TRICYCLIC ANTIDEPRESSANTS AS UPTAKE BLOCKERS

The biogenic amine hy~othesis of affective disorders ascribes a functional depletion of neurotransmitters within critical regions of the brain as either the cause or, at the very least, a concomitancy of the depressive syndrome. Much indirect evidence has accummulated to suggest that the therapeutic effect of tricyclic antidepressants (TCA's) is mediated by their ability to block the re-uptake of physiologically released neurotransmitter leaving more neurotransmitter available within the synapse to interact with the post-synaptic receptor, thereby correcting the posited functional de-

The evidence upon which the proposed mechanism of TCA's therapeutic activity is based is largely indirect. ~ost of the evidence comes from in-vitro studies of the ability of tricyclics to block neurotransmitter uptake into either brain slices, minces or preparations of pinched-off nerve endings (synaptosomes). The therapeutic response, however , cannot be demonstrated upon acute aQministration of these drugs and clinically effective activity is usually not ob-·served until after several weeks of chemotherapy. The uptake inhibitory characteristics which have been demonstrated in-vitro, therefore, cannot explain the latency to the therapeutic effect.
A time-course for the effect of protriptyline administration upon n3 -l-norepinephrine (E 3 -l-NE) uptake into hypothalamic crude synaptosomal homogenate (CSH) was determined iii after acute, sub-acute and chronic treatment . In addition, the ability of a number of TCA's to block the uptake of three biogenic amines, the catecholamines, norepinephrine and dopamine, and the indoleamine, serotonin, was assessed using a CSH in-vitro test system.
The results support the biogenic amine hypothesis as well as the proposed mechanism of action of TCA's . Uptake blockade could be demonstrated after acute and chronic administration but not after sub-acute administration. An attempt to describe some of the characteristic effects of individual TCA's as well as effects common to the class of drugs was approached on the basis of predominant neurotransmitter influences.
iv Acknowledgement The traditional acknowled~em ent must b e reserved for t h ose who ma k e t he traditional -sojour n through t h e educational process -for me t h e experience has been anyt hing ·out traditional . Th e time invested , the financial and emotional cost represented an insurmountab le barrier and wa s overcome only with t h e encourag ement and assistance of many people . To all of t h em I off er my sin cerest a:?preciation and ment ion them here in chronological order .
Tl:anks to my r· ' i om and Dad for kn owing not to pu s h me i nt o anythin e; and alway s "b eing willing to pull me out of troublei t ' s because of them I took t he chances I did . Than.Ks to Bob and Delores Oland er wh o provided cont i nual encourager.ient and t h e necessary g ood tines .
Thanks to Dr . Gary P . Carlson for the encougaging att itude h e expressed in t h e early years of my interest in graduate work at U. R.I .
Thanks to my friend and advisor Al Sw onger who cont i nu es to set an example of a unique comb ination of superior i nve sti gative intellect and deep inter-and i ntra-personal communicative prowess .
Thanks to the State of Rhode Island f'or t h eir financial support and t h e fac ulty of U.'R . I. for their understanding during my most difficult periods .
Thanks to ~I enry Pedro for providing me with mu c:1 of the necessary inspiration .
Thanks to Ra y Sci en zo for h is long hours of hel p in setting up t h e ori ginal experimental protocol and for introducing me to H. P.
Thank s to the b est family a man could h op e to have -my sons Erett and Keit h who almost alway s respected my ri ght to privacy and my need for t h eir g ood behavior during the long h ours I studied in their presence and worked in the lab in v their absence.
Thanks to X:a.t.b_ryn E . Vale o, a woman like no othermore a mother to my sons than most , more a friend to me than mo st, an excellent secretary, an excellent lab technician, an unbelieveable person from whom I must continually fight to keep from taking all she has to give .
Thank you, all.
In addition, I would like to ac kn owl edg e the unfailing assistance of Rob erta Doran a.nd Vicki Burnett at t h e ti . P. . I . inter-library loan office for all t h eir hel p and to r.Irs. :.Jady lis ;,food who so graciously consent ed to do t he fi gures in t h is t h esis.    InteBrity of radioactive neurotransmitter sub strates determined by thin lay er chroma- The biogenic a~ine hypothesis of affective disorders, admirably and extensively reviewed by Baldessarini (1975), was first proposed by Jacobsen in 1959 and popularized by Schildkraut and Kety (1967). hypothesis abound (Davis, 1970;Schildkraut, 1973;Leonard, 1975;Weil-Malherbe, 1972) and even some dealing selectively with either norepinephrine (Schildkraut, 1965), dopamine (McClure, 1973) or serotonin (Lapin and Oxenkrug, 1969) as the critical neurotransmitter exist.
Tricyclic antidepressants have been shown to be effective inhibitors of the primarily neurotransmitter-specific, "hi-affinity", uptake process which many investigators believe is responsible for the termination of activity of physiological concentrations of neurotransmitter at the synapse. The ability of these antidepressants to block the uptake of neurotransmitter from the synapse may result in more neurotransmitter available within the synapse to interact with the post-synaptic receptor. This property of the drug would logically tend to reverse the effect of a lowered level or lowered potency or, in terms of the biogenic amine hypothesis, a functional depletion of neurotransmitter and can account for a reversal of the depressive symptoms.
The onset of clinical antidepressant activity, however, cannot be explained by correlations with antidepressant plasma levels (Oswald, 1972). A lag of several weeks of treatment before antidepressant activity becomes apparent has been reported in clinical trials of tricyclic antidepressant therapy (Hammer, et al., 1962;Asberg et al., 1970). Uptake studies to date deal primarily with in-vitro effects of antidepressants and in doing so cannot uncover any significant alterations in the drug effects on the uptake systems which might be coincident with and possibly related to the clinical antidepressant activity. Although one recent study (Ross and Renyi, 1975b) reports the effect of uptake blockade in animals treated for one day orally, there has not been a study reporting the effect of chronic administration of tricyclics on the hi-affinity uptake system of biogenic amines in the central nervous system. The design of many antidepressant drugs and the animal screening models for potential new antidepressants relies heavily upon the uptake inhibitory property of the tricyclics. Although the property of uptake inhibition fits the heuristic biogenic amine hypothesis, it has yet to be established that the therapeutic effect is mediated by this mechanism either entirely or in part.
A test of the hypothesis that tricyclic antidepressants mediate this therapuetic activity by inhibiting the uptake of biogenic amines can be approached experimentally using isolated pinched off nerve endings (synaptosomes) as a model of nerve endings functioning in-situ (Kuhar, 1973;Bradford, 1975 (Burn, 1953). Koelle and Valk (1954) reported that unlike acetylcholinesterase in cholinergic neurons, MAO is not selectively associated with adrenergic neurons. Kamijo et al. (1955), Balzer and Holtz (1956) and Corne and Graham (1957) all reported the inability of a variety of MAOI's to significantly potentiate the actions of catecholamines either administered exogenously or released by stimulation of the sympathetic nerve.
Considerable evidence has accumulated to suggest that the uptake of catecholamines occurs into post-ganglionic nerve endings. Kopin et al. (1964) maintained that if allowances are made for differences in regional blood flow through various tissues studied, there is a good correlation between the accumulation of norepinephrine and the level of endogenous norepinephrine. The levels of endogenous norepinephrine may be taken to reflect the extent of innervation of the organ or tissue by sympathetic postganglionic terminals, although turnover may be judged a better indicator by some (Costa, 1969). Additional evidence that the uptake of norepinephrine occurs primarily into sympathetic post-ganlionic neuronal endings became available when Hertting et al. (1961) reported almost complete disappearance of the ability of norepinephrine and epinephrine to accumulate in tissues in which the sympathetic postganglionic nerves innervating the tissue had been cut and the terminals allowed to degenerate. This approach was modified by Zaimis et al. (19 65), Iversen et al. (1966) and Sjoqvist et al. (1967) whereby sympathetic innervation was obliterated by immunosympathectomy at birth; the results obtained were similarly interpreted.
A number of techniques have been employed to conclude that localization of accumulated radiolabeled norepinephrine occurs predominantly in neuronal endings include studies using density gradient centrifugation (P otter and 1963), autoradiography and electron microscopy (Werle et al., 1962;~arks et al., 1962;Samorajski et al., 19 64) and histochemical flourescence techniques (Hamberger et al., 1964). Although the evidence implicates the neuronal ending as the site of uptake, methodology at that time was not available to accurately separate and describe the component of uptake into tissue other than neuronal endings. Fischer and Kopin (1964), however, did present evidence to indicate that the uptake of catecholamines was not limited to neuronal tissue in their report describing uptake into denervated salivary glands of the rat.
Further elaboration of the characteristics of uptake systems of peripheral tissues came about as a result of attempts to study the effects of drugs. Macmillan (1959) proposed the "nerve uptake hypothesis" ascribing the potentiating properties of cocaine to its ability to interfere with the catecholamine uptake mechanism. Adrenergic blocking agents were found to inhibit the uptake of 3 Hnorepinephrine including phenoxybenzamine and dicholroi soproterenol (Axelrod et al., 1962b). Tricyclic antidepressants, neuroleptics of the phenothiazine class as well as guenethidine, bretylium and ouabain were shown to be effective inhibitors of the noradrenergic uptake mechanism (Axelrod et al., 196lb;Herhitting et al., 1962;Dengler et al., 1962) as well as the sympathomimetic amines tyramine, amphetamine and ephedrine (Axelrod and Tomchick, 1960;Axelrod et al., 196la, Heritting et al., 1962;Dengler et al. 1961).
M uscholl (1960), Axelrod et al. (196lb) and Heritting et al. (1962) all reported that reserpinized tissues failed to accumulate epinephrine and norepinephrine. Dengler (1961) found that the accumulation of epinephrine and norepinephrine in tissue slices was prevented when reserpine was present in the incubation medium . As a result of these experiments there was relatively wide acceptance that reserpine inhibited the uptake of catecholamines (Brodie and Beaven , 1963). After an extensive review of the literature, however , Shore (1963) concluded that the actions of reserpine were predominantly centered around the intracellular uptake and storage mechanism and not the neuronal membrane uptake system. Direct evidence to support this hypothesis came from Lindmar and M uscholl (1964) where in both normal and reserpinized is~lated rat heart preparations, they infused 0.5 ug of norepinephrine over a 10 minute period and collected and analyzed the perfusate for unchanged norepinephrine. Analyzing the norepinephrine content of the heart after the perfusion interval, they determined that in normal hearts the catecholamine taken up from the medium was almost entirely accounted for as unchanged norepinephrine accU!Ilulated into the tissue . In reserpinized hearts, however, the amount of norepinephrine in the perfusate was the same as in the perfusate of normal hearts, but tissue analysis revealed very much lower levels of catecholamine ·;i i th very high levels of M AO metabolites in the perfusate . These These results suggested that norepinephrine accumulation was 1 1 not affected but its subsequent storage was. Iversen (1965aIversen ( , 1965b (Iversen 1965a(Iversen , 1965b. The accumulation of catecholamines under these conditions could not be explained in terms of the uptake system described when using low catecholamine concentrations in the perfusate, however, the characteristics of this uptake process could be described using classical enzyme kinetic models. This second uptake system, a process for which the term Uptake 2 (u 2 ) has been applied, showed a much lower affinity than could be ascribed to the initial uptake system described (referred to as Uptake 1, abbreviated u 1 ).
Several properties of u 2 serve to distinguish it from u 1 • Catecholamines accumulated by u 2 disappeared rapidly from the tissue if the perfusion was continued using a catecholamine-free medium, although 2 ug. of catecholamine per gram of tissue remained even after prolonged periods of washout. A comparison of the kinetic constants showed that unlike u 1 , u 2 exhibited no stereospecificity; also, although u 2 accumulated norepinephrine more rapidly, epinephrine exhibited a greater affinity for uptake with the Km being about five times lower than that for norepinephrine.
Although u 2 appeared inoperative at low substrate concentrations and thought to be activated where a critical threshold of substrate concentration is reached (Iversen, 1965a), a more recent study reported evidence to conclude that both systems operate at all substrate concentrations with u 1 having the greater influence at low and u 2 at high substrate concentrations (Lightman and Iversen, 1969).
Another property serving to distinguish between u 1 and u 2 includes substrate specificity and the effect of drugs on inhibiting the transport effected by each system. Burgen and Iversen (1965) reported the results of a variety of substituted B-phenylethylamines on the u 2 system. Interestingly, 0-methylation as well as N-substitution greatly enhanced that affinity for u 2 ; whereas the effect of these compounds on u 1 are exactly opposite in nature (Horn, 1973).
Cocaine, imipramine and desipramine while potent inhibitors of u 1 were found to be only weakly active with respect to u 2 (Iversen 1965a;Salt, 1972). Phenoxybenzamine is a potent inhibitor of u 2 as is a variety of steroids including B-estradiol, corticosterone, testerone, deoxycorticosterone and androsterone (Iversen and Salt, 1972).
Originally it was suggested that u 2 might represent a sepq rate neuronal membrane transport system at sites along the entire neuron; that is, cell body, axon and nerve terminal. Evidence for this concept was provided by the histochemical studies of Norbert and Hamberger (1964) and Hamberger et al. (1964) who reported that the accumulation of norepinephrine was localized to terminal regions of postganglionic sympathetic neurons when low doses of norepinephrine were administered and amine throughout the cell body and axon as well as the terminals when high doses were administered. This hypothesis, however failed to explain the washout phenomenon reported to exist for u 2 • Another possibility offered was that u 2 may represent an uptake of catecholamines by storage granules within adrenergic neuronal terminals (Iversen, 1965a). Evidence for this hypothesis included the indisputable similarity of properties between u 2 and eith er isolated adren ergic nerve granules or adrenal medullary granules incubated in-vitro (Euler and Lishajko, 1964;Carlsson et al., 19 63). The particle uptake system is saturable at amine concentrations of 80 ug/ml. which is very close to that described for u 2 , also, both are relatively insensitive to inhibition by cocaine, desipramine or metraminol and both are inhibited by tyramine and p k enoxybenzamine. Granular uptake lacks specificity for the optical isomers of epinephrine and norepinephrine. Finally, the uptake of catecholamines in isolated granules occurs at concentrations of 1 ug/ml. or higher in the absence of magnesium and adenosine triphos-phate and u 2 is known to begin to exert substantial influence at this concentration of amine. One important difference between u 2 and the isolated particles is reflected by the fact that no difference in affinity can be shown for epinephrine and norepinephrine as can be observed in the u 2 system.
The hypothesis which has gathered the most support by far is that u 2 represents an extra-neuronal uptake mechanism (Iversen, 1975). Support has come from the results of histochemical studies of the distribution of catecholamines in various tissues after exposure to high concentrations of exogenously administered norepinephrine. The work of Fisher and  cited earlier may have provided that needed impetus to investigate the association between non-neuronal or extra-neuronal uptake and u 2 • After perfusion of rat hearts with medium containing high concentrations of norepinephrine, intense flourescence was observed in cardiac muscle cells and unidentified connective tissue cells as well as nerve terminals (Ehinger and Sporrong, 1968;Farnebo and Maimfors, 1969;Clarke et al., 1969;Jacobowitz and Brus, 1971). Extraneuronal uptake, like u 2 , was found to be sensitive to normetanephrine and relatively insentive to desimpramine (Farnebo and M alfors, 1969;Clarke et al., 1969). Numerous reports of uptake of catecholamines into nonneuronal tissue including salivary gland parenchyma (Hamberger, 1967), smooth muscle of cat spleen (Gillespie 1968(Gillespie , 1973, vas deferens (Gillespie et al., 1970), chick amnion (Burnstock et al., 1971), perfused rabbit ear artery (Avakian and Gillespie, 1968), cat nictitating membrane (Draskoczy and Trendlenburg , 1970) and guinea pig trachea (O'Donnell and Saar, 1973), appeared. Kinetic constants for the extra-neuronal uptake into arterial cells was shown to have characteristics similar to u 2 (Gillespie and Towart, 1973). The uptake of norepinephrine into smooth muscle also showed susceptibility to phenoxybenzamine and washout was inhibited by normetanephrine (Gillespie, 1968).
Although u 1 has been considered the principal mechanism responsible for the inactivation of physiological concentrations of norepinephrine (Iversen, 1971), the preponderance of sympathetic innervation is clearly a consideration in determining the importance of either uptake mechanism in isolated tissue preparations, as is the concentration of catecholamine in the medium. Smooth muscle of the vasculature contains relatively little sympathetic innervation and it would seem reasonable to conclude that u 2 is most important in terminating the action of circulating catecholamines in this tissue. The evidence to support the hypothesis that neuronal uptake isthe principal inactivating mechanism of depolarize-released neurotransmitter comes from reports that organ response to sympathetic nerve stimulation is greatly increased by drugs shown to be effective inhibitors of the neuronal uptake mechanism (Thoenen, 1964a(Thoenen, , 1964bBrown , 1965). Based on results reported by Langer (1970), u 1 may account for the recapture of as much as 70-90% of the transmitter released by low frequency sympathetic nerve stimulation in the cat nictitating membrane.
B. Characterization of kinetically distinct neuronal uptake mechanisms.
i) Norepinephrine, dopamine and tyrosine: Norepinephrine uptake as an active process described using Michaelis-Menten kinetics was dissociated from the contribution of passive diffusion by using low substrate concentrations (l0-7 M) in the medium of brain slice preparations (Dengler, 1962). The active component of uptake into brain slices was shown to be temperature dependent, occur against a concentration gradient, be dependent upon Na+ in the medium, require oxygen and glucose, can be lowered by metabolic inhibitors (Dengler et al., 1962;Dengler, 1965;Renyi, 1964, 1966), and does not require calcium or magnesium ions (Hamberger, 1967). Limited accumulation occurs when incubation is carried out in a sucrose medium (Mirkin et al., 1963).
Similar results have been reported with the use of a crude _ synaptosomal homogenate. Davis et al. (1967), Bogdanski and Tissari (1967), Bogdanski et al. (1968) Colburn et al. (1968 and White and Keen (1971) all showed an absolute requirement for 143 mM Na+ concentrations and inhibitory effects of increasing K+ concentrations above 5mM, as well as temperature dependence and reduction in uptake by metabolic inhibitors. Conclusions of these studies suggest that uptake by brain slices, at low substrate concentrations similar to that used by Dengler (1962), occurs primarily into neuronal endings. Harris and Baldessarini (1973) showed similar require-ments for dopamine uptake into rat corpus striatum with one notable exception. Whereas Snyder and Coyle (1969) reported no substantial effect on initial uptake of catecholamines using reserpine pretreatment, Harris and Ealdessarini (1973) reported a considerable effect.
Additional support for the hypothesis that amine accumulation occurs into nerve endings comes from the work of Snyder et al. (1965) who showed that intraventricular injection of labeled norepinephrine was found localized to a great extent in the synaptosomal fraction upon differential centrifugation. Histochemical techniques confirmed that the uptake of monoamines into rat cerebral cortex was confined to varicosities indistinguishable from those containing endogenous norepinephrine (Eamberger and M asuoka, 1965;Hamberger, 1967). Eenn (1967) using electron microscopic autoradiography reported that 8 0% of the H 3 -norepinephrine accumulated by brain slices was localized to neurons. More recently Lidbrink and Jonsson (1974) showed a reduction of uptake of norepinephrine into cerebral cortices after noradrenergic terminals were destroyed by lesion of the dorsomedial reticular formation with 6-hydroxydopamine. Snyder and Coyle (1969) studied the stereospecificity of norepinephrine and dopamine uptake in different areas of the rat brain. The M ichaelis constant of 0.4 uM for norepinephrine uptake into extrastriatal areas is in good agreement with the 0.56 uM Km reported by Colburn et al. (1968) using whole rat brain. Snyder and Coyle (1969) extended the range of catecholamine concentration from 0.05 uM using 1.2 uM as the upper limit. They reported that the striatum had a higher affinity for dopamine than norepinephrine and both neurotransmitters display only a single uptake component over the ra.~ge of substrate concentrations studied. All other brain areas showed two components of dopamine uptake, one considered a high affinity the other a low-affinity transport mechanism, whereas norepinephrine continued to be described adequately by a straight line using the Lineweaver-Burk method suggesting only one transport system exists for this amine. In a later paper , they reported the Ki values for mutual inhibition of dopamine and norepinephrine uptake in all regions of rat brain to be identical with their respective Km values and concluded that these two catecholamines use the same transport systems (presumably dopaminergic) and would not require such a characteristic.
The uptake of precursors of norepinephri:ooand dopamine, namely tyrosine and dopa, have been described (Naeme, 1961;Guroff et al., 1961;Dengler et al., 1962;Yoshida et al., 1963aYoshida et al., , 1963bYoshida et al., , 1963cYoshida et al., , 1965 but delineation of two transport systems has not been the focus of attention and wh eth er these systems exist remains to be sh own. Chiri g os et al., (1960) have reported stereospecificity of 1-tyrosine for brain tissue and illustrated its uptake inhibition by the amino acids leucine, isoleucine, valine, histadine and cysteine. They also noted that stereospecificity was lacking in muscle preparations. Interestingly, Ross and Renyi (1966) studied the uptake of tyramineand 1-dopa at concentrations similar to those reported for high affinity uptake of dopamine  and reported Km's for dopamine and tyrosine of 0.7 uM and 0.4 uM, respectively, in slices of whole brain. These values are similar to those reported for extra-striatal norepinephrine uptake Snyder and Coyle, 1969). It would appear that these Km values adequately describe a high affinity component of uptake when compared with other amino acids with Km's in the millimolar range (Cohen and Lajtha, 1972). In addition, Wofsey et al. (1971) present evidence to indicate that tyrosine may become selectively incorporated into a population of synaptosomes that can be separated from those which accumulate norepinephrine by a technique known as incomplete equilibrium centrifugation.
ii) Seroton"in and tryptophan: Fuxe and Ungerstedt (1967) described the localization of intraventricularly administered serotonin to serotonergic neurons using histochemical flourescence techniques.
Although a number of attempts were made to illustrate serotonin accumulation in brain slices (for a review see Katz and Chase, 1970)  recovered in the synaptosomal fraction upon subcellular fractionation using differential centrifugation techniques.
Similarly, Kuhar et al. (1970) studied the subcellular localization of serotonin after incubation of brain slices with the amine in concentrations ranging from 10-5 to 10-8 M and found the major portion of accumulated serotonin localized to synaptosomes.
The kinetics of serotonin accumulation into slices of rat striatum and hypothalamus revealed two distinct transport processes with KmH=l.7xl0-7 M and Km 1 =8 .0xl0-6 M in t h e -7 -6 striatum and KmH=l.4xlO M and Km 1 =8.0xl0 M in the hypothalamus (Shaskan and Snyder, 1970). The Ki values for t h e competitive inhibition of norepinephrine uptake by serotonin approximated the Km for low affinity serotonin into catecholaminergic neurons. These kinetically distinct components were confirmed by Wong et al. (1973). Kuhar et al. (1974) review the results of a number of studies illustrating the reduction of serotonin uptak e associated with the selective destruction of serotonin nerve terminals by midbrain raphe lesions suggesting that uptake occurs predominantly into serotonergic nerve endings wh en low substrate concentrations are employed. Histochemical flourescence as well as the measurement of tryptophan hydroxYlase activity and endogenous serotonin levels support the hypothesis that high affinity uptake process is specific for neurotransmitter inactivation upon depolarized release (Kuhar et al., 1972). M assari and Sanders-Bush (1975) have utilized this concept of high-affinity neurotra.nsmitterspecific uptake to compare the effects of para-choloroamphetamine or B-9 lesions with the distribution of serotonergic neuronal endings in different areas of the brain.
Initial studies of tryptophan transport into synaptosomal preparations utilized substrate concentrations limited to the low affinity component of transport (Grahame-Smith and Farfitt , 1970). In this study the concentrations of sodium ions in the media were varied from 0 to 112 mM by osmotic replacement with either choline or sucrose and resulted in no effect on tryptophan uptake, however, cyanide and ouabain did significantly reduce the uptake of substrate.
Interestingly, preloading the synaptosomes with 1-tryptophan resulted in an increase in the uptake of either 1-tryptophan or 1-phenylalanine. L-Tryptophan preloaded synaptosomes were also sh o~n to greatly accelerate the rate of efflux of tryptophan by either 1-tryptophan or 1-phenylaJanineimplying that a faciltated exchange process was occuring. A number of amino acids were reported very active in stimulating the efflux of tryptophan from preloaded synaptosomes. It would appear that this low affinity transport system represents the same mechanism by which other amino acids gain entry into the neuron and is not specific for tryptophan.
A more recent study revealed a high affinity component of the uptake of 1-tryptophan as well as a low affinity component (Parfitt and Grahame-Smith, 1974). Alth ough the affinity M ichaelis constant (KmL) originally reported by Grahame and Parfitt (1970) as l.Oxl0-3 M is considerably disparate from their more recent data ( KmL 3.2xl0-4 M); use of different tissue preparation, i.e. whole brain vs. cerebral cortex, can account for the results. The KmH, however, is only lower than the the KmL by a factor of three reported in this study, (Parfitt & Grahme-Smith, 1974), whereas, other investigators have reported differences between high and low affinity uptake systems differing from a minimum of 21 (in cerebral cortex) to over 300 (glycine in the spinal cord) orders of magnitude which would appear to represent physiologically more distinguishable processes (Snyder et al., 1973).
iii) Other neurohumors: a) Acetylcholine and choline: Unlike the catecholamines and serotonin, acetylcholine appears to be inactivated largely by the intrasynaptic degradative enzyme acetylcholinesterase and diffusion rat h er than an uptake mechanism (for a review see Katz and . 1 1iledi, 1973). Early work aimed at characterizing an uptake process for acetylcholine did not prove fruitful (Guth, 1962;Burton, 1964). Even in the presence of the reversible cholinesterase inhibitor physostigmine, negative results were reported (Elliot and Henderson, 1951;Schuberth and Sundwall, 1967) although it was suggested that physostigmine may have strong inhibitory actions on the uptake mechanisms (Polak and Meeuws, 1966). Use of the organophosphate cholinester-. ase inhibitors including paraoxon (Diethyl-4-nitrophenyl phosphate) have resulted in significant accumulations of acetylcholine into brain tissue (Polak and Meeuws, 1966;Schuberth and Sundwall, 1967;Liang and Quastel 1969;Heilbronn, 1969) with the uptake requirement for oxygen, reduced by metabolic inhibitors and described using M ichaelis-Menten kinetics (Schuberth and Sundwall, 1967;Polak and M eeuws, 1966;Liang and Quastel, 1969).
Acetylcholine uptake seems unimportant physiologically for it has been shown that acetylcholine uptake into synaptosomes is neither readily incorporated into vesicles ( M archbanks, 1968) nor released by electrical stimulation or potassium depolarization . Further evidence leading to a conclusion that acetylcholine uptake is not physiologically important comes from the results of M archbanks (1969b) and Adamic (1970) suggesting that both molecules utilize the same carrier. Similarly autoradiographic techniques have been employed using slices of rat cortex incubated in labeled acetylcholine and the distribution of label w~s reported as diffuse with no obvious preference for specific cell types or structures (Polak, 1969). Finally,  attempted to collect t h e excess acetylcholine which might be produced by brain stimulation in the presence of an acetylch oline uptake inhibitor if the acetylcholine transport process were physiologically active. They report that no additional acetylcholine is collected in the presence of uptake inhibitor and conclude that acetylcholine uptake may not be physiologically operational. These results may be explained by considering the transport mechanism responsible for acetylcholine uptake to be identical with the choline carrier; the low affinity choline transport mechanism being common to all cells requiring choline as substrate for synthesis of phasphatidyl-or phosphorycholine. Hodgkin and Martin (1965) (Potter, 1968) to 440 uM (Hensworth and Bosmann, 1971).
Green and Haubrich (1971) using synaptosomal preparations incubated with low concentrations of choline, reported a significant amount of acetylcholine formed and suggested that more than one uptake system may exist for choline accumulation in the brain. Yamamura and Snyder (1973) and Dowdall and Simon (1973) reported two kinetically distinct carrier-mediated systems for choline transport having a K~ of 1-5 uM and KmL less than 25 uM. The two systems showed clearly distinguishable effects of Na concentration (Haga and Noda, 1973) with the high affinity component more dependent on alterations in sodium concentration in the media.
Although it has been suggested that the high affinity component represents a mechanism of cholinergic neurons which may serve to recapture the product of intrasynaptic acetylcholinesterase for the synthesis, storage and subsequent reutilization of acetylcholine, conclusive evidence of this is not available as yet (Kuhar and M urrin, 1978). Kuhar , et al. (1973) have provided evidence to indicate that choline uptake occurs into cholinergic neurons primarily by the high affinity uptake process. Septal lesions, known to result in reductions of acetylcholine in the cerebral cortex and hippocampus, resulted in a reduction of choline uptake by 6alo in synaptosomes prepared from these cholinergically innervated areas, whereas, tryptophan and glutamatic acid accumulation were unaffected although GABA and norepinephrine uptake were affected slightly. b) gamma-Aminobutyric acid (GABA) GABA was shown to be taken up by slices of rat cerebral cortices when a low concentration (0.05uM) of substrate was used in the medium (Iversen and Neal , 1963). The electron microscopic autoradiographic experiments of Bloom and Iversen (1971) suggest that more than 7cY~ of the GABA taken up into brain slices is localized to the nerve terminals. It has also been suggested that at this low concentration, reminiscent of that used to detect high affinity uptake, a distinct population of nerve terminals are responsible for the uptake of labeled GABA (Iversen and Snyder, 1968;Iversen and Johnston, 1971). Clearly distinguishable high and low affinity components for GABA uptake into synaptosomes and brain slices have been reported Raiteri, 1973a, 1973b).
As with other high affinity transport systems there is an absolute requirement for physiological concentrations of electrolytes and uptake is inhibited strongly by low concentrations of Na+ (Iversen and Neal, 1968). Inhibition of GABA uptake has been reported with 2,4-dinitrophenol, ouabain and malonate (Kuriyama et al., 1969;Snodgrass et al., 1973). c) Glutamate, aspartate and glycine: , Johnston (1972a, 1972b) and Bennett et al. (1972) have studied the uptake of glutamate and aspartate and have reported that transport occurs by both high and low affinity mechanisms with the high affinity system having an absolute dependence on sodium ions. Furthermore, the metabolic requirements of both glutamate and aspartate uptake are indistinguishable with azide, eyanide and omission of glucose from the media having minimal effects and it appears as though glutamate and aspartate share the same transport carrier. Wofsey et al. (1971), using subcellular distribution studies, have associated the high affinity uptake with a unique population of nerve terminals suggesting that the high affinity mechanism may be directly related to the function of replenishment of neurotransmitter.
A high affinity uptake system specific for glycine has been shown in slices of rat spinal cord, pons and medulla ( Neal, 1971;Jolmston and Iversen, 1971), slices of cat spinal cord (Balcar and Johnston, 1973) as well as in crude synaptosomes of rat spinal cord Bennett et al., 1972) and in purified synaptosomes of cerebral cortex of the rat (Peterson and Raghupathy, 1973).
Localization of glycine uptake into nerve endings by the high affinity system has been shown by autoradiographic analysis (~atus and Dennison, 1972;Iversen and Bloom, 1972;Ehinger, 1972;Hosli and Hosli, 1972;Ljungadhl and Hokfelt, 1973) as well as subcellular fractionation teclmiques Arregui et al., 1972). Glick (1972) has shown inhibitors of Na+/K+ dependent ATP-ase to be effective inhibitors of glycine uptake; however, no inhibitors has yet been described as being more effective in inhibiting either the high or the low affinity uptake. The low affinity mechanism of glycine uptake seems to be relatively no~-specific in that 1-alanine,l-serine and other small neutral a,~ino acids compete for the transport site (Cohen and Lajtha, 1972).

d) Histamine and histadine:
Although the probable mechanism of histamine inactivation is via the degradative enzyme histamine methyltransferase, subcellular distribution studies show the incorporation into the P 2 (synaptosomal) fractions and after subsequent treatment in a hypo-osmotic medium histamine was shown to be localized to the vesic11lar fraction (Snyder 2~ et al., 1974). Young et al. (1971) reported an ontogenic related change in the distribution of histamine among subcellular fractions. Older animals (21 days) showed greatest accumulation in the synaptosomal fraction whereas younger animals appeared to concentrate histamine in the nuclear pellet.
A stereospecific uptake of L-histidine into brain slices has been demonstrated ( Neame, 1964) although the substrate concentrations were not low enough to differentiate a high affinity component and in all probability the low affinity system is shared by other basic amino acids such as tryptophan.
c. Effects of tricyclic antidepressants on the hi-affinity uptake process of serotonergic noradrenergic and dopaminergic neurons.

i) in-vitro
The ability of tricyclic antidepressants to block the uptake of norepinephrine into brain slices was described by Dengler et al. (1961) using concentrations of radioactively labeled norepinephrine (2.8 -14.0xl0-8 M) well within the concentration range where hi-affinity transport is responsible for a large part of the uptake of nor~pin ephrine into nerve endings. The concentration at which the uptake of norepinephrine into nerve endings is inhibited by fifty percent, (rc 50 ), varies depending upon the preparation used, i.e. brain slices versus synaptosomes, as well as by brain region. The rc 50 for desmethylimipramine (DM I) varies from 0.003 uM in rat brain synaptosomes (Ross and Renyi, 1975a) to 0.11 uM in rat brain slices (Salama et al., 1971). Buus Lassen et al., (1975) reported an 1c 50 for DM I of 10 uM in rat forebrain synaptosomes as opposed to 0.001 uM in synaptosomes prepared from rat hippocampal tissue.
The large difference is believed to be due to the concentration of noradrenergic neuronal endings within brain regions (Squires, 1974;Buus Lassen et al., 1975).
It is also true that studies utilizing different species as well as different preparations reported identical rc 50 values for the tricyclics tested. Ross and Renyi (1967a) utilizing mouse cortex slices reported an 1c 50 value of 0.03 uM for the inhibition of norepinephrine uptake by DNI whereas an identical value was reported by Shaskan and Snyder (1970) using a crude synaptosomal homogenate of rat hypothalamus. A good correlation between the partition coefficient and the Ic 50 values for a number of chemically different uptake inhibitors was found to exist in brain slices (Carmichael and Israel, 1973).
All studies report DM I to be the most potent tricyclic tested with respect to its ability to block norepinephrine uptake (Ross and Renyi, 1967a;Shaskan and Snyder, 1970;Horn et al., 1971;Salama et al., 1971;Carmichael and Israel, 1973;Squires, 1974;Maxwell et al., 1974;Ross and Renyi, 1975a;Buus Lassen et al., 1975). M any studies indicate that the demethylated form of the tertiary amines, i.e. the secondary amines, are more potent with respect to their ability to inhibit norepinephrine uptake whereas the tertiary amines are more efficacious in its effect on serotonin uptake (Blackburn et al., 1967;Ross and Renyi 1967b;Carlsson, 1970;Shaskan and Snyder, 1970;Kannengiesser et al., 1973;Horn and Trace, 1974;Renyi, 1975a andBuus andLassen et al., 1975).
Structure activity relationships have been studied with respect to both the noradrenergic ( Horn and Snyder, 1972;Ferris et al., 1972;Horn, 1973a;M axwell et al., 1974) and serotonergic ( Horn, 1973b) transport system in the central nervous system. It is on the basis of these studies that evidence can be found to substantiate the concept that there exists a chemically distinguishable transport mechanism for each neurotransmitter.
The uptake inhibitory characteristics of tricyclics as they apply to dopamine have been studied (Ross and Renyi, 1967a). Their effect on this transport system has not generally been considered as important due to the relatively large 1c 50 values reported (Horn et al., 1971). There is no correlation with the inhibition of uptake of dopamine into either rat hypothalamus or whole brain synaptosomes with respect to methyl substitution on the side chain nitrogen of any of the tricyclic antidepressants ( Horn et al., 1971;Halaris et al., 1975). Alternatively, antiparkinsonian drugs have been shown to be very potent in their ability to block dopamine uptake in the central nervous system (Coyle and Snyder, 1969b).

ii) in-vivo
Imipramine was shown to reduce the amount of labeled epinephrine found in homogenates of whole mice when compared with saline treated controls (Axelrod and Tomchick, 1959).
Similarly, imipramine increased the rate of disappearance of labeled norepinephrine in the whole animal (Axelrod and Tomchick, 1960). Hertting et al. (1961) reported reduced levels of H 3 -norepinephrine in heart, spleen, adrenals, liver and muscles with elevated normetanephrine found in muscle tissue after treatment with imipramine. Glowinski and Axelrod (1964) administered imipramine DMI or amitriptyline to rats and after 1 hour administered H 3 -norepinephrine by an intra-cerebroventricular (ICV) injection. All three compounds were reported to be effective in inhibiting the uptake of H 3 -NE into rat brain. It is interesting to note that Schanberg (1963) could not show imipramine to be effective in inhibiting the uptake of 5-hydroxytrptophan, the precursor of serotonin, into brain slices when the drug was administered i.p. 1 hour prior to sacrifice with subsequent testing in-vitro.
As with the in-vitro data, many studies measuring the effectiveness of tricyclics on either norepinephrine or serotonin uptake report tertiary amines as being more effective then the secondary amines in blocking the uptake of 5HT in the central nervous system (Ross and Renyi, 1975b).

D. Effects of tricyclics on t he metabolism of biogenic amines: i) Serotonin
Da Prada and Pletscher (1966) reported a significant reduction in the levels of 5-hydroxyindoleacetic acid, (5-HIAA) the major metaboliteof serotonin in the CNS, for both imipramine and amitriptyline. The ratio of product to precursor indicates that imipramine is slightly more efficacious in reducing the turnover of serotonin and this is reflected in the in-vitro data as well (Ross and Renyi, 1969;Kannengiesser, 1973). Palaic et al., (1967), using an intracerebroventricular perfusion of H 3 -5HT at 55 uM concentration for 1 hour, demonstrated a reduction in endogenous levels of 5-HIAA but not of H 3 -5EIAA. At this concentration the predominant idoleamine inactivation mechanism is presumably a low affinity transport system.
It seems possible that the effect of DMI on inhibiting the uptake of 5HT may have been overshadowed by the low affinity transport mechanism as well as diffusion. A reduction in endogenous levels of 5HIAA imply decreased activity of the neuron which may come about as a result of feedback mechanisms in the event DMI is inhibiting 5HT uptake. Subsequently increases in the intrasynaptic amine concentration and hence its interaction with post-synaptic receptors is the postulated mechanism for feedback inhibition (Costa and Meek, 1974).
c 1 4-serotonin was administered intracisternally 15 minutes prior to an i.p. injection of either DM I or I M I (25mg/kg) and 2 hours after the intracisternal injection brains were assayed for H 3 -5HT and deaminated metabolites (Schildkraut et al., 1969b). suggests that both the secondary and tertiary amines have the same effect on norepinephrine metabolism when administered chronically; that of inhibiting uptake and increasing turnover as measured by the rate of disappearance of H 3 -NE from the brain (levels of H 3 -NE at 6 minutes vs. levels at 3 1/2 hours after label).
In a more recent study of the effect of chronic administration of DU and Dl\1I, turnover of the biogenic amines including norepinephrine was reported as being decreased although the decrease was not statistically significant (Leonard and Kafoe, 1976). In these experiments turnover was measured as the ratio of specific activity of labeled product to labeled precursor after administration of H 3tyrosine and H3-tryptophan. The results are obviously at variance with that reported by Schildkraut et al. (1970Schildkraut et al. ( , 1971) with respect to the turnover of norepinephrine, and the authors could not offer a reason for the apparant discrepancy. The effects of chronic administration of tricyclics on the turnover of norepinephrine awaits further study before a conclusion can be drawn.

iii) Dopamine
Dopamine has been the least studied of the catecholamines with respect to the effects of tricyclic antidepressants on its metabolism. Da Prada and Pletscher (1966) reported a slight but insignificant decrease of homovanillic acid (HVA) after an acute administration of imipramine whereas amitriptyline caused a significant increase in HVA.
Similar results were obtained by Nielsen (1975) in a study of the affect of acutely administered amitriptyline. Levels of dopamine were shown to be increased although the increase did not achieve statistical significance. DMI, however, did increase the levels of dopamine significantly.
Chronic administration of both imipramine and DM I decreased the turnover of dopamine (Leonard and Kafoe, 1976). The incubation medium (IM) originally used in preliminary experiments was Kreb's bicarbonate (Umbreit et al., 1964), as modified by Snyder and Coyle (1969)  were added to increase the stability of the neurotransmitters by preventing auto-oxidation shown to occur in oxygenated media (Iversen, 1972).
One milliliter (ml.) of CSH was introduced into 6-8 ml. The brains of six aminals were pooled and aliquots were placed in media that was identical to the media of Snyder and Coyle (1969) except that calcium and EDTA were modified as follows. For the first condition, l.2mM calcium was replaced with lOmM ca++ and EDTA was omitted. The calcium concentration was then lowered to 2.5mM in the second condition. The third condition employed was that of Snyder and Coyle (1969)

vii) Regional distribution of neurotransmitter uptake
The uptake of each neurotra~smitter into each brain region employed in this study was investigated. The method employed was similar to that outlined above in methods except that all these brain regions were studied for each neurotransmitter. The media used was that described by Snyder and Coyle (1969).
The results are expressed as Particulate: Medium ratio calculated as CPM/mg protein/Incubation time: CPM /ml. supernatant.
viii) Effect of calcium in 1-norepinephrine release induced by hi-potassium depolarization The role of calcium in the release of norepinephrin'e 51 and its consequent effect on uptake was studied. The hypothalami of ten animals were pooled and divided into equal aliquots. Preincubation media used were as follows: (1-2) hi -K+ (45mI~) plus ca++ ( usually not considered by investigators. There is sufficient evidence to suggest that both hetero-exchange as well as homo-exchange processes do occur (Cohen & Lajtha, 1972;Levi et al., 1976). M any studies use short incubation times to maximize the magnitude of unidirectional flow of substrate and prolo:1ged incubation usually results in a deviation from linearity. "Re-uptake" refers more to the process in-situ and i :s inappropriately used as a synonym for uptake.
This thesis utilized the term uptake to represent an apparent accumulation of amines and assumes that exchange processes are not significant over the short incubation times used. General: The reproducibility of the brain dissection procedure is reported in table 1 and compares favorably with the values reported by Glowinsky and Iversen (1966). Since the boundaries differ some, the difference in values between those published and reported here appear to be reasonable. The weighed brain regions were carried through the homogenization procedure and the functional relationship between the wet weight of brain tissue for a given brain region and the  Mean weights for various brain regions dissected from 24 animals. A method for the detection of specifically inhibitory contaminants in radioactive substrates is described by Timpton et al. (1977). The advantage of this procedure is   Snyder and Coyle, 1969). Serotonin uptake was linear for six minutes and this value agrees exactly with that reported by Shaskan and Snyder (1970).  Effect of synaptosomal concentration in the incubation medium upon the uptake of H3-l-NE into hypothalamic crude synaptosomal homogenate.
-·-y s1•c.  1967). When calcium was removed from the medi-u:r: and 0.1 : mM EDTA added, the uptake into hypothalamic synaptosomes was shown to be significantly greater than the value obtained using the mediUJ:Jof Snyder and Coyle (1969). Higher concentration of EDTA resulted in attenuation of the response.
As has been pointed out earlier in this report, preliminary experiments were performed using the mediumdescribed by Snyder and Coyle (1969). The rationale for its use was  Kreb's. The authors suggested that EDTA and ascorbate reduced auto-oxidation and pargyline prevented the formation of deaminated metabolic products of NE, but calcium levels were not reduced. Subsequently, Snyder and Coyle (1969) published the use of their calcium reduced mediu~and it has been used in subsequent studies with no attendant discussion.
EDTA is a chelator of divalent cations. The molar ratio of divalent cation to chelate molecule is, at the theoretical limit, equal to two. Since EDTA possesses four carboxylic acid groups it is theoretically possible for two calcium ions to electrically neutralize one EDTA molecule .
Complex ion-chelator formations may also occur in acidic media by protonation of the amine groups. Therefore , it is with considerable conserative bias that the theoretical processes are known to occur in transport systems of neurotransmitter amino acids (Cohen and Lajtha, 1972). ,1 Preventing the calcium dep· endent process would serve to reduce   (Roberts, 1977).
In recent years a growing number of investigators have be~ come concerned with the validity of the methods employed. 72 The most popular method by far is that of Lineweaver -Burk (1934), although Dowd and Riggs (196~) demonstrated that this method led to least accurate determinations of kinetic constants whether the curve was plotted by eye or using the r method of least squares. The Hofstee plot (S/v vs. S) was considered the most favorable since it was viewed that of the two variables Shad less error associated with it than v. Estimates of kinetics constants can be obtained using non-linear statistical techniques (Cleland, 1967;Wentworth , 1965a, b)  Recently, a graphical method for the ·analysis of kinetic data called the direct linear plot (DLP) has been 73 re· ported by ~isenthal and Cornish-3 owden (1974). _Ton-para metric statistical considerations supporting the use of t his method have been describ ed (Cornish-Bowden and Eisenthal, 1974;Trag er and Porter , 1977). Th e advantage to the direct linear plot , over the other methods as pointed out by the authors is that it is simple to construct with kinetic constants read directly off the graph without calculations as is done with reciprocal plots. It is extremely sensitive to outliers or abberant observations and provides accurate determinations of kinetics constants.
The kinetics data for 1-NE uptake into hypothalamic CSE was analyzed using a number of different methods , some graphi cal and some statistical.     Ross and Renyi, 1964Salama et al., 1971Snyder et al. , 1968Ross and Renyi, 1975Ross and Renyi, 1975Shaskan and Snyder, 1970 This study  Ross and Renyi, 1969Wong et al., 1973Shaskan and Snyder, 1970Ross and Renyi, 1975Ross and Renyi, 1975 This study Remarks mid-brain mouse slices rat brain synaptosomes rat brain synaptosomes rat brain synaptosomes rat brain slices Medium of Snyder and Coyle ( 1969) N/A The information was not available in the cited paper. vi) Subcellular distribution of H 3 -norepinephrine uptake An attempt to characterize t h e functional integrity of the synaptosomal transport system was made by separating some of the constituents of the CSH. The synaptosomes are known to be confined to a region on the sucrose gradient corresponding to 1.1 molar (Gray and W hittaker, 1962).
Mitochondria are known to sediment below 1.2 M sucrose and monoamine oxidase activity is taken to be a mark er for mitochondria (Laduron, 1977). In figure 7 it can be observed that the radioactive neurotransmitter concentration is highest at the synaptosomal fraction and some label can be seen associated with myelin and microsomes as might be mitochondrial or microsomal components. vii) Regional distribution of neurotransmitter uptake The selection of brain regions for studying the uptake of different neurotransmitters was based on the rationale that selection of brain regions with a high concentration of nerve terminals would result in a greater degree of uptake compared with non-specific binding. Snyder and Coyle (1969) used hypothalamus for norpeinephrine and striatum for dopamine uptake studies. Kuhar et al. (1972) used forebrain for serotonin uptake studied. Confirmation of this selection of brain regions was attempted by studying the uptake of each neurotransmitter in each brain region. Figure 8 depicts the results. Each value and its associated variation represents the results obtained from 3 animals. Dopamine uptake is unquestionably greatest into the striatal tissue although a fair amount of dopamine can be taken up into hypothalamic tissue presumably into noradrenergic as well as dopaminergic neurons to some degree . N orepin-, phrine uptake, although large in the striatum, may represent uptake into dopaminergic and other nerve terminals.
Hokefelt (1970) estimated the noradrenergic contribution towards total catecholamines in the striatum to be 5% based on histochemical observations. The selection of tissue for uptake is similarly clear with greatest values obtained for serotonin uptake into forebraintissue.
The choice of NE into hypothalamus appears to be governed more by tradition and concentrations of all three

SEROTONIN NOREPINEPHRINE DOPAMINE
neurotransmitters are shown to be equivalent in the hypothalamus (Anden et al., 1966).
viii) Effect of calcium in 1-norepinephrine induced release by hi-potassium depolarization The results appear predictable. Removing neurotransmitter from the synaptosomes may reasonably result in greater uptake by increasing the physical capacity to accumulate neurotransmitter taken up. Kant and Meyerhoff (1977) demonstrated at 17.5% increase in NE released by Hi-K+ in the absence of ca++ which can be used to explain the reason for an increase in uptake over normal Kreb's.
The fact that uptake is increased in normal ca++ Hi-K+ as opposed to ca++ free Hi-K+ media suggests that exogenously administered NE may equilibrate with both neurotransmitter compartments (i.e. vesicular and cytoplasmic).
The increase in uptake measured in the refilling condition appear inconsistent with notions regarding the distribution of neurotransmitter within the synaptosome.
Classical theory considers norepinephrine to be bound with a protein, chromagranin A, ATP and M g++ (Cooper,Bloom & Roth,197~). It is also postulated that a mobile pool exists within the vesicle (Kirshner, 1962). uptake. There appears to be no obvious separation of effects  (2) Reference Ross and Renyi, 1967aShaskan and Snyder, 1970Horn et al., 1971Carmichael and Israel, 1973Maxwell et al., 1974Ross and Renyi, 1975 This study (number of experiments) *Ic 50 1 s expressed in micromolar (uM)  Renyi, 1967 Horn et al., 1971 This study (number of experiments) *Ic 50 1 s expressed in micromolar (uM) shown to be high (Asberg et al . ,1971)    The ar gument is difficult to refute on t he oretical grounds and no attemut to do so will be persued . Instead, these experiments will be repeated with post-pubertal a.Di mals prior to publication .  (Sayers and Burki , 1976;Snyder and Yammamura , 1977 (Schmidt et al., 1977).
Other clinically effective antidepressants exhibit different pharmacological profiles than the tricyclics .
Mianserin is not a very potent inhibitor of catecholamine or serotonin uptake (Goodlet et al., 1977). Th e in-vitro comparisons then are performed with the understanding that they might not reflect the actual relative potencies which may occur upon c~ronic administration of the drug, however , it represents a starting point and a source of comparison for data which will no doubt become * wean% s.e.m. tal~ulrted+f%om indtviduat igvjfti~a.,orf' lesults pres.rnt3c Tn ~al)Jes7--g.  To the left of the vertical doub l e line in table 10 a.re the uptake inh i bitory potencies of t he t ricy clics with r espe ct to three neurotransmitters a s well a s the relative (to i miuramine) antic ho linergic potencies . The relativ e potency ratios for serotonin , norepinephrine and dopamine, r eferred to as t h e biogenic a.n ine ratios, can be comnared wit h one another but t h e antic h olinergic:biogeni c ami ne ratios, in t he last three columns of table 10, cann ot be compared directly with t h e bio g enic amine ratios simpl y because a different response measure was used i n determining t he antic holinergic influence. The values obtai ned for the anticholiner gic:biog enic amine ratios become more meani ngful when viewed as a percent of t h e total a ntic h olinergi c influence with resuect to each of t h e bi ogenic a mines. ~he values placed within parentheses directly below the anticholinergic: biogeni c a.mine ratios were obtained by summing the t hr ee ratios for a single drug and expressing each ratio as a percent of t h e total.
Several portions of this table demonstrate interesting correlations with lmown effects of tric yclics. Based upon the biogenic amine comparisons in the first three columns to t h e ri ght of t h e double line, t here appears to be n o diff erence between the effects of amitriptyline an d doxepin.
Clinical studies do not demonstrate any clear differenc e s between doxepin and amitriptyline as antidepressants (Beck , 1973). Doxepin has been reported to be well tolerated by older patients because of fewer incidences of anticholinergic side effects . This difference is clearly reflected in the potencies of anticholinergic effects where amitriptyline is ten times more uotent than doxepin. This may sugg est that the anticholinergic properties are not i mp ortant for t he clinical therapeutic effect . Cairncross et al . (1 963 ) sugg ested t hat the a."'1.ticholinergic effect was imuortant for therapeutic activity but :B lac kwell ~al . (1972) conclud ed that the antic~oliner g ic effect was related more to the sedative properties of the drugs . This is difficult to reconcile since stimulation of locomotor activity , a behavioral resuonse measure which could be vie·wed as an inverse indicator of sedation , has been s hown to be centrally mediated by anticholinergic drugs (Aquilo:'.li us et a.l. , 1972) .
It may be that the difference in antic holiner gi c potency is only reflected in the periphery with these two drugs . The particular proportional indluences upon the biogenic amine syste:ns may serve to mask any psychostimulatory properties these drugs possess if they were purely anticholinergic .
This may occur as a result of the lack of proportional influences within the periphery that these neurotransmitter systems may possess within the central nervous system . A look at the anticholinergic: biogenic amine ratio 11 perc ent" figures of table 10 sugg ests that there are no differences in the relative effects of a.Dticholinergic to biogenic amine influences between amitriptyline and doxepin. It may be as a. result of this balancing influence that doxeuin and aoitriptyline are sedative.
On the other end of the pharmacological suectrum is the psychostimulatory effects of protriptyline. Protr iptyline is the only tricyclic antidepressant recommended by a manufacturer for use in anergic :patients . Since dopaminergic influences are believed to be responsible for stimulating effects on locomotor activity (?ibiger et al., 1973;Creese and Iversen , 1973;~seug and Loh , 1974;Tseug et al., 1974) and cholinergic -dopaminergic interactions are important as well (Klawans, 1968 ;Hornykiewicz , 1966;Friehoff & Alpert , 1973) , it seems reasonable to conclude that a cho linergic -dopaminergic interaction ma.y mediate the psychostimulatory effect of this drug . Compared with desmethylimipramine, the percent of anticholinergicdopa.minergic interaction is 22 times greater for protriptyline whereas the effects with respect to anticholinergicnorepinephrine or anticholinergic-serotonin influences are norepinephrine or a.nticholinergic-serotonin influences are comparable. This may reflect the mechanism of psy chostimu lation occuring in-vivo.
This approach may offer a reasonable, although admittedly speculative, interpretation for the different effects observed with the clinical use of tricyclics, but most important is the therapeutic effect. Review of the relative potency ratios for biogenic amines demonstrates the common factors which may suggest how all these drugs ma.y work with respect to the neurotransmitters studied. All dopamine:norepinephrine ratios a.re greater t han one and all serotonin : dopamine ratios are less than one. Con sidering only the b iog enic amines, t h is is t h e onl y factor common to all tricy clics reflected in table 10 . It may b e sugg ested t hat t h e relative effects of serotonin with respect to dopaoine and norepinephrine with respect to DA are involved .i n the antidepressant effect of t h ese drugs .
Cont inuing with t h e concept t hat a balance of effects upon critical neurotransmitters may be responsible for the t h erapeutic effect , t h ese observations are remi n isent of t he elements of the "permissive hy pot he sis of depressionP (Prang e et al ., 1974) . This hy pot he sis stat es that s erotonin acts as a gating or controlling influen ce for the expression of either man ia or depression by :ncreased or decreased levels of norepinephrine , respectively . Agents af f ecting t h ese systems perhaps with res pect to a third buffer or reference system such as t he dopaminergic system may account for t h e comm on di mension requir ed for a unified neurotransmitter hypothesis of affective disorders .
An attempt to explain t h e mechanism of action of t h ese drugs using only four of possibly scores of neurotran s mitters is admittedly premature . Certainly oth er me chanisms may be responsible for the t herapeutic effect both within as well as outside the frame work of the biogenic amine hypothesis.
It is i mp ortant to nore that t h e develo pm ent of the tricy clics came out of investi gations with antihistaminic drugs are tricyclics possess antihista~inic properties .
Research directed towards evaluating these properties may reflect more light on even more complex interactions then we dare to expect. Knowledge of the role of neurotransmitters in behavior is still at its infancy (Ealdessarini, 1975). Unt il more data is accu:nula.ted with respect to the pharmacokinetics , physical properties and phar::na.codynamics of tricyclic antidepressants , table like that of table 10 represent a way in which leads toward a particular line of investigation may be obtained. A glimmer of hope in unravelling the complex interactions of a number of variables in biological systems is the increasing applications of statistical methods. Techniques of utilizing parametric and nonparametric statistical methods are becoming more accessible and easier to apply. Pattern recognition tecrL~iques are available to a.id in the analysis of complex interactions using cluster analysis, path analysis, principal components and other multivariate approac h es (Rarper, et a~, 1977).

c. Conclusion
Should the limitations noted above prove to be n egliei b le, t hen it would appear that protript:rline bl ocks t h e uptake of norepinephrine up on chronic adoinistration whereas when administered sub-acutely uptake blockade is not clearly discernable . These findings demonstrate a te~poral correlation of untake blockade with clinical effectiveness in humans and lends support to both t he catecho lamine hypo t hesis and the propo sed mechanism of action of tricyclics as uptake blockers . In addition it is i n teresting to note that there is a ris k of attemp ted suicide in deuressives during t he initial days of ch emotherapy (Beck , 1973) and sub-acute admi n istration displays hi ghl y variable effects and per haps even some potentiation of uptake t h ereby enhancing t h e functional depletion of neurotransnitter at the level of the uost synaptic receptors . This effect could exulain t he clinical observation of increased suicidal tenden cy during initiation of tri cyclic chemotherapy .
In support of the sub-acute data obtained is the evidence that twelve hours post-protr iptyline using either serotonin or tryptarnine as substrate , MAO activity is elevated ( Ed Kaiser-pers onal c ommunication) . An increase in uptake provides more substrate for r~AO t herefore t he se results appear c orroborative of the subacute data presented here .
It appears that the relative antich olinergic properties can be predictive of the psych ostimulatory effects of pr otriptyline . Comparisons of the effects of drugs using in-vitro biochemical techniques may be useful in formulating hypotheses regarding the mechanism of action of the tricyclic antidepressants . The lack of psychostimulatory action of the most potent anticholinergic of the tricy clics, amitriptyline , may be explained by a relative interaction between anticholinergic and dopaminergic influences .