the serotonin precursor, 5-hydroxytryptophan, produced a behavioral disruption in pigeons or rats working on approach schedules of reinforcement and caused an elevation of serotonin levels in the forebrain structures during the same time period that the behavior was depressed, with both parameters returning to normal IeveIs at the same time. However, the behavior of rats on an avoidance schedule was not changed after a 5-hydroxytryptophan injection even though the brain serotonin was elevated. The administration of a-methyl-m-tyrosine to pigeons on approach schedules resulted in a differential depression of brain serotonin, norepinephrine, and dopamine levels along with a reduction in response rates to about 20%of normal. The time course of the behavioral change appeared to follow the time course of the serotonin change more closely than that of the other amines measured. In additional studies on avoidance behavior, acetylcholine content of brain (parts) rapidly decreased during the excitation period following administration of tetrabenazine to iproniazid pretreated rats. f i e duration of increased response rates correlated best with the duration period of lowered acetylcholine levels in the telencephalon. In addition, serotonin and norepinephrine concentrations remained elevated except in the midbrain where norepinephrine showed a slow continuous decreasing trend toward naive control levels. These data suggest that changes in a telencephalic cholinergic system and a midbrain noradrenergic system probably operate in the maintenance of the behavioral excitation. Recent data with atropine support the involvement of the cholinergic system in behavioral excitation, since low doses enhance the effect whereas high doses block it. REFERENCES Aprison, M. H. (1958). J. Neurochem. 2, 197. Aprison, M. H. (1962). Recent Aduan. Biol. Psychiat. 4, 133. Aprison, M. H. (1965). Progr. Brain Res. 16, 48. Aprison, M. H., and Ferster, C. B. (1960). Expen'entia 16,159. Aprison, M. H., and Ferster, C . B. (1961a). J. Neurochem. 6, 350. Aprison, M. H., and Ferster, C. B. (1961b). Recent Advan. Biol. Psychiat. 3, 151. Aprison, M. H., and Ferster, C. B. (1961~).J. PhamcoZ. EzptZ. Therap. 131, 100. Aprison, M. H., and Hingtgen, J. N. (1964). Federation Proc. 23, 456.

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