THE INTEGRATED RELATIONSHIPS OF PROTRIPTYLINE, THYROID STATUS AND NORADRENERGIC MECHANISMS IN RAT BRAIN

Reasons for the observed lag-time for clinical efficacy of the tricyclic antidepressant drug, protriptyline, have been investigated. t-, • Our results demonstrate that chronic protriptyline administration resulta in several compensatory neural mechanisms: (1) changes in endogeneous cytoplasmic norepinephrine levels, (2) changes in norepinephrine turnover rate/recovery rate following the last dose in a chronic series of test drug administrations and (3) adaptations of the pre-synaptic receptor. The time course for these adaptive changes parallel the time required for the onset of their clinical effectiveness. We propose that acutely the action of the tricyclics on norepinephrine re-uptake is largely negated by compensatory adjustments in turnover rate of the neurotransmitter; but with chronic administration, adaptations in pre-synaptic alpha-receptors occur. This phenomena in turn reduces the extent to which the norepinephrine neurons can offset or compensate for the blockade of norepinephrine re-uptake. Specifically, pre-synaptic alpha-receptors become hyposensitive, and receptor tolerance develops; not to the direct action of the tricyclic drug on neurotransmitter re-uptake, but to the compensatory neural adjustments which offset the action of the acute tricyclic administration and delay their clinical efficacy.

ACKHOWLEDGEMEMT I would like to express deepest gratitude to my advisor, Dr. Al.vin K. Swonger, for hia unending support and encouragement throughout the entire course of thia research.
I am also grateful to Dr. Rupert Hanaond for allowing me to · use his facilities at Rhode Island Hospital, and also for his valuable professional evaluations.
I would also like to acknowledge the following individuals for their assistance in the preparation of this dissertation: Mr. Girolamo A. Ortolano, Mr. Kenneth R. Wunschel, Jr. and Mr. Stephen Wallace.
Lastly, a special and sincere "thank you" is extended to Dr. Milton w. Hamolsky, Chief of Medicine, at Rhode Island Hospital, who was always able to make time to "CHAT" with an overanxious graduate student.
Financial support for this investigation was provided by the Rhode Island Hospital Fund for Basic Research. iii.

DEDICATION
This theala ls dedicated -to ay father ED, for hi• inexh&uatlble ambition and unyieldin1 integrity. iv.

PREFACE
In the last 25 years many observations have documented the suggestion that a relationship exists between central norepinephrine function and affective disorders. The catecholamine hypothesis of affective disorders is one theory advanced by researchers. It propos• that some, if not all depressions are associated with an absolute or relative deficiency of catecholamines, in particular norepinephrine (ME), at the functionally important adrenergic receptor sites in the brain.
The tricyclic antidepressants (TCA), which normalize a depressed mood, block the active re-uptake of norepinephrine after release frOll the presynaptic nerve terminals. Thus both the ti.me and the concentration of norepinephrine availability is increased in the synaptic cleft for post-synaptic attachment. It is likely, however, that the ability of the tricyclics to block norepinephrine re-uptake, though the primary mechanism of action, may not be the only mechanism involved. This is evident frOll the observation that re-uptake blockade of NE by the tricyclics occurs within minutes after their administration; however, clinical efficacy in patients is not obvious until these drugs have been administered chronically (10-20 days). This observed lag-time for clinical efficacy suggests the existence of an alternate mechanisa of action for this class of antidepressants.
My research was designed to investigate three possible alternate mechanisms of action for the tricyclics, which might correlate bett91' with the requirement for chronic administration. v.
OUr results demonstrated that tricyclic antidepressant drugs, when administered chronically, do not alttr monoamine oxidase activity in vivo in any of the three brain regions we examined (corpus striatua, forebrain, and hypothalamus). Similarly, we were not able to identify a noradrengeric relationship other than those that were both toxic and lethal when thyroid hormone was administered with the tricyclic antidepressant drug Protriptyline. We did, however, observe that in the hyperthyroid condition norepinephrine turnover is decreased in hypothalamus when compared to control animals.
The results we obtained from chronic protriptyline administration and its effects on norepinephrine levels and turnover, as well as the results we obtained when yohimbine was employed are enlightening.
Chronic protriptyline administration produced decreased norepinephrine levels in hypothala11Us, and decreased norepinephrine turnover, when compared to saline administered controls. The alpha-receptor antagonist (yohimbine} produced only minor effects on norepinephrine levels and turnover when experimental animals were pretreated chronically (18 days) with protriptyline.
These results suggest an adaptive response (hypo-sensitivity) of the presynaptic alpha-receptor (autoreceptor), and this phenomena also occurs at the post-synaptic alpha-receptor. These results, suggesting receptor adaptation, indicated the existence of another mechanism of action for the tricyclic antidepressants. vi.

LIST OF TABLES
Honoaaine oxidase activity levels in three regions of rat brain undar control conditions.
The effect of triiodothyronine on monoamine oxidase activity in three brain regions.
In vivo effects of protriptyline administration on hyperthyroid animals.
In vivo toxic effects of simultaneous administration of triiodothyronine and protriptyline on the rat.
The effect of thyroidectomy on monoamine oxidase activity in three brain regions.
A comparison of slope and linear regression coefficients for saline treated animals following the administration of alpha-methyl-para-tyrosine (400 mg/kg, I.P.). 3.

LIST or ILLUSTRATIONS
The effect of thyroidectomy or administration of triiodothyronin• on norepinephrine levels in the hypothalamus.
Norepinephrine levels in hypothalamus following alpha-methyl-para-tyrosine administration. A dose-response curve.
Norepinephrin• depletion in hypothalamic tissue in control animals following the administration of alpha-methyl-para-tyrosine (400 mg/kg -I.P.).
Turnover of norepinephrine in the hypothalamus of rats treated with triiodothyronine (l mg/kg/day, 7 days, I.P.).
Norepinephrine depletion in hypothalamus as a measure of \ initial concentration, three hours after alpha-methyl-para-tyrosine administration (400 mg/kg/day, I.P.).
Turnover of norepinephrine in the hypothalamus of thyroidectomized animals.
Norepinephrine levels in hypothalamic tissue following acute, subchronic and chronic protriptyline administration.
Norepinephrine depletion in hypothalamic tissue following acute protriptyline administration.
Alpha-methyl-para-tyrosine dose-response curve in animals administered protriptyline chronically.
Norepinephrine depletion in hypothalamic tissue of animals receiving chronic protriptyline administration.
A comparison of turnover rates in hypothalamic tissue of rats administered protriptyline or saline chronically.

18.
Norepinephrine depletion as a measure of \ initial. concentration in the hypothalamic tissue of chronically treated protriptyline or saline animals.
Turnover rate/recovery rate for norepinephrine in rat hypothalamic tissue following acute, subchronic and chronic protriptyline administration.
Norepinephrine depletion in hypothalamic tissue following yohimbine administration.
Norepinephrine depletion in hypothalamus following chronic protriptyline administration, yohimbine administration and alpha-methyl-paratyrosine administration.
xi. This eviderx:e has implicated the involv .. ent of brain neural. systema which employ norepinephrlne (NE) aa their neurotransaitter Kobayashi,.!!_!!_., 1974;Ross and Reis, 1974;and Malbon, 1979). From these studies and theories the "catecholamine hypothesis for affective disorders" has been derived (Bunney and Davis, 1965;Schildkraut and Kety, 1967;Segal, !!_al., 1974;Baldessarini, 1975;Leonard, 1975;de la Fuente, 1979;and Stern, _!!al., 1980). This theory states that elevated mood is associated with an increased interaction of brain NE with its respective receptors; whereas, a depressed mood. is associated with a decreased interaction between NE and respective receptors. In addition, it is also proposed that some, if not all, depressions are associated with an absolute or relative deficierx:y of catecholamines, in particular NE, at the functionally important ad.renergic receptor sites in the brain (McClure, 1971;McClure, 1973;and Barchas, !.!. al., 1978). Srudies in man have also shown that drugs which cause depletion and inactivation of NE centrally produce sedation and/or depression, while drugs which increase or potentiate brain NE levels are associated with behavioral stimulation or excitation and generally exert an antidepressant effect.
An additional building block for this theory lies in the fact that tricyclic antidepressants (TCA's), which normalize a depressed mood, l 2 block th• active reuptake of NE after its release frOll the presynaptic n·erTe t ... inala (Rossi, 1976;Kant and Meyerhoff, 1977;and Sulser, et ~·, 1978). The reuptake process is believed to be the primary means for t8l'11inating the post-synaptic action of NE, and the tricyclics are able to increase the time for released NE to interact with respective target cell receptors (Wurtman, 1965;and Iversen, 1973).
Other evidence (Alpers andHimwich, 1972: Frazer, et al., 1978; and de Montigny and Aghajanian, 1978) suggests that the ability of the tricyclics to block NE reuptake, though the primary mechanism of action, may not be their only mechanism of action. Blockade of NE reuptake can be demonstrated after a single injection of a TCA drug (Callingham, 1967;Tod.rick and Tait, 1969;Schildkraut, et al., 1970;and Oswald, et al., 1972). Paradoxically, tricyclics do not exert a clinically observable antidepressant effect unless given chronically for two to three weeks. Therefore, blockade of the reuptake mechaniS11 cannot be reconciled with the time course (lag-time) for the chronic clinical efficacy of these drugs.
As a further building block adding support to the idea that tricyclics act by another mechanisa is the observation that TCA drugs inhibit monoaaine oxidase (MAO) activity in vitro (Gabay and Valcourt, 1968;Halaris, et !!_., 1973;Roth and Gillis, 19744;and Roth and Gillis, 197~b).
If specific enzymatic parameters regarding NE degradation diff9l' between normal individuals and patients prone to endogeneoua depression, then 3 tricyclica may exert their antidepressant influence• by altering or returnin1 to normal certain enzyme imbalances. These imbalances may only be changed during long-term tricyclic treat'llent, Le. MAO inhibition (Nies, et al., 1971;Youdia am Holzhauer, 1976;and Sullivan, et al., 1977).
Since NE availability at post-synaptic receptor sites is one of the fundamental correlates for mood changes (depression or anxiety) it has also been suggested that cellular NE levels rna.y decrease in individuals prone to endogeneous depression, and chronic administration of tricyclics reduce or prevent these changes (Schildkraut, et al., 1970;Schildkraut, et al., 1971; and Leonard. and Kafoe, 1976).
Contrary to these findings however, Neff and Costa (1967) and Alpers and Himwich (1972) have reported that chronic TCA treatment with imipramine or protriptyline did not induce changes in cellular NE levels in rat brain.
The effect of chronic tricyclic administration on NE turnover has also been suggested as a possible mechanism or parameter which may be altered by chronically administered tricyclics ( Schildkraut, et al., 1970;Schildkraut, et al., 1971; and Leonard and Kafoe, 1976); but this hypothesis is not supported by other studies (Neff and Costa, 1967; and Alpers and Himwich, 1972).
Another hypothesis this research investigated dealt with the effects and interrelationships between thyroid hormone administration and TCA drugs; and their individual and/or combined abilities to alleviate several depressive synaptoms observed in clinically depressed patients. Studies by ), Wheatley, (1972 and Slushc-, (1975), have reported confirming results that both patient mood and behavior improved when thyroid hormone was administered in conjunction with tricyclics, but the biochemical reasons for this improvement in mental health remains unresolved.
It has been documented that dysfunction of the thyroid gland has long been associated with mental illness (Gibson, 1962;Slusher, 1975;Singhal and Rastogi, 1978;and Bain and Walfish, 1978). Consequently, symptoma of depression are frequently associated with hypothyroidism; whereas, anxiety, fatigue and irritability are associated with hyperthyroidism. It may be proposed that endogeneous affective disorders are linked specifically to overall thyroid status. This hypothesis has been suggested and states that in hyperthyroidism there is increased receptor sensitivity (Emlen, et al., 1972;Engstran, et al., 1974;Engstra1, et al., 1975;and Strombom, et al., 1977), which induces, through canpensatory mechanisms, a reduced neuronal activity and decreased NE turnover, both of which result from a negative feedback mechanism (Strombom, !! al., 1977;Oppenheimer, 1979;and Sterling, 1979). Conversely, the opposite would occur in hypothyroidism. Therefore, concurrent administration of thyroid analogs with tricyclics should hasten the biochemical mechanisms of action elicited by chronic tricyclic treatment. This would yield a shorter time interval for clinical efficacy for these drugs and would result in an elevated mood (Earle, 1970;Wilson, et al., 1970;and Wheatley, 1972). 5 This aspect of this research study was designed t_o investigate and identify any possible central nervous system (CNS) biochaaical 111echanit111 which might be altered following both concurrent thyroid hormone and tricyclic administration which may accelerate improvement in a depressed patient.
In summary, this study investigated several hypotheses which were suggested as alternative mechanisms of action associated with tricyclic administration, and possibly resolve the existing controversy ovsr the occurrence of a lag-time, between drug administration and clinical efficacy. Also, this study was designed to identify the relationship between thyroid hormone administration, tricyclics and accelerated clinical improv .. ent in depressed patients.
The several hypotheses this research attempts to test follow: (l) that the requirement for chronic treatment with TCA drugs before clinical efficacy is observed is related to an action on mitochondrial MAO, and that the time course for this effect parallels the time course for clinical response.
(2) that simultaneous administration of thyroid hormone potentiates the action of TCA drugs through an effect on MAO activity, NE turnover or NE cellular levels • (3) that the time course in clinical efficacy of TCA drugs parallels the time course for development of presynaptic receptor subsensitivity (arxi concommitant changes in neurotransmittezo turnover).  Hare, (1928) as tyramine oxidases, and were further characterized by Blaschko, _!!al. (1937Blaschko, _!!al. ( , 1954. Pugh and Quastel (1937) were the first researchers to study MAO activity in nerve tissue. MAO activity varies greatly in different regions of the brain, with highest activity being observed in the hypothalamus and basal ganglia Youdi11, l973A, Youdim, 1973B;and Hazama, !!_al., 1976). The first studies on the intracellular localization of MAO were performed by Hawkins (1952) and Blaschko (1957). These independent investigators observed that in adult rat liver homogenates 70-80\ of the total MAO activity is found in the microsomal fraction of the subcellular fractionation preparation.
Evidence that MAO is localized. in the outer mitochondrial membrane was first observed and reported by Schnaitman, !!. al. (1967), with additional support added by de Champlain, !!_al. (1969). This close association between MAO and the mitochondrial membrane has complicated the purification of the enzyme, and the study of its pure chemical nature 7 and charact .. lstic enzymatic paraaet_.s can only be examined using subcellular mitochondrial preparations employing unsolublllzed partially purified enzyme preparations.
There is substantial evidence available which states that MAO exists in more than one form (Gorkin, 1966;Sandler and Youdi11, 1972;Houslay and Tipton, 1973;Neff and Yang, 1974;Neff, et al., 1974;and Youdim and Collins, 1975). Multiple forms of human brain mitochondrial MAO were first described by . The presence of different forms of MAO in tissue hccaogenates, mitochondrial preparatlona and in carefully prepared outer mitochondrial membrane preparations has been supported by the discovery of substrate selective inhibitors of MAO. The two drugs employed for the specific characterization of MAO forms were clorgyline and deprenil (Johnston, 1968;Knoll and Magyar, 1972;Houslay and Tipton, 1974;Neff, et al., 1974;and Bakhle and Youdim, 1975). Johnston, (1968), . proposed that clorgyline allows the investigator to distinguish with in vitro and in vivo rnet~odologies between two forms of brain MAO; termed Type A and Type B enzyme. Type A enzyme is .thus very sensitive to clorgyllne and preferentially deaminates 5hydroxytryptamine and norepinephrlne. Type B enzyme is specifically inhibited by deprenil and ls relatively insensitive to clorgyllne (Knoll and Magyar, 1972). For the Type B form of MAO preferential substrates include benzylamlne and phenylethylamine (Houslay and Tipton, 1974).
Dopamine, tyramine and tryptamine are substrates for both enzyme forms (Full_., 1972;Squires, 1972;Yang and Neff, 1973;and Neff and Yang, 1974). 8 There is also significant evidence suggesting that intraneuronal MAO plays an important role in regulating the functionally active poola of NE in the nerTe cell cytoplasa in the neurons of the CNS (Kopin, 1964;Bloom and Giarman, 1969;Green and Grahame-Smith, 1975;Youdim, l975a;Youdim, 1975b;and Youdim, l97Sc). The exact mechanism by which regulation for these homeostatic processes is accomplished by MAO has not yet been resolved. Intraneuronal MAO may metabolize neurotransmitter monoamines before and/or after reuptake into the presynaptic nerve ending, following stimulated release.
This degradative process appears to depend solely upon the activity of the MAO enzyme present in the nerve terminals and postsynaptic structures. The reuptake process also appears to be governed or regulated by the relative concentrations of free intra-and extraneuronal NE. MAO is apparently essential for keeping cytoplasmic NE in the neuron low, and within a specific steady-state equilibrium.
Under conditions when MAO activity is decreased (HAO-inhibitor administration) the concentration of total cytoplasmic NE rises. It has also been suggested (Bloom, 1963;Eiduson, !!_al., 1964;Kordon and Glowinski, 1970;and Gripois, 1975) that NE which is present in the presynaptic neuron cytoplasm may react with the MAO enzyme in the cytoplasm and cause a change in the steric conformational structure of the MAO enzyme. This may or may not be reversible. This staric conformational 9 change in MAO results in an activity cha.nge (i.e. increasing or decreasing MAO activity in the cytoplasm) and has been further documented by Iversen, et al. ( 1975). This change in enzyme structure, which haa been substrate induced, may alter the specific activity of MAO toward a specific substrate, and paradoxically not change the affinity for this substrate (NE).
Possibilities for these conformational changes in the enzyme molecule could occur by "folding" of MAO chains, since the enzyme exists as a monomer, dimer and tetramer (Collins and Southgate, 1970;Gerkin, 1972;Southgate, 1972;and Yasunobu and Oi, 1972). Each MAO chain has a molecular weight of 75,000. Folding of these chains could result in reducing the activity of the enzyme (i.e., less accessibility of the substrate (NE) to the enzyme's active sites); or folding may initiate a self-catalytic process, or enzyme inactivation process which may or may not be reversible. Similarly, it has been suggested (Eiduson, et al., 1964), that the MAO degrading mechanis11 consists of a three-point attachment to the enzyme surface by the catecholamine, which very likely involves the amine, one alpha hydrogen, and either the other alpha hydrogen or the beta-methylene, once again substantiating the complexity of this structure-activity relationship, .and the possible steric variations for inactivity that are available. It appears there is a self-regulatory mechaniSlll in the nerve cell cytoplasa which can increase intraneuronal NE concentrations or decrease NE levels. This process is dependent upon signals which are increased or decreased, sequential to neural firing rates.

lO
In moat theories of brain dysfunction and function, HAO is of significant importance. Any change in the HAO activity usually alters th• function of a specific CNS neurotransmitter, and in particular NE.
ll Since the possibility exists that alterations in the metabolism of central ta0noaminea cause several mental disorders, many investigatara have studied HAO activities in the CNS (Leonard, 1975;Youdim and Holzbauer, 1976;Ananth and Luchins, 1977;and Barchas, et al., 1978). Ashcroft and Sharman (1960) ware the first researchers to measure the metabolites of biogenic amines in the cerebral spinal fluid (CSF) of mentally ill patients. In certain types of endogeneous depression low CSF concentrations of 5-Hydroxy Indole Acetic Acid (5-HIAA) ware observed (Daneker, et al., 1966;van Praag, et al., 1970;and Sjostrcm, 1973). These findings also agree with the lower 5-hydroxytryptamine and 5-HIAA concentrations found in post-mortem brains of patients with depressive illness who committed suicide (Shaw, !!_al., 1967;and Bourne, et al., 1968).
Further supporot for a possible link between MAO and mental illness is documented by the beneficial effects achieved with drugs which inhibit MAO (Shaw and Hewland, 1973;Davidson, 1974;Neff, et al., 1974;Ananth and Lucins, 1977;and Campbell, et al., 1979). Since alterations in monoamine metabolism can occur during their degradation, variations in the function of MAO could ultimately result in a dysfunction of the neural pathways in which the respective monoamine (NE) plays a role as a specific transmitter substance. It is therefore important that the influence of tricyclics on HAO, when administered both acutely and chronically be examined (Roth and Gillis, 1975;Spiker and Pugh, 1976;Gabay and Achee, 1977;Honecker and Hill, 1977;Ponto, !!_al., 1977;Roth, 1978;and Ach" and Gabay, 1978). These studies are imperative if we are to understand the interrelationship between tricyclics and delayed clinical efficacy.
B. PROCEDURE AND METHODS l. General experimental procedures.
Male albino rat (Charles River Laboratories, C.D. Strain, COBS) weighing 250-400 grams were used for the determination of HAO activity levels, NE pool size levels and NE turnover studies, as well 12 as for all studies involving thyroid hormone interrelationships. All animals were housed in the animal care facility at Rhode Island Hospital and were maintained at the ambient temperature of 20-22° c. These animals were given Purina Rat Chow and watal' !! libitwn and were subjected to a 14-hour lighting cycle ( 0500-1900 hour · , ; • 2. Drugs employed.
Radioactive substrates for monoamine oxidase included c 14tryptamine bisuccinate and cl 4 -5-hydroxytryptamine. These chemical were purchased from New England Nuclear, Waltham, Mass. and are listed under the following catalogue numbers: NEC 259, tryptamine; NEC 225, 5hydroxytryptamine.
The tricyclic antidepressant drug employed in these studies was protriptyline (Vivactil) manufactured by Merck Sharp & Dohme, Pennsylvania. Protriptyline was chosen as our experimental drug since it is a secondary amine tricyclic which preferentially blocks reuptake of the catecholamine NE (Ross and Reny!,l975a;Ross and Renyi,l975a;Ros• and Reny!,l975b). Desipramine, desmethylchlorimipramine and nortriptyline are other similar secondary tricyclics. In contrast, 13 imipramine, amitriptyline and doxepin are classified as tertiary amines, and preferentially block th• reuptake of serotonin (Rossi, 1976).
MAO activity was measured by the method of Wurtman and Axelrod (l 963). Rats were decapitated and brain tissues were surgically excised.
The surgically removed ~issues were weighed by flotation in 0.5 M potassium phosphate buffer (pH 7.4), 15 ml total volume. Tissues were then homogenized by a polytron cell disrupter (Modal PT-10/20, with PT-10, ST generator, Brinkman, Switzerland) for two minutes at high speed (dial setting 10). All preparations were kept in an ice bath during cell disruption. Following cell disruption, 100 ul aliquots of each tissue sample was placed in assay tubes and reacted with labelled tryptamine bissuccinate or 5-hydroxytryptamine (New England Nuclear,NEC 259,and NEC 225), which were used as the substrates for the MAO enzyme.
The total assay volume was 1.2 ml (0.5 ml potassium buffer, pH 7.4; 0.5 ml distilled water; O.l ml tissue homogenate; and 0.1 ml substrate solution). Assay tubes were incubated for 20 minutes in a shaker water bath at 37° c. At the end of the incubation period, the reaction was quenched with 0.2 ml of 2.0 N HCL. This caused denaturing of MAO and prevented further substrate degradation (deamination). The deaminated radioactive products, 5-HIAA and Indole Acetic Acid (IAA), were then extracted by vigorous shaking with 6.0 ml of a toluene/ ether solution (l:l). This procedure is a modification of the original Wurtman and Axelrod (1963) method, and allows a more efficient extraction of radioactive products ( 5-HIAA and IAA). Efficiency was increased frcm 62+ 2\ to 82+ 4\ and was used in all subsequent MAO determinations (Kaiser, 1975A). Following a 10 minute centrifugation, (IEC International Centrifuge, Model PR-2) of this mixture (2000 rpm, 280 x g), a 4.0 ml aliquot of the organic phase was transferred to a liquid scintillation counting vial containing 10.0 ml phosphor [0.4 gram p-Bis-(2-5-phenyloxazolyl-benzene)] (POPOP), and 4.0 grams 2,5diphenyloxazole (PPO) per liter of toluene (Bray, 1960). These samples were then counted in a liquid scintillation counter (spectrophotometer) (Searle Analytical, Mark III) for 10 minutes. A small amount of 1 4 ctryptamine or 1 4 c-5-hydroxytryptamine (less than 0.3\) (Kaiser, 19758) was extracted by this procedure, but it creates a negligible error in the extracted radioactive samples.

DNA methociology.
Homogenized tissue aliquots of O.l ml. were taken from the cell preparations previously described (assay for MAO), placed in 15 .o ml graduated conical centrifuge tubes, and frozen until assayed. A standard DNA stock solution was prepared containing 200 mg of deoxyribonucleic acid sodium salt (highly polymerized, grade A, Cal-Biochem, La Jolla, CA.) per 100 ml of 1.0 N arrnoniwn hydroxide (NH 4 0H). This solution was stored at 4° c.
Standards of l.O, 2.0, 4.0, a.o and 12.0 ug DNA per sample were run with each assay in order to check their reproducibility and precision. All samples were then assayed by the method of Kissane and Robins (1958), which has been slightly modified to allow the use of larger tissue samples (Wunschel,personal co1DJ1unication). Modifications 15 involved the use of proportionately larger volWRea of reagents for the extraction procedures. All samples were assayed in triplicate and averaged.
The calculations involved in determining the DNA standard Curve, the linear regression analysis to obtain the regression coefficient, and a graphical representation of the data can be found in the Appendix sect ion, Figure   5. Scintillation counter efficiency adjustment and DNA calculations. After completing the enzymatic kinetic parameter studies, MAO activity levels were measured in the same three regions of rat brain in control animals. From these results it is apparent that in these brain The results fro• these exp.r!ments report that when the concentration of protriptyline in the preincubation media approaches l0-6 M, MAO enzyme activity in the corpus striatum is inhibited when either c 14 -tryptamine or c 14 -serotonin are used as substrate. Similar results were observed in both forebrain and hypothalamus. We interpret these results to be a non-specific inhibitory effect. daily injections) and chronically (18 daily injections}. A dose of 10 mg/kg/day protriptyline hydrochlcride dissolved in physiological saline was administered for this regiment of injections. This dose of protriptyline was selected since it appears extensively in the literature (Schildraut, et al., 1971;Leonard and Kafoe, 1976;Rossi, 1976).
This dose of protriptyline is between 10 and 20 times the dose used in treating clinical depression (Long, 1977).
The results of these experiments are presented in the appendix sect ion (Tables 3, 4, 5 and 6} • Our observations from these stud iea indicated that .!E_ vivo protriptyline administration had no consistent effect upon MAO activity in the brain tissues we examined regardless of length of administration (l, 3, 6 or 18 consecutive injections) and regardless of whether MAO activity was examined l, 3, 6, 12 or 24 hours after the last ~rug administration.

D. DISCUSSION
The activity of monoamine oxidase, one of the degrading enzymes in the metabolism of the neurotransmitter NE, has been measured under various experimental procedures in several regions of rat brain. This area of investigation was pursued, since the mechanism of action of the tricyclics remains controversial. Specific actions of the tricyclics on MAO activity may explain why chronic treatment is required to produce clinical efficacy.
We began our studies by initially developing and optimizing both our methodology and technique for isolating and quantitatively measuring tissue MAO. The numerical data we obtained for our enzymic kinetic parameters is supported by other investigators, and this adds support and validity to our procedures.
Examining the effects of acute (one daily injection), subchronic (three or six daily injections) or chronic (eighteen daily injections) protriptyline administration in vivo on MAO activity, we found no consistently significant effect of protriptyline on MAO activity in the brain regions we examined. 20 Having extensively researched the first hypothesis of this study.
and concluding that no specific in vivo effect for protriptyline on MAO activity could be substantiated, we proceeded to test our second hypothesis, "that thyroid hormone interacts with the tricyclics and may potentiate their effect on MAO activity, NE turnover, and on cellular NE levels." C H A P T E R III C HA P T E R III.
A. THE THYROID HORMONES: Thyroid status and its interrelationship with monoa.mine oxidase, norepinephrine biosynthesis and the tricyclic antidepressant drugs. 21 The mechanism of action of the thyroid hormones (T3 and T4) is of considerable interest because of the amazing diversity of their effects (Fleischmann, 1947;Fischer, et al., 1968;Prange, !!. al., 1971;Breese, et al. , l 974;Strombom, et al" 1977;Singhal and Rastogi, l 978 ) • These hormones exert profound effects on many enzymes and on al.most all organ systems. Th~ also play an important role in the complex biological processes involved in growth and cell differentiation (Oppenheimer, 1978). These hormones are thought to cause their actions by modulating or regulating the actions of other endogenous hormones or enzymes (Barchas, et al., 1978). This is accanplished by influencing thyroid hormone receptors associated with the nuclear chromatin (Sterling, 1979); and by al taring mitochondrial function (Sterling, et al., 1978). The concept of an intracellular, nuclear receptor for thyroid hormone is based on earlier receptor models, which have been theorized for the action of steroid hormone action on respective target cells.
In these models hormone action occurs via nuclear transcription (Samuels and Tsai, 1973;De Groot and Stausser, 1974;Samuels, et al., 1974;De Groot and Torresani, 1975;Mac Leod and Baxter, 1976;Chan and O'Malley, 1976). Other researchers (Gardner, 1975;Oppenheimer, 1975;Kurtz, et al., 1976;Surks and Oppenheimer, 1977), suggest that the hor110ne penetrates the plasma aaabrane and is bound by a specific cytosol receptor. This hormone-receptor complex enters the nucleus, and increased transcription of a genetic message for increased mRNA occurs. This in turn results in mRNA directing increased synthesis of specific nuclear proteins.
Ample evidence is available to support the view that an association exists between mental disturbances and altered levels of hormones such as adrenal corticoids, thyroid hormones, androgens and the estrogens (Rubin and Mandell, 1966;Mandell and Mandell, 1967;Dewhurst, et al., 1969;Glass, !! al., 1971;Wheatley, 1972). Only recently neuroendocrinologists have become interested in studying the effects of hormones on the functioning of the brain. Psychiatrists, however, have felt for many years that the solution to several etiological problems in psychiatry would becane manifest only after a better understanding of neuroendocrinological mechanisms became apparent, and in particular those involving thyroid hormone mechanisms .
Since the earliest descriptions of both hyperthyroidism and hypothyroidism, it has been suggested that certain psychiatric disorders may be manifestations of thyroid dysfunction. The clinically observable symptoms of thyroid dysfunction are sometimes of such severity that they bring the patient to the attention of the psychiatrist, and occasionally demand psychiatric hospitalization.
In clinical studies conducted (Jefferys, 1972;Thomson, et al., 1972;Mc I.arty, !!al., 1978;Nusynowitz and Young, 1979;Cohen and 23 Swigar, 1979), the prevalence of thyroid dysfunction in a psychiatric population (1320 patients) was 1.2\ for males and 2.0\ for females. It is doubtful whether these figures are significantly different from the prevalence of thyroid dysfunction in the general population (Whybrow, ~al., 1969;Henschke and Pain, 1977;Tunbridge, !!_al., 1977). This suggests that the importance of thyroid disease and mental disturbances may not be of important clinical significance. Therefore, the value of thyroid function testing in psychiatric patients is still unclear.
There are two criteria however, which must be discussed. First, thyroid dysfunction, (hypothyroidism or hyperthyroidism) may manifest itself as a psychiatric illness, and consequently some of the signs and symptans of mental derangement mimic those observed in thyroid dysfunction de la Fuente, 1979). Thus, the diagnosis of these conditions on overt clinical parameters is sometimes very difficult. The occurrence or prevalence of thyroid dysfunction in a psychiatrically ill population is uncertain, and has been reported to be higher than the gen9:1"al population (Nicholson, !!_al., 1976;Weinberg and Katzell, 1977). It has also been reported to be the same as the general population (Bursten, 1961;Gibson, 1962;Clower, !!al., 1969). It is also clear that the stress caused by various types of acute and chronic organic illness profoundly affect thyroid function tests (Johansson, et al., 1972;Mason, et al., 1973;Mason, 1975}. Although psychiatric illness may produce similar alterations in thyroid status, the magnitude, duration, and frequency of these thyroid changes are unclear and require examination.

24
It was our intent to study the interrelationships between MAO activity in various brain regions following both in vitro pre-incubation with thyroid hormone {T3 and T 4 ) and also following in vivo subcutaneous injection of thyroid hormone far varying time periods. Also, we studied the effects of thyroidectomy on MAO activity. The thyroid hormones are known to exert important influences on the central nervous system {CNS) {Fleischmann, 1947; Gibson, 1962;Harrison, 1964;Bain and Walfish, 1978;Oppenheimer, 1979;and Sterling, 1979), ani in particular on both peripheral and CNS MAO activity {Novick, 1961;Fisher, et al., 1968;Callingham and Lyles, 1974;Moonat, et al., 1975;Lyles and Callingham, 1976;Asaad and Clarke, 1978). It has been suggested by Novick, (1961) and Youdim and Holzbauer, (1976), that the thyroid hormones modify MAO activity, but the specific mechanisms by which these changes occur have not been resolved. There exists the possibility of modulating the biogenesis of mitochondria. This theory has been advanced by other researchers . (Novick, 1961;Youdi.m ani Holzhauer, 1976). Also, ·it has been suggested that the thyroid hormones can increase the synthesis of MAO itself (Fischer, et al., 1968).
This theory has been docwnentad for heart tissue Lyles ani Callingham, 1976). Al.so, it has been suggested that the thyroid hormones activate an inactive cytoplasmic MAO in brain tissue (Asaad and Clarke, 1976). Conversely, however, rat liver MAO activity was decreased following thyroid hormone administration (Moonat,et al.,197 5). Thus, any apparent thyroid hormone induced influences on MAO activity still remain unclear, and also tissue specific.
Similarly, emotional or mental stress may also be related to possible HAO-thyroid relationships, and these relationships should be investigated.
Also, Kennedy et al., (1977) reported increased NE turnover in brown adipose tissue (BAT) pads of the rat with statistically significant increases in NE levels in this tissue with T 4 treatment. Contradictory to these studies however, , has reported that thyroxine pretreatment caused a decrease in NE turnover and levels in the rat in both brain and heart tissue. Lastly, studies by Rastogi and Singhal (l974a,l974b) have reported an increase in NE levels in brain and total animal body weight when T 3 was administered.
Since this controversy exists, regarding the effects of T3 and T4 administration on NE levels and turnover, it was imperative to include the effects of the thyroid hormones on these NE parameters in this study.
Also, since a similarity exists between the clinical manifestations of depression and those of hypothyroidism, the effects of thyroidectomy 26 were investigated in relation to NE levels and turnover. Evidence from many clinical and animal studies indicate that a number of metabolic ar¥i psychic disturbances are common to both thyroid deficiency ani affective illness (Oppenheimer, 1979;Sterling, 1979).
Biochemical studies have demonstrated that both hypothyroidism and depression show a diminished response to infused NE (Schneckloth, et al., 1953;Prange, et al., 1967). Furthermore, psychological studies have suggested that the symptoms of myxedema, a severe form of hypothyroidism, makes an insidious appearance and is generally characterized by listlessness, lack of energy, slowness of speech, reduced sensory capacity, impairment of memory, social withdrawal and altered sleep patterns (F.ayrs, 1960;Kales, !!_al., 1967). Several of these psychological symptoms are commonly observed in depressed patients; slowness of speech, reduced sensory capacity, lack of energy, social withdrawal, and altered sleep patterns (Libow and Durrell, 1965;Whybrow, !!._al., 1969;Davenport and Dorcey, 1972;Davenport,.!!_ al., 1976).
Even though there appears to be sufficient evidence to implicate thyroid dysfunction with associated depression, it is still impossible at present to implicate abnormal thyroid function as a result or a cause of affective disorders. In order to possibly clarify this hypothesis and gain insight into whether or not alterations in neurotransmitter mechanisms (i.e. pool size and/or turnover) cause suppressed behavior and learning deficiencies, which are viewed in both depressed and hypothyroid patients, we felt that !t was imperative to investigate NE parameters during hypothyroidism.

27
Effective pharmacological treatments for mental illness have existed only during the last 30 years. This period has also witnessed a revolution regarding the care of psychiatric patients and has resulted in a decrease in the number of patients in both state and county mental facilities. This combination of both drug therapy and psychological and socioenvironmental treatment is responsible for the vastly improved prognosis for patients with mental illness (Barchas, et al., 1977;Berger, et al., 1977;Gold and Pottash, 1981;Spiker, 1981).
Since a high prevalence of mental dysfunction exists, and also a concommitant economic loss due to work disability and/or hospitalization, as well as the ever present danger of suicide, a persistent as well as compelling reason to search for a treatment for depression, that is both rapid, safe and convenient becanes necessary. Since treatment with thyroid hormones has been shown to enhance the pharmacological actions of several of the barbiturates (Conney and Garren, 1961;Prange, et al., 1966), and also since other clinical studies (Prange, et al., 1970A;Earle, 1970;Prange, !!_al., 19708;Wheatley, 1972;Whybrew, et al., 1972;Slusher, 1975;and Schmidt, 1977) as well as some animal investigation (Prange and Lipton, 1962; have documented an increase in drug efficacy, it has been theorized that the thyroid hormones may accelerate the antidepressant actions of the tricyclics or these other pharmacological agents (Cavalieri and Pitt-Rivers, 1981).
Keeping in mind these observations we designed our experimental protocol to incorporate both protriptyline and T 3 administration. We planned to 28 investigate their possible synergistic interrelationships with regard to MAO activity, NE levels and NE turnover in three tissues of the rat brain.
The occurrence of psychiatric symptoms in patients suffering frail hypothyroidism is well established (Asher, 1979;Rubin and Mandell, 1966;Oppenheimer, 1979;Sterling, 1979). Also, the similarity between the manifestations of anxiety states and hyperthyroidism is also well established (Dewhurst, et al., 1969;Singhal and Rastogi, 1978;Youdim and Holzbauer, 1976). Therefore, whether as a cause or an effect, there is evidence which implicates thyroid dysfunction with psychiatric illness.
Since this strong implication exists, between thyroid status and mental illness, it becomes more critical that effective, fast-acting pharmacological treatments for mental illness become available. These treatments will become available only after sufficient animal research has been conducted and only then will there be immense benefits for both patients and the general population.

B. PROCEDURES AND METHODS
l. General experimental procedures.

29
Hypothyroidism was induced by surgical thyroidectomy at 7 weeks of age (appraximately 175 grams body weight). a time at which thyroid function has stabilized to the adult level (Hammond, 1968;Kennedy, et al., 1977;Hamolsky, personal communication). Studies based on measurements of protein bound iodine (PBI) levels and basal metabolic rate have revealed that significant hypothyroidism does not develop in the rat until 7 to 8 weeks post-thyroidectomy (Hammond, 1968;Kennedy, et al.• 1977). Therefore, no measurements other than body weight recordings were made until after this time period had passed.
Also, it is appropriate to note that an age diffat'ence existed among the experimental groups being examined, and this partially accounts for the observed differences in total body weight. The thyroidectomized rats were approximately 18 weeks of age when sacrificed, whereas, thyroxine treated animals were 12 weeks of age. In each instance corresponding euthyroid animals of equivalent age served as paired controls for each experimental group. Also, to adjust for varying total body weights and animal age differences, DNA determinations were conducted on all brain tissue fractions used in MAO activity level measurements.
2. Assay methodology for norepinephr ine levels and turnover.
Norepinephrine (NE) in brain tissue was assayed by being extracted into o.~ N perchloric acid (PCA) with subsequent purification over alumina columns. The NE was oxidized to its trihydroxyindole derivative and assayed fluorometrically on a spectrophotofluorometer (Aminco-Bowman) by the method of Lund (1950).

30
The method for NE determinations is aa follows: brain tissue samples were hcmogenized in 0.4 N PCA (total volume, 10 ml) on a Brinkman polytron, high speed for 30 seconds. Following hemogenization another 25 ml of 0.4 N PCA was added to the homogenate (total voluae -35 ml). The homogenate was allowed to stand for one hour at 4° C, and shaken vigorously every 15 minutes until the hour had elapsed. The The alumina is now activated and can be used for NE adsorption. ~.
Alpha-methyl-para-tyrosine {methyl ester, Sigma Chemical Co.) was used to measure NE turnover. This agent canpetitively inhibits tyrosine hydroxylase, the rate-limiting enzyme in the synthetic pathway for NE biosynthesis. At zero time, 400 mg/kg alpha-methylpara-tyrosine {Brodie, et al., 1966) is injected interperitoneally (Ip) into both experimental and control animals, and these animals are sacrificed by decapitation at varying time intervals after alphamethyl-para-tyrosine administration. Depletion rates for all groups of animals were determined from 0 to 9 hours after alpha-methyl~para-tyrosine injection.
The validity of this method rests on several assumptions.
First, NE is assumed to be maintained at a steady state level, existing in a single depletable compartment within the nerve ending. In this situation only, can efflux rate be said to be equal to synthesis rate. 32 Second, that tyrosine hydroxylase ls and remains totally inhibited over the entire course of the turnover measurement. Third, that alphamethyl-para-tyrosine acts only by blocking NE synthesis, does not intarfere with tyrosine uptake (or required cofactors). Also, that the mechanisms for release or re-uptake of neurotransmitters are not altered.
Fourth, that the neuronal pools of both DOPA and dopamine are verry small, and thus cannot serve as a reservoir for NE synthesis after tyrosine hydroxylase has been inhibited. The fifth, and last criteria, is that alpha-methyl-para-tyrosine does not interfer with the experimental drug which is being studied. Similarly, the experimental drug cannot affect the enzyme inhibiting characteristics of alpha-methyl-paratyrosine on tyrosine hydroxylase activity.
In addition, an alpha-methyl-para-tyrosine dose-response curve for tyrosine hydroxylase was performed, resulting in a dose for alphamethyl-para-tyrosine which no longer produced an increase in the slope of the depletion curve for NE. It was at this dose (400 mg/kg) that all synthesis of NE by tyrosine hydroxylase had been inhibited ( Figure   2 ), (Nagatsu, et al., 1964;Spector, et al., 1965; Volicer and Reid, --_ _... Costa, 1971;Ostman-Smith, 1979).

C. RESULTS
l. Monoamine oxidase activity in three brain regions.
The effects of prelncubation with tissue homogenate with triiodothyronine (T3} on MAO activity in three rat brain regions (corpu• striatwa, forebrain and hypothalamus) has been measured using in vitro techniques and employing either cl4_tryptamine or cl4_ serotonin aa substrate. In these studies concentrations of T3 of lo-9 M and hightr significantly reduced MAO activity in all brain regions studied. HAO activity was reduced by 20-40\, regardless of length of preincubation time with r 3 (O, 15, 30 or 60 minutes) (Appendix section, Figure 7 and Tables 29-34). In the hypothalamic preparation at least 30 minutes of preincubation with T3 was required for HAO inhibition.
Similar results were obtained when preincubation times were O, 15 or 30 minutes; and whether using c 14 -tryptamine or cl4_serotonin as substrate.
At the ti.Dle of sacrifice T3 and T4 administered animals were found to have a decreased body weight ( 24 + 5 grams), whereas saline administered animals gained 54 !.. 11 grams .
One to eight days of S.C. administra~ion of T3 (l mg/kg/day) resulted in no consistent changes in HAO activity when the animals were sacrificed 24 hours after the last T3 injection, when cl 4 -serotonin was used as substrate (Table 2). Similar results were obtained when cl 4 -tryptamine was used as substrate and when thyroxine (T 4 ) was employed instead of T3 (Appendix section, Tables 35, 36, and 37).
At the conclusion of these in vivo T 3 and T4 studies, we --investigated the interactions of T3 administration and subsequent protriptyline administration on MAO activity in corpus striatum, forebrain and hypothalamus. We chose to use only T3 in these studies since it is the more active form of the thyroid hormones and is more potent, faster acting and more rapidly metabolized (Tata, 1964;; ., The effect of triiodothyronine on monoamine oxidase activity in three brain regions. Corpus Str iatum Forebrain Hypothalamus  Oppenheimer, 1979). No further data involving T~ administration are presented.

35
The T 3 and protriptyline studies were performed since previous investigators Wilson, et al., 1970;Earle, 1970;Whybrow, et al., 1972) have documented that thyroid hormone, when administered with tricyclics, prcxiuces a quicker clinical improvement in depressed patients. In these studies animals were made hyperthyroid by seven consecutive S.C. injections of T 3 (1 mg/kg/day), a regiment previously shown to prcxiuce physiological changes analogous to hyperthyroidism (Hammond, 1968;Kennedy, !_!al., 1977). On the seventh day following their last T 3 injection, animals received one injection of protriptyline (10 mg/kg -S.C.);three hours after receiving protriptyline all animals had perished;, convulsing prior to death (Table 3).
The next group of experiments involved simultaneous administration of r 3 (1 mg/kg -S.C.) and protriptyline (10 mg/kg -S.C.). Six hours after the second simultaneous injection of T3 and protriptyline all animals perished, convulsing prior to death (Table 4).
These results (Tables 3 and 4) agree with those of other investigators, and suggest that TJ pretreatment enhances the toxicity of many centrally acting drugs (Carrier and Buday, 1961;Conney and Garren, 1961;Prange and Lipton, 1962;Park, et al., 1972;. Animals surviving simultaneous administration of protriptyline (5 and 1 mg/kg) and T 3 (O.l mg/kg), resulted in MAO activity levels which   were not reproducible, between animals of the same group, and were inconsistent when one group was compared to another group. 38 Our thyroidectomized (Tx) and sham-operated animals underwent surgery at seven weeks of age and eight weeks prior to sacrifice.
During the eight week time interval, total body weight recordings of animal growth were maintained. During this interval Tx animals gained approximately 30 .!. 8 grams; paired controls (sham-operated) gained 150 to 200 grams. This observation, that Tx animals exhibit deficient growth patterns, has been previously documented (Fleischmann, 1947;Barker, 1949;Eayrs and Taylor, 1951).
The data obtained for MAO activity levels in the three brain regions we examined are presented in Table 5. As illustrated in Table 5 the Tx group clearly exhibits a significant reduction in MAO activity levels regardless of substrate employed or brain region examined, when these values are compared to age-matched sham-operated controls.
Since Tx decreased MAO activity in the brain regions we examined, our next group of experiments investigated possible changes in MAO activity in both Tx and sham-operated animals following protriptyline administration (10 mg/kg/day-S.C.). Animals were administered protriptyline acutely (one injection) and subchronlcally (3 or 6 consecutive daily injections, S .C.). Animals were sacrificed at varying time intervals following the last administration of protriptyline. These results are presented in the Appendix section (Tables ll, 12 and 13).   Numerical values for these data as well as for corpus striatum and forebrain are presented in the Appendix section (Table 38).
Prior to assessing NE turnover with MT a dose-response curve for tyrosine hydroxylase inhibition was conducted. Literature values (Brodie, et al., 1966;Spector, et al., 1967)   is significantly less than control (p ~.01, t-test).
in Figure 2. The nW'llerical values for this plot can be found in the Appendix section (Table 39). Figure 2 illustrates NE levels in the hypothalamus 3 hours after a. MT adrninistrat ion of 200, 4 00, 600 and 8 00 mg/kg -I.P •• We chose to use the 400 mg/kg dose in NE turnover studies.
A similar MT dose-response curve was perfarmed for TJ administered animals ( l mg/kg/day -7 days). Triiodothyronine administration increases tyrosine hydroxylase activity (Jacoby, 1975;Strombom, et al., 1977;Kennedy, !!. al., 1977), and this study was conducted to insure complete tyrosine hydroxylase inhibition at the 400 mg/kg dose. All data are presented in the Appendix section (Table 40)    sham-operated. controls are illustrated in Figure 6. Eight weeks after Tx, NE turnover is increased.; however, repletion has also occurred more quickly. Numerical data for these points, as well as for corpus striatwn and forebrain are listed in the Appendix section (Tables 43 and 44).

D. DISCUSSION
Our second hypothesis was that simultaneous administration of thyroid hormone potentiates the action of TCA drugs through an effect on MAO activity, NE turnover and/or NE cellular levels. Our data indicate that r 3 administration (l mg/kg/day -7 days) produces no inhibition of MAO activity in vivo, although MAO inhibition was observed. in vitro in these brain tissues. Similar results have been reported by other investigators for rat .heart and kidney (Callingham and Lyles, 1974;Lyles and Callingham, 1976;Asaad and Clark, 1978). Triiodothyronine administration produced. a significant decrease (p ~O.Ol) in NE levels and NE depletion (turnover) in these brain regions when canpared to saline administered animals.
Thyroidectomy, on the other hand, was associated with a decrease in MAO activity in these tissues. NE levels in these tissues were unchanged, however, an increase in both depletion and repletion rates was observed following MT administration.

LEGEND
In control anir:ials (-0-) NE has been d~pleted 58\ three hours after MT administration. In hyperthyroid animals (-x-) NE has been depleted 35\ three hours after MT administration (a 23\ difference in NE turnover rate). At-test for depletion rate resulted in a statistically significant difference between these two slopes (p <. 0 .01).
HOURS SACRIFICED AFTER CXMT ADMINISTRATION

SS
Also it was observed that when rats are made hyperthyroid by r 3 administration and are then administered a single injection of protriptyline, the animals begin to convulse and die within six hours. Similar toxic results have been viewed by other researchers when employing · imipramine (Prange and Lipton, 1962;. This observation remains unresolved. A possible suggestion for increased toxicity may be due to an increased interaction between T3 and increased receptor sensitivity in heart tissue to catecholamines (Bax, ~al., 1980;Chang and Kunos, 1981), thus accounting for the observed convulsions prior to death.
Reviewing our results, any interaction between thyroid hormone (T 3 ) and MAO activity in these brain regions is inconclusive, except for significantly decreased MAO levels following thyroidectomy. Zile, (1960), has reported that T 3 administration does not effect whole brain MAO activity; we are in agreement with these results. MAO activity in peripheral tissues (heart, liver and kidney) following TJ administration however, has been shown to increase (Callingham and Lyles, 1974;Lyles and Callingham, 1974;Asaad and Clark, 1978), indicating organ specificity.
We feel our results indicate that T 3 does not effect brain MAO activity, and any alterations in MAO activity are non-specific. Thus, any interaction between the tricyclics and thyroid hormone is not related to MAO.
NE levels and turnover following r 3 administration or after thyroidectomy indicate a decrease in turnover in hyperthyroid rats 56 in the brain regions we examined. These results are in agreement with . We observed decreased NE levels in the brain areas of hyperthyroid animals and these results are in agreement with Engstrom ( 1974). Landsberg and Axelrod (1968) presented data in heart tissue which is in agreement with our data for brain tissue, that A. NORLPINEPHRINE: Norepinephrine and its functional relationship with the presynaptic receptor.

57
The catecholamines (norepinephrine, epinephrine and dopamine) are low molecular weight substances that contain a catechol nucleus and an amine group. Norepinephrine (NE), one of these catecholamines, is synthesized and secreted by mamalian nerve tissue and serves an important function both in neural and endocrine integration.
The amino acid tyrosine (the substrate precursor to NE) is normally present in the circulation in levels between 10-15 mg/l (Spector, ~ al., 1963). Tyrosine is taken up from the bloodstream and concentrated within the brain and other neural tissues via an active transport mechanism (Chirigos, !!_al., 1960 Norepinephrine is primarily stored in a bound form in nerve cells within chrc:naffin granules or dense-core vesicles (Kirshner, 1974). 58 This bound catecholamine further interacts with adenosine triphosphate (ATP) and forms a tetracatecholamine-ATP complex. This salt is further bound to soluble proteins, the chromgranins, which are located within the storage particle. The inability of labelled exosenuous catecholamines to enter these storage granules suggests that the endogenous ATPamine complexes are still further combined with macromolecular components within the granules and form a still further stable complex (Weiner, 1970).
Results also suggest that NE is not located in a single pool, but that only a small percentage of neuronal NE is necessary for normal neural function Sulser and Sanders-Bush, 1970).
NE thus exists in a large 'storage pool' and a much smaller 'functional pool' both of which are located within the nerve cell axoplasm.
Currently it is suggested that the functional pool contains newly synthesized NE, while the sitorage pool contains catecholamine which has been in the nerve cell for longer periods of time. This assumption is based upon the observation that newly synthesized NE is preferentially neleased during enhanced neural firing. Kopin, ~al. (1968) has danonstrated that newly synthesized NE is preferentially released during stimulation of isolated cat spleen. In vivo studies have reported that utilization of newly synthesized NE is increased in response to certain stressful conditions; whereas, utilization of NE stored for longer time intervals was not affected (Thierry, et al. 1971;Glowinski, et al., 1972). 59 Evidenc:e also exists to suggest that in addition to containing newly synthesized NE, the functional pool also contains NE which has been accumulated via the re-uptake process. Therefore, both newly synthesized NE as well as conserved NE are preferentially utilized through release in response to i11111ediate stressful conditions (Potter, et al., 1962;Chidsey and Harrison, 1963). NE released presynaptically by stimulation is primarily transported back into the presynaptic neuron (80\) and is then once again bound in these presynaptic vesicles. This prevents degradation by MAO and preserves intracellular NE (Kopin, 1966;Titus and Dengler, 1966).
The re-uptake of NE by active transport and binding into the presynaptic vesicles protects NE from depletion and destruction by MAO.
MAO, therefore plays an important role in the regulation of the "free" intracellular levels of NE. Very small quanta of the presynaptically released NE reaches the post-synaptic target tissue. Synaptic NE may be 0-methylated and excreted, or can be excreted "free" or conjugated as glucuronides or sulfonates (Kopin, 1964;Axelrod, 1966).
Catechol-0-methyl-transferase (COMT), another enzyme responsible for the degradation of catecholamines, is apparently unimportant in the metabolization of intraneuronal NE. COMT is significantly involved in the catabolism of circulating catecholamines, which occurs chiefly in the liver and kidney.
The third hypothesis this research attempts to investigate relates changes in cellular NE levels and turnover to acute subchronic and chronic tricyclic administration, and these changes parallel their delayed clinical efficacy. As stated earlier the "catecholamine hypothesis for affective disorders" proposes that some, if not all depressions are associated with an absolute or relative deficiency of catecholamines, particularly NE, at the functionally important adrenergic receptor sites in the brain. Consequently, studies have shown that drugs which cause depletion and inactivation of NE in brain produce sedation or depression, whereas, drugs which increase or potentiate brain NE are associated with behavioral stimulation or excitation and. generally produce or exert an antidepressant effect in man.
Because a lag-time ls evident between tricyclic administration and clinical efficacy (Schildkraut, et al., 1970;Schildkraut, .!! al., 1971;Sulser and Sanders-Bush, 1971;Leonard, 1975;Leonard and Kafoe, 1976;and Berger, 1978),this aspect of this study will examine possible changes in intracellular NE and also turnover rates and attempts to observe if these changes parallel the time course for clinical efficacy for this class of antidepressant agents.
Because brain NE levels are maintained constant in the face of conditions which alter nerve firing rate, it has been suggested that NE which is utilized during enhanced neuronal firing is replaced by an increase in NE synthesis (Weiner, 1970;Costa and Meek, 1974;Bjorklund, ~al., 1976;and Ostman-Smith, 1979). This compensatory mechanism has been termed "the theory of steady-state kinetics." The mechanism by which increased noradrenergic nerve activity results in the stimulation of increased NE synthesis is not known. The most concrete explanation however, is that nerve stimulation releases NE from a nerve terminal, which consequently results in decreased intraneuronal NE concentrations. Since NE competetlvely inhibits its own synthesis by interfering with the pteridine cofactor required by tyrosine hydroxylase (Udenfriend, et al., 1965;Gordon, !! al., 1966;Kennedy, et al., 1977), nerve stimulation would result in a decrease in negative feedback inhibition of tyrosine hydroxylase. The converse also applies 62 (Spector, !!_al., 1965;Spector, 1966;Spector, et al., 1967;and Weiner, 1970). It is therefore appropriate for one to conclude that NE synthesis is controlled by a small, chemically undetectable pool of intraneuronal cytoplasmic NE.
The term "turnover" refers to a process of renewal of a substance in an organ. The concept of turnover implies that the substance being renewed exists at some steady-state level, which is balanced by identical rates of influx and efflux. In the case of brain NE the rate of NE influx equals the rate of NE syntehsis, since endogenous NE cannot enter the brain via the circulation due to the blood-brain barrier (Dobbing, 1961;Guroff and Udenfriend, 1962;Bertler, et al., 1966;and Oldendorf, 1974 give the investigator an indication of the rate at which brain NE is synthesized and utilized, which then, may be used as an indicator of central noradrenergic activity. Many methods for measuring NE turnover exist and have been reviewed extensively by others (Costa and Neff, 1968;Costa, 1970;Costa and Neff, 1970;and Weiner, 1974). The method employed here is a measure11ent of the rate of decline of endogenous brain NE after inhibition of tyrosine hydroxylase by alpha-methyl-para-tyrosine. This method was first described by Brodie, et al., (1966). 63 Synthesis inhibition is accomplished by employing alpha-methylpara-tyrosine, (MT), an in vivo reversible inhibitor of tyrosine hydroxylas• (TH), the rate-limiting step in NE synthesis (Spector, et al., 1965;and Spector, 1968). After blockade of NE synthesis, brain NE levels decline in a monoexponential fashion (Brodie, et al., 1966), i.e. the concentration of NE declines at a rate that is proportional to the NE concentration remaining in the neuronal cytoplaSTI at any given time.
This phenomena may be viewed as "the rate of NE utilization," and at the steady-state, "the rate of synthesis," which is proportional to cytoplasmic NE concentrations.
Therefore it was imperative that we investigate the noradrenergic effects caused by tricyclic administration during acute, subchronic and chronic treatment schedules, and thus possibly identify biochemical changes in NE levels and turnover rates. These data may then add support in identifying noradrenergic mechanisms necessary for latent clinical efficacy.

Presynaptic autoreceptor modulation of noradrenergic mechanisms.
Before introducing the concept "presynaptic alpha-receptors" (Norberg and Hamerger, 1964;Langer, 1974;Berthelsen and Pettinger, 1977;Malbon, 1979;U'Prichard and Synder, 1979), it is first necessary to examine the processes involved in NE release from presynaptic noradrenergic nerve terminals. NE is synthesized from the amino acid tyrosine, and is stored in vesicles in the neuroplasm. These vesicles in turn migrate to the nerve cell membrane, and release transmitter when an action potential arrives. It has also been demonstrated that an 64 influx of calcium into the neuroplasm causes these NE containing vesicles to migrate to the neuronal membrane (Rubin, 1970;Blaustein, et al., 1972;Phillis, 197~). The mechanism involved in this migration -is still unclear. At the manbrane, fusion occurs. and these vesicles discharge their contents into the synaptic cleft. These vesicles then re-form after discharge and are again filled with transmitter fra1 the neuroplasm.
Brown and Gillespie (1957) first suggested that this release may be modulated by another mechanisa, in addition to the arrival of an action potential. They also noted that the alpha-blocking agent phenoxybenzamine increased the overflow of NE in cat spleen caused by repeated nerve stimulation.
Other studies conducted by Starke, et al., ( 1971); and Enero, et al., (1972) using other alpha-receptor blocking agents also caused an increase in overflow of transmitter following repeated nerve stimulation. These observations led to the hypothesis that alpha-receptors are present in the outer surface of adr~ -nembranes of neurons and are involved in the regulation of NE relaase via a negative feedback mechanism. Thus, as NE is released into the synaptic cleft, it tends to limit its own release, by stimulating a presynaptic inhibitory alphareceptor {Kirpekar and Puig, 1971;Starke, 1972;Starke and Schumann, 1972;Rochette, et al., 1976;Lorenz, et al., 1979). This phenomenon occurs in peripheral tissues as well as in the CNS (Bunney and Aghajanian, 1975;Carlsson, 1975;Strombom, 1975;I.anger and Dubocovich, 1977;Baraban and Aghajanian, 1980). When presynaptic alphareceptors are stilllulated by an appropriate agonist or blocked by an appropriate antagonist (oxymetazoline, phenoxybenzamine, or phentolamine) (Kapur and Mottram, 1978;l<alsner and Chan, 1979;Werner, et al., 1979;Baraban and Aghajanian, 1980) the negative feedback mechanism inhibiting or facilitating NE release is altered. The sequential release of NE resulting in transmitter overflow thus occurs "or" is prevented. It also appears that the presynaptic alpha-receptors modulate the release of NE by controlling the influx of calcium into the presynaptic nerve ending (Langer, et al., 1975). In summary, the presynaptic alphareceptor mediates NE release, which is dependent upon synaptic neurotransmitter concentrations, and a functionally operative negative feedback mechanism. Activation of presynaptic alpha-receptors (autoreceptors) leads to a decrease in transmitter release, while blockade of these alpha-receptors results in an increase of NE release following nerve stimulation (overflow) (Langer, 1974;Langer, 1977;Starke, et al., 1977).
The physiological and biochemical events reulting from the activation or inactivation of alpha-receptors in the CNS remains unclear.
Some evidence (Segal, et al., 1975;Greengard, et al., 1976;Skolnick and Daly, 1977;l<anof and Greengard, 1978;Hall and Ogren, 1981) in tu=n activates cyclic-AMP generating systems. Other studies (Glauhiger and Lefkowitz, 1977;Williams and Lefkowitz, 1977) indicate that the number of CNS alpha-receptors can be altered in .a variety of brain areas by chronic treatment with specific drugs and hormones, which in turn results in altered CNS mechanisms. Still other studies suggest that drugs and hormones may sensitize or desensitize these pre-and post-66 synaptic alpha-receptOPs (Comsa, 1950;Thibault, 1956;Potter, et al., 1962;Haggendal and Lindqvist, 1964;Lee, et al., 1967;Lipton, et al., 1968 ;Prange, et al. , 197 0) • It was our intent in this study to examine the effects of the alpha-receptor antagonist (yohimbine) on NE levels and turnover following chronic tricyclic administration.
We felt that if receptor adaptation occurs with chronic tricyclic treatment, employing yohimbine would allow us the opportunity to observe these changes in both NE levels and turnover rate. These results would then allow us to specifically test whether adaptive changes in presynaptic receptors might be an important component of the mechanism of chronic tricyclic action.

B. METHODS AND PROCEDURES
Initial experiments involving NE levels and turnover were performed in three areas of rat brain (corpus striatum, forebrain, and hypothalamus) in control animals {saline injected), protriptyline administered (10 mg/kg/day for l, 3, 6, and 18 days) and in thyroidectomized animals which also received protriptyline acutely, subchronically and chronically.
With the conclusion of these studies, the examination of both corpus striatWri and forebrain was discontinued, since the primary neurotransmitter in the corpus striatum is dopamine (Hillarp, et al., 1966;Snyder and Coyle, 1969;Iversen, 1973;Hornykiewicz, 1973;Harris and Baldessarini, 1973), and 5-hydroxytryptamine {serotonin) for forebrain (Schwark and Keesey, 1975;Saavedra, et al., 1976;Bjorklund, et al., 1976). It was felt that further examination of these tissues would be unnecessary, and unrelated to the effects of chronic protriptyline administration on norepinephrine turnover. All studies involving yohimbine are performed only with hypothalamic tissue preparations.
We chose to examine only the hypothalamus in our receptor-interaction studies because we considered the hypothalamus an important target tissue for the actions of protriptyline. This area of the brain is responsible for many neuroendocrine transmitter interactions (Stokes, !!._al., 1981) as well as regulating or modulating both physiological and emotional responses (Kordon and Glowinski, 1970;Dewhurst, et al., 1968;Kobayashi, et al., 197~, de la Fuente, 1979).

Yohimbine our presynaptic autoreceptor antagonist was obtained from
Sigma Chemical Company, St. Louis, Missouri.
Our experimental protocol for drug administration involving our norepinephrine turnover studies is complicated and has been diagrammed 68 below for better understanding. This procedure allowed us to examine norepinephrine turnover when protriptyline and or our test drug, yohimbine, were at maximal plasma levels and optimally effective. results have been reported by others examining whole brain preparations (Schildkraut, et al., 1970;Schildkraut, et al., 1971;Roffler-Tarlov, et al. , 197 3 ) • Figure 8 presents data for NE depletion in hypothalamic tissue following acute protriptyline administration (10 mg/kg -S.C.), and followed by MT administration (400 mg/kg -I.P.). MT was administered three hours after protriptyline. These animals were sacrificed at various times after MT administration. A t-test for comparison of depletion rates between saline administered ( Figure 3) and acute protriptyline administration resulted in a statistically significant difference in turnover rate (p4'_0,005). This result is consistent with Leonard and Kafoe (1976). Numerical values for these data are listed in the appendix section (Table 47).  (Table 48).  (Table 51). These data suggest that tyrosine hydroxylase (TH) is completely inhibited three hours after MT administration at any of the above doses. Thus, the 400 mg/kg dose was employed for our turnover studies in chronically administered animals.  (Table 52).  TITLE: A comparison of turnover rates in hypothalamic tissue in rats administered protriptyline or saline chronically LEGEND Animals were administered protriptyline (10 mg/kg/day, 18 days) (-x-) or saline (-0-). Three hours after the final injection of protriptyline or saline, animals were administered MT (400 mg/kg -I.P.). Animals were sacrificed at various times after MT administration (0, 1, 2, 3, 4.5, 6 and 9 hours). All values represent the

HEAN + S.E. for at least 3 experimental determinations. A t-test
for comparison of depletion rates between 0 and 3 hours after HT administration indicates taht a statistically significant difference existed in depletion rate between these two groups (p L0.01). difference in NE depletion is recorded between these treatment conditions. Tables 6 and 7 list both the slope and regression coefficient for saline and protriptyline treated animals. Table 57 in the appendix section presents the statistical results when NE depletion was compared between these two groups. Figure 14 is a turnover rate/recovery rate plot for the data presented in Tables 6 and 7. These data suggest that adaptations in NE turnover rate/recovery rate have resulted from chronic protriptyline administration.
A t-test for the comparison of turnover rate/recovery rate indicated that acute turnover rate/recovery rate is significantly different when compared to either subchronic or chronic turnover rate/ recovery rate (p"90.001). No statistically significant difference was observed when subchronic administration was compared to chronic administration for turnover rate/recovery rate.

Yohimbine studies and norepinephrine turnover.
Studies determin~ng NE levels and turnover following yohimbine, protriptyline and MT administration were conducted. The first plot   TITLE: Turnover rate/recovery rate for NE in rat hypothalamic tissue following acute, subchronic and chronic protriptyline administration LEGEND The turnover rate/recoveryrate for NE in rat hypothalamic tissue following acute, subchronic and chronic protriptyline administration has been plotted and the slope for these 3 injection schedules has been determined. The acute turnover rate/recovery rate slope (-t:::I-) is significantly different (pL..0.001) from either subchronic turnover rate/recovery rate (-x-) or chronic turnover rate/recovery rate (-o-).  (Table S3).   Figure 11).
This study indicates that the effects of yohimbine appear nullified 99 when protriptyline has been administered chronically. In control studies, it should be recalled that NE was depleted 56% and 70% three hours after yohimbine administration (Figures 15 and 16). Numerical values for each time point are listed in the appendix section (Table 56).

D. DISCUSSION
To test our third hypothesis "that chronic protriptyline administration may induce changes in specific NE ?arameters and the presynaptic autoreceptor" studies were designed to evaluate changes in NE levels and turnover, employing the alpha-receptor antagonist yohimbine after chronic protriptyline administration. The results of these studies strongly suggest that chronic administration of protriptyline alters presynaptic alpha-receptors. Chronic protriptyline administration decreases NE levels in the hypothalamus; a finding which at first seems contradictory to the "catecholamine hypothesis of affective disorders", which suggests that increased NE at post-synaptic receptor sites is required to alleviate depression. Increased neurotransmitter levels would indicate increased release following an action potential, and decreased NE levels would indicate decreased neurotransmitter release. 100 Secondly, chronically administered protriptyline decreases NE turnover when compared to control animals, and exhibits increased turnover when compared to acute protriptyline administration. This result also seems contrary to the "catecholamine hypothesis of affective disorders" since one would predict that increases in turnover (i.e. synthetic rate) are necessary (to alleviate depression) to maintain the constant efflux of NE to the post-synaptic receptor.
Thirdly, yohimbine administration {which in control animals illicited NE depletion), to chronically administered protriptyline animals has no effect or minimal effects on NE depletion ( Figure 17).
This suggests that chronic protriptyline administration may produce subsensitivity of alpha-2-(presynaptic)-receptors, which in turn modify NE release to the synapse.
These results suggest the existence of presynaptic alpha-2receptors are undergoing adaptation when protriptyline is administered chronically to experimental animals. The implications of these adaptive mechanisms and their relationships to the catecholamine hypothesis of affective disorders will be discussed in Chapter v.

A. SUMMARY AND CONCLUSIONS
Our studies attempted to identify possible alternate mechanisms of action in the CNS when the tricyclic, protriptyline, was administered chronically to rats. These studies also examined the effects of both protriptyline and thyroid hormone administration on monoamine oxidase (HAO) activity. The following results were observed: (1) In vitro preincubation with protriptyline inhibits MAO activity in all three brain regions examined.
(2) In vivo administration of protriptyline has varying and InconsTstant effects on MAO activity.
(3) Monoamine oxidase activity is inhibited in vitro by preincubation with triiodothyronine.
(4) Triiodothyronine administration has no in vivo effect on HAO activity in the three brain regions-examined.
( 5) Thyroidectomy decreases MAO activity in all three brain regions examined.
(6) The simultaneous administration of protriptyline and triiodothyronine have toxic lethal effects on animals when administered at the dose ranges we examined, and in those animals which survived simultaneous administration, monoamine oxidase data was inconsistent and inconclusive.
(7) Protriptyline administration to thyroidectomized animals results in variable and inconsistent effects on MAO activity.
Several of these observations (1, 3, 4) have been documented by others (Gabay and Valcourt, 1968A;Roth and Gillis, 1974A;Roth, 1977). In addition, other animal studies have documented toxic effects of concurrent triiodothyronine administration with the tricyclic lmipramine, or with other pharmacological agents (Carrier and Buday, 1961;Conney and Garren, 1961;Prange and Lipton, 1962;. No documentation could be found identifying toxic interactions when protriptyline was administered with triiodothyronine.
At the completion of these studies it was concluded that any interaction between protriptyline and MAO activity was non-specific and our first hypothesis "that the requirement for chronic treatment with tricyclics before clinical efficacy is observed is related to an action on mitochondrial MAO, and that the time course for this effect parallels the time course for clinical response" was incorrect.
Similarly, the toxic interactions of simultaneous triiodothyronine and protriptyline administration, though of interest, also remain unresolved.
Continuing, we next examined the effects of thyroidectomy and both triiodothyronine (T3) and protriptyline administration on NE levels and turnover. We observed the following results: (1) That seven days of triiodothyronine administration (1 mg/kg/day) results in decreased NE pool size in the brain areas examined.
( 2) That thyroidectomy induces ~ change in NE pool size in these same brain regions.
(3) That both acute and chronic protriptyline administration (10 mg/kg/day) result in decreased NE pool size.
( 4) That NE turnover is decreased in tr iiodothyronine treated animals when compared to saline administered control animals.
(5) That NE turnover in thyroidectomized animals is increased when compared to Sham-operated control animals.
(6) That NE turnover in the hypothalamus of both acutely and chronically administered protriptyline rats is decreased when compared to saline administered control animals.
The possible physiological significance of alterations in brain NE levels and turnover after triiodothyronine administration, and following acute and chronic protriptyline administration may be postulated to be related to compensatory and adaptive mechanisms, resulting from changes in noradrenergic alpha-receptor sensitivity. Decreases in both NE levels and turnover were observed after seven days of T3  (Engstrom, et al., 1974;Engstrom, et al., 1975;Strombom, et al., 1977).
Increased receptor sensitivity may also be responsible for the toxic side effects of protriptyline. Long (1977) has reported animal results in which heart palpitations and arrhythmias have occurred when T3 was administered with other pharmacological agents. Possibly T3 increases receptor sensitivity and sequential protriptyline administration results in an increase ' in toxic effects.
Both acute and chronic protriptyline administration induced changes in NE levels and turnover in hypothalamus. The acute effects of the tricyclics on brain noradrenergic parameters have been documented by many investigators (Schildkraut, et al., 1970;Schildkraut, et al., Roffler-Tarlov, et al., 1973;Leonard and Kafoe, 1976). Decreases in NE levels have been postulated to result from the negative feedback inhibition by NE on tyrosine hydroxylase (TH) activity, via the pteridine cofactor site. Decreased turnover also results from negative feedback inhibition. Alternations in both of these noradrenergic parameters result from the tricyclics primary mechanism of action (re-uptake blockage) and may be viewed as compensatory noradrenergic neural adjustments.
In our chronic protriptyline studies we observed decreased hypothalamic tissue levels of NE and also a decrease in NE turnover rate compared to chronic saline administered controls. These results suggest a decrease in NE synthetic rate. Other investigators have also demonstrated changes in other noradrenergic parameters which also suggest decreased synthetic rate. Handel, et al., (1973) demonstrated that chronic imipramine treatment decreases midbrain tyrosine hydroxylase (TH) activity. Schildkraut (1965) and Schildkraut, et al., (1972Schildkraut, et al., ( , 1975 demonstrated that chronic treatment with amitriptyline or imipramine decreased the excretion rate of norepinephrine metabolites in the CSF.
We postulate that the observed decrease in NE levels and turnover rate is an adaptive response resulting from increased efficiency of transmission as a consequence of re-uptake blockade.
Our studies suggest that adaptations in the presynaptic alphareceptor in the hypothalamus have occurred following chronic protriptyline administration. In our final studies we employed yohimbine in an attempt 105 to further examine whether alpha-receptor adaptations have occurred and whether such changes correlate with the lag-time observed for clinical efficacy.
The following results were obtained when yohimbine was employed: (1) Yohimbine administration (20.0 mg/kg, I.P.) produced a 56% decrease in total cytoplasmic NE levels in control animals three hours after test drug administration.
These results can be explained in the following manner. Yohimbine, agents. Post-synaptic receptor subsensitivity has already been documented in peripheral systems (Langer and Luchelli-Fortis, 1977).

B. SUMMARY
We have documented several compensatory neural mechanisms which occur during the chronic treatment period with tricyclics: (1) changes in endogeneous cytoplasmic total NE levels, (2) changes in NE turnover rate/recovery rate following the last dose in a chronic series of test drug administrations, and (3) adaptations in the pre-synaptic receptor.
1. Adaptations in total NE tissue levels.
Decreases in cytoplasmic NE levels following chronic protriptyline treatment ( Figure 7) suggest a more efficient utilization of NE (less neurotransmitter is required to maintain normal physiologic function).
Clinical studies support this postulate and report increases in H 3 -NMN (a COMT metabolite) and decreases in H 3 -DCM (MAO metabolies, 3, 4dihydroxymandelic acid and 3, 4-dihydroxy-phenyl-glycol) following chronic tricyclic administration. These data indicate that more neurotransmitter is available to synaptic receptors (and COMT), despite an observed reduction in total endogeneous cytoplasmic NE levels.

Adaptations in NE turnover.
Acute protriptyline administration results in a decreased slope for NE turnover~ three and six hours after tricyclic administration, when compared to saline treated animals. Twenty-four hours after protriptyline administration the slope for NE turnover has returned to control values (T~bles 6 and 7). This observation has been observed by others when measuring H 3 -metabolites (H 3 -NMN) followin g H 3 -NE infusion, and employing acutely imipramine or DMI pretreatment. Since re-uptake blockade by tricyclics results in an accumulation of NE in the synapse, this accumulation would stimulate pre-synaptic alpha-receptors and in turn a negative feedback mechanism would become operative; thus resulting in a decrease in the release of pre-synaptic NE .. This mechanism is responsible for the decrease in NE turnover following acute protriptyline administration.
With subchronic (6 day) protriptyline treatmer.t, the slope for NE turnover appears to be elevated at both the three and six hour time points after the last protriptyline injection, when these slopes for turnover rate are compared to the turnover rates for acute protriptyline administration. Similarly, the slopes for turnover rate for chronically administered animals also suggest an increase in NE turnover rate, when compared to acute protriptyline administration. These data suggest the development of receptor adaptations following a chronic tricyclic treatment schedule. Figure 18, a turnover rate/recovery rate plot, depicts this comparison of turnover slopes for acute, subchronic and chronic treatment groups. This figure explicitly demonstrates the modifications in neurotransmitter turnover rate/recovery rate in the three treatment sehedules.
Pre-synaptic alpha-receptor adaptation (hyposensitivity) following chronic protriptyline administration is suggested by our data. Since yohimbine prevents pre-synaptic alpha-receptor stimulation, and allows NE overflow to the adrenergic synapse to occur, chronic protriptyline administration modifies this mechanism. Control of NE release by these receptors (which is dependent upon synaptic NE concentration) is altered, thus the negative feedback mechanism for facilitating or suppressing the controlled release of NE to the synapse becomes modified.
When animals were administered protriptyline chronically and subsequently administered yohimbine, a decrease in endogeneous NE levels was not observed in our studies ( Figure 17). These results are contrary to earlier studies involving yohimbine administration to control animals ( Figure 15). This observation suggests that chronic protriptyline administration induces pre-synaptic hyposensitiivity.       with the values reported by Collins, (1972) andTipton, (1972) in pig brain, rat liver and beef adrenal medulla. Similarly, the Vmax value obtained is 1.1362 nmoles of IAA formed/hr/ug DNA.
x .... ('") ....  (Collins, 1972;Tipton, 1972;Fowler and Oreland, 1979;Suzuki, 1979).                           Numerical values for NE levels in hypothalamus following alpha-11ethyl-para-tyrosine administration and sacrificed three hours after the administration of alpha-methylpara-tryosine. Data shown graphically in Figure 9.        TABLE 45 Korepinephrine levels in three brain regions following a single acute administration of protriptyline or. following 3 consecutive administrations of protriptyline. All 196 animals vere sacrificed 3 hours after the final protriptyline injection. *Significantly lower than controls when N=70 9 however. the difference is not significantly lower than its paired saline control. Therefore this diffC'ence may be due to injection stress. and .. y not really be a difference.    TABLE 49 Korepinephrine levels in three brain areas of thyroidectomized rats and Shall-operated controls following 3 and 6 consecutive days of protriptyline administration. Aniaab were aacrif iced 12 hours after the final protriptyline administration       Prot.