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Relationship between regional distribution of imipramine and its effect on learned helplessness in the rat
RELATIONSHIP BETWEEN REGIONAL DISTRIBUTION OF IMIPRAMINE AND ITS EFFECT ON LEARNED HELPLESSNESS IN THE RAT A. D. SHERMAN Neurochemistry
Research
and G. L. ALLERS
Laboratory. Department of Psychiatry. Iowa City, IA. U.S.A.
University
of Iowa,
~--The regional distribution of imipramine and its major metabolite. desipramine, was determined in relation to drug effects in a “learned helplessness” paradigm. This model for depression has been shown to parallel the antidepressant action of imipramine in humans. Although 4 days of drug administration were required for a significant number of animals to show drug effects. some animals were responsive to a single dose of imipramine. Beginning with the first day. the desipramine levels in the hippocampi of animals in which imipramine prevented learned helplessness were significantly higher than in those animals which did not respond to imipramine. No such relationship was found for the other regions studied. The data suggest that the delay in onset of the effects of imipramine is related to the rate of hippocampal transport of its demethylated metabolite. Summary
delayed onset in the clinical effectiveness of tricyclic antidepressants (TCAs) has been well documented (Klein and Davis, 1969) and differences between chronic and acute effects of TCAs have been reported in many studies (deMontigny and Aghajanian, 1978; Segal. Kuczenski and Mandell, 1974). In these studies, it has been assumed that changes induced by acute administration of TCAs are non-specific and probably unrelated to the antidepressant action of the drug since they occur in the brain at a time when the drugs are not clinically effective. In general, the distributional aspects of these agents are not directly considered in relation to the delayed onset of action of the TCAs. Plasma levels of imipramine have been reported at 210 _t 110 ng,/ml following a 1.2 mg/kg dose in humans (Perel, Shostak, Gann. Kantor and Glassman, 1976) and nortryptylene at 140 + 50ng/ml (Kragh-Sorensen, EggertHansen and Larsen. 1974) with a 5510-fold range between high and low values. The exceptionally high variability between subjects suggests that pharmacokinetic parameters may be relevant to the delayed onset 0r clinical effectiveness. In animals, Hafliger (1959) found higher levels of imipramine in rabbit brain, heart and kidney than in liver. In contrast, high tissue levels have been reported (Dingell, Sulser and Gillette, 1964) in rabbit spleen, heart, lung and kidney, with brain levels substantially lower. This pattern was also described in rats. Radioautography performed on mice following intravenous administration of [“VI-imipramine (Cassano and Hansson. 1966) revealed the time-dependent nature of cerebral distribution of imipramine. especially in the neocortex and hippocampus. Their The
Key words: distribution.
imipramine.
learned
helplessness.
data revealed an unusually high concentration of labelled TCA in rat hippocampus, with the level in frontal neocortex higher than that m the temporal or occipital cortex. Retention of [14C]-TCA by hippocampus was also shown to be substantially longer than in other areas of the brain. The relationship between tissue levels of TCA and therapeutic effects of these agents has not been investigated because of the lack of an animal model for depression which would provide both an estimate of antidepressant action and the availability of brain tissue for analysis. Recently, the “learned helplessness” model of depressant (Seligman and Maier, 1967) has been subjected to pharmacological analysis (Sherman, Allers, Henn and Petty, 1979) and has been shown to represent a useful animal model for depression. Briefly, this behavior is (a) sensitive to imipramine, but not to chlorpromazine or lorazepam. (b) affected in a highly dose-related manner and (c) requires several days of exposure to imipramine before antidepressant activity can be demonstrated in a significant proportion of the animals. The present paper describes the relationship between the regional distribution of imipramine and its “therapeutic” action in preventing the development of learned helplessness. METHODS Male SpragueeDawley rats were used in all studies. All were maintained on food with drinking water that contained imipramine.HCl (Sigma Chemical Co.) at a concentration equivalent to 100 mg base/l. Based on an average daily intake of 19 ml/day, this represents a dose of approximately 7.5 mg/kg/day. Animals tested on the first day of drug administration were given
regional
I.59
160
A. D. SHERMAN and G. L. AI.LERS Tahlc 1. Total free TCA (imipramine plus desipramine) levels in regions or rat brain Day Region ~_I___ Hippocampus Frontal neacortex Temporal neocortes Rest of brain
1 1718 i: 613 563 * 280 519 * 142 313 & 166
2
3
1533 i: 308 788 & 459 790 jI 444 240 2 92
1689 i 572 770 + 287 423 $: 47 579 :t 173
4 2238 513 476 234
f + + +
344 126 187 72
Data are mean rig/g rt SD for 7-8 al~imais~group. All animals received imipramine WC1 equivalent to 100mg base/l in drinking water except group 1 which was given 7.5 mg;kg intraperitoneall}~ about i hr before initial testing.
at 7.5 mg/kg (i.p,.) 1 hr before learned heIpiessness training. All groups (7--g animals each) are identi~ed by the number of days of imipramine availability at the time of learned helplessness training. Initially, the animals were exposed to an 0.7 mA shock on a 10 set on/50 set off schedule for 1 hr in the apparatus used for later testing. The apparatus consisted of a 12 x 18 x 12” box with a grid floor. After this learned helplessness training, the animals were allowed 2 hr in their home cage with tap (drug-free) water to drink. After 2 hr, the animals were returned to the same apparatus for Ien trials of escape testing. For escape testing, a 3 x 3” platform 9” above the grid floor was inserted through the side of the box and shock (0.4 mA) initiated. If. after 10 see, the animal had not escaped onto the platform. the shock was terminated. Animals escaping onto the platform were allowed to stay there for the remaining time until 10 set had elapsed and were returned to the grid floor. After an intertrial interval varying around 20 set, the trial was repeated. After 10 trials, the animals were sacrificed by cervical fracture and brains removed on a cold plate mainThe foll~)w~ng sections were tained at -6-C. obtained: hippocampus, frontal neocortex (anterior to the posterior limit of the septum), temporal (remaining) neocortex, and remainder of brain exclusive of the cerebellum. Each section was homogenized in 4 vol of 5?/, trichloroacetic acid, amj~rip~y~ene*H~~ (Merck, Sharp & Dohme) equivalent to I ,ug base was imipramine
added, and the sample centrifuged at 16,OOOg for 10min at 4°C. The supernatant was passed over a 4 x 0.5 cm Amberiite XAD-2 column which had been washed with 10 vo1 of water. chloroform, and water successively. The column was washed with 0.7 ml of water, then imipramine, desipramine and the internal standard amitriptylene were eluted with 2 ml of chIaroform. The chloroform was concentrated to less than 50 ~1 and a 5 ~1 aliquot was injected onto a I m x 2 mm column of OV-17 in a Bendix 2500 gas chromatograph operating at 210°C. The injector and detector temperatures were 22W and 245” respectively. Amitriptytene, imipramine and desipramine eluted at 3.4, 4.1 and 4.9 min respectively, and peak areas were determined on a lT1 digital integrator. Recovery as determined for 3H-imipramine was 84 i 8%.
RESULTS
Free drug levels (Table 1) were essentially constant from the first day onward, possibly affected by seIfregulation of intake by the animals since the total amount of drug received would depend on total water consumption. On each day, animals were classified into two groups; “responders” to imipramine intervention had 0 or 1 escape failures (1Osec) on the final five trials of escape testing, while “non-responders” had 4 or 5 failures. No animals had 2 or 3 escape failures.
Table 2. Regional drug levels (imipramine plus desipramine) in two areas of brains of “responders” and “non-responders” Region -“-__ Frontal neocortex
._.--~
Day
Responders
Non-responders
1
717 * 387 (3) 586 + 315 (6) 508 t 61 (4) 513 I: 126(X) 2133 + 453 (4) 1712 rt 321 (6) 2184 k 288 (4) 2238 & 344(8)
448 It. 123 (4) 1394 & 8 (2)$ 1032 f 71 (4)*
2 3 4 Hippocampus
to imipramine intervention in learned helplessness
1 2 3 4
1165 1131 (3)t 1294 ir 28 (1) 1194 ) 169 (4)t
* Significantly higher than responders; P -c 0.05 by Mann-Whitney U-test. t Significantly lower than responders; P < 0.05 by Mann-Whitney U-test. Data are mean -fr SD ngig. Number of animals in parentheses. Conditions as in Table 1.
Imipramine Table
3. Hippocampal
Day
Responders
1 2 3 4 All
ND 49 * 147 + 531 *
161
distribution
drug levels in responders and non-responders intervention in learned helplessness Imipramine Non-responders
(3) 30(5) 90(6) 104(8)
244 + 102 (22)
Responders
to imipramine
Desipramine Non-responders
146 f 97* (4) 325 k 129* (3) 1I2 k 56 (2)
2133 1663 2037 1707
453 (3) 261(5) 316(6) 441 (8)
1019 + l96* (4) 969 + 489* (3) 1081 f 20* (2)
198 f 72(9)
1833 f 405 (22)
1036 k 205* (9)
+ + k *
Data are mean ngig k SD. Number of animals in parentheses. * P < 0.05 vs responders by Mann-Whitney U-test. Other conditions
Based on this classification, no reliable differences in temporal neocortical levels or levels in the “remainder” of the brain were observed in comparing responders and non-responders. In the hippocampus and frontal neocortex (Table 2) however, differences reached statistical significance. Beginning with the second day, lower drug levels in the frontal neocortex were associated with response to drug. From the first day, higher hippocampal levels were seen in responders as opposed to non-responders. The presence of unmetabolized imipramine (Table 3) at higher levels in the hippocampi of “non-responders” as opposed to “responders” supports earlier suggestions (Dingell, er al., 1964) that the demethylated metabolite desipramine might be the active agent following imipramine administration. Accordingly, for all days, the desipramine level in hippocampi of responders was significantly higher than levels for nonresponders to imipramine intervention in learned helplessness. A hippocampal concentration of 5 x 10m6 M (I 335 ng desipramine/g) divided responders and nonresponders exactly for the first 3 days, with responders exceeding this level and non-responders failing to reach this level, e.g. Day 1: 4/4 responders and O/3 non-responders (P = 0.029); Day 2: 616 vs O/2 (P = 0.036); Day 3: 4/4 vs O/4 (P = 0.014).
DISCUSSION
The typical delayed response to TCA administration can be seen when all animals are taken together. On Day 1, 4/8 were classified as “responders” (i.e. 0 or 1 escape failures on the last five trials) resulting in a probability of 0.50. On day 4, however, all animals acquired the task and had 0 or 1 escape failures on the final five trials (P = 0.04). This 4-day delay in response to TCAs is in contrast to the 2 weeks normally considered necessary before full therapeutic response is established in humans. There are several possible explanations for this difference. First, the present data are based on a model system which may, in fact, be based on variables quite different from those related to depression in humans. While the model system parallels human depression
as in Table
I.
in several aspects, there may be important differences as yet untested. Second, the lack of a “placebo effect” may be very relevant, Clinically, those patients which appear to respond to TCAs within the first few days may be classified as actually having had no response since many patients improve without therapy during this time. In the model system, it is most unusual for an undrugged control not to acquire the learned helplessness and thus not performed well on the escape responding task which follows it. Thus, it may not be possible to differentiate those patients who actually respond to TCA therapy within a few days from while this represents a minor “placebo responders,” problem in the model system. Thirdly, the average dose of imipramine (7.5 mg,ikg per day) is substantially higher than that given clinically, which only rarely exceeds 4 mg/kg per day. Finally. differences in hepatic metabolism rates or transfer rates from the CSF may be different enough to produce a difference such as this. Reference to Table 1 and autoradiographic data (Cassano and Hansson. 1966) reveals a reasonably good overlap considering the differences in methodology. In both cases, the ratio of drug in the frontal neocortex to temporal and occipital neocortex exceeds I.0 and averages 1.25 & 0.39 in the present study. This difference appears to be due to a regionally high concentration in the most frontal areas (Cassano and Hansson. 1966). The higher level in hippocampus was also confirmed with hippocampal levels 224 times higher than other areas measured, This confirmation (in a different species) tends to support this hypothesis that the increased level and retention of TCAs in the hippocampal formation may be related to the slow diffusion of drug into the hippocampus from the CSF. The large hippocampal surface exposed to the lateral ventricle makes this an attractive explanation. Further support comes from studies involving acetazolamide in which high hippocampal concentrations of drug were attributed to this mechanism. The finding of statistically-reliable differences in drug level between responders and non-responders appears unlikely to represent a random difference in membrane permeability to TCAs. For example. no
162
A. D. SHEKMAN and G. L. ALLERS
reliable differences were observed in the temporal neocortex or the “rest of brain” sections where drug levels were found to be lower than the other two sections. In the frontal neocortex, the lower level in responders appears to be due to the accumulation of TCA in non-responders. In the hippocampal formation. where the highest TCA levels &e found, the situation appears quite different. From the first day. higher levels of desipramine were present in the hippocampi of responders than in non-responders. In those cases in which either reduced demethylation of the parent compound by liver microsomes or decreased uptake of the secondary amine metabolite resulted in a lower level of desipramine, the animals failed to respond to imipramine administration. In considering these distributional studies, it should be stressed that the animals tested on the first day received drugs via intraperitoneal injection while all other animals received drug orally in drinking water. The presence of unmetabolized imipramine in the hippocampus followed a time-dependent course in those animals which responded to TCA administration. Beginning with the second day, levels in the hippocampus increase in a logarithmic manner and represent a substantial proportion of the total drug present by the fourth day. In non-responders. levels are essentially constant. As related to TCA therapy of depression in humans, these data suggest that hippocampal drug levels may be a critical variable in establishing the efficacy of these agents in a clinical situation. Furthermore. the extremely high ratio of its secondary amine metabolite to imipramine supports the hypothesis of
Dingeil et rtl. (1964) that desipramine may be the effective antidepressant following imipramine administration.
REFEREILCES
Cassano. G. B. and Hansson. E. (1966). Autoradiographic distribution studies in mice with C14-imipramine. I,I~. J. A’rwops~chiut.2: 269-278. Dingell. J. V., Sulser. F. and Gillette. J. R. (lY64). Spccles differences in the metabolism of imipramine and desmethylimipramine (DMI). J. Phclrn~trc. c’\-p. Thc>r. 143: ., __
I +‘!‘L.
Hafliger. F. (1959) Chemistry of Tofranil. Curl. Psydlrut. Assn. J. 4 (supplement): S69%S74. Klem, D. F. and Davis. J. M. (1969). Diuyruks td Dr~tq Trrutmrnt of Psydkmk Disorders. Williams & Wilkens. Baltimore. Kragh-Sorensen. P., Eggert-Hansen. C. and Larsen. N. E. (1974). Long-term treatment of endogenous depression with nortriptvlene with control of nlasma levels. P.s~~c~/Io/. Merl. 4: l74-1x0. deMontigny, C. and Aghajanian. G. K. (107X). Tricyclic antidepressants: long-term treatment increase\ rcsponsivity of rat forebrain neurons to serotonin. .‘cir~cc 202: 1303--1306. Perel. J. M.. Shostak. M., Gann. E., Kantor. S. J. and Classman, A. A. (1976). In: Pkur,rluc,okinc,ti~s of P.\yc~hoclcfil,u Drugs (Gottschalk. L. A. and Merlis. S.. Eds), p. 229. Spectrum. New York. Segal, D. S.. Kuczensky, R. and Mandell. A. J. (lY74). Theoretical implications of drug-induced adaptive regulation of a biogenic amine hypothesis of affective disorder. B&l. P,syc,llicrr.9: l47- 159. Seligman. M. E. P. and Maier. S. F. (lY67). Failure to escape traumatic shock. J. GYP.P.~ydw/. 74: 1-Y. Sherman, A. D., Allers, G. L.. Henn. F. A. and Petty, F. (1979). A neuropharmacologically-relevant animal model of depressjon. ~Vclrrophun)luco/(J~~~ 18: X9 I ~93.
Research
and G. L. ALLERS
Laboratory. Department of Psychiatry. Iowa City, IA. U.S.A.
University
of Iowa,
~--The regional distribution of imipramine and its major metabolite. desipramine, was determined in relation to drug effects in a “learned helplessness” paradigm. This model for depression has been shown to parallel the antidepressant action of imipramine in humans. Although 4 days of drug administration were required for a significant number of animals to show drug effects. some animals were responsive to a single dose of imipramine. Beginning with the first day. the desipramine levels in the hippocampi of animals in which imipramine prevented learned helplessness were significantly higher than in those animals which did not respond to imipramine. No such relationship was found for the other regions studied. The data suggest that the delay in onset of the effects of imipramine is related to the rate of hippocampal transport of its demethylated metabolite. Summary
delayed onset in the clinical effectiveness of tricyclic antidepressants (TCAs) has been well documented (Klein and Davis, 1969) and differences between chronic and acute effects of TCAs have been reported in many studies (deMontigny and Aghajanian, 1978; Segal. Kuczenski and Mandell, 1974). In these studies, it has been assumed that changes induced by acute administration of TCAs are non-specific and probably unrelated to the antidepressant action of the drug since they occur in the brain at a time when the drugs are not clinically effective. In general, the distributional aspects of these agents are not directly considered in relation to the delayed onset of action of the TCAs. Plasma levels of imipramine have been reported at 210 _t 110 ng,/ml following a 1.2 mg/kg dose in humans (Perel, Shostak, Gann. Kantor and Glassman, 1976) and nortryptylene at 140 + 50ng/ml (Kragh-Sorensen, EggertHansen and Larsen. 1974) with a 5510-fold range between high and low values. The exceptionally high variability between subjects suggests that pharmacokinetic parameters may be relevant to the delayed onset 0r clinical effectiveness. In animals, Hafliger (1959) found higher levels of imipramine in rabbit brain, heart and kidney than in liver. In contrast, high tissue levels have been reported (Dingell, Sulser and Gillette, 1964) in rabbit spleen, heart, lung and kidney, with brain levels substantially lower. This pattern was also described in rats. Radioautography performed on mice following intravenous administration of [“VI-imipramine (Cassano and Hansson. 1966) revealed the time-dependent nature of cerebral distribution of imipramine. especially in the neocortex and hippocampus. Their The
Key words: distribution.
imipramine.
learned
helplessness.
data revealed an unusually high concentration of labelled TCA in rat hippocampus, with the level in frontal neocortex higher than that m the temporal or occipital cortex. Retention of [14C]-TCA by hippocampus was also shown to be substantially longer than in other areas of the brain. The relationship between tissue levels of TCA and therapeutic effects of these agents has not been investigated because of the lack of an animal model for depression which would provide both an estimate of antidepressant action and the availability of brain tissue for analysis. Recently, the “learned helplessness” model of depressant (Seligman and Maier, 1967) has been subjected to pharmacological analysis (Sherman, Allers, Henn and Petty, 1979) and has been shown to represent a useful animal model for depression. Briefly, this behavior is (a) sensitive to imipramine, but not to chlorpromazine or lorazepam. (b) affected in a highly dose-related manner and (c) requires several days of exposure to imipramine before antidepressant activity can be demonstrated in a significant proportion of the animals. The present paper describes the relationship between the regional distribution of imipramine and its “therapeutic” action in preventing the development of learned helplessness. METHODS Male SpragueeDawley rats were used in all studies. All were maintained on food with drinking water that contained imipramine.HCl (Sigma Chemical Co.) at a concentration equivalent to 100 mg base/l. Based on an average daily intake of 19 ml/day, this represents a dose of approximately 7.5 mg/kg/day. Animals tested on the first day of drug administration were given
regional
I.59
160
A. D. SHERMAN and G. L. AI.LERS Tahlc 1. Total free TCA (imipramine plus desipramine) levels in regions or rat brain Day Region ~_I___ Hippocampus Frontal neacortex Temporal neocortes Rest of brain
1 1718 i: 613 563 * 280 519 * 142 313 & 166
2
3
1533 i: 308 788 & 459 790 jI 444 240 2 92
1689 i 572 770 + 287 423 $: 47 579 :t 173
4 2238 513 476 234
f + + +
344 126 187 72
Data are mean rig/g rt SD for 7-8 al~imais~group. All animals received imipramine WC1 equivalent to 100mg base/l in drinking water except group 1 which was given 7.5 mg;kg intraperitoneall}~ about i hr before initial testing.
at 7.5 mg/kg (i.p,.) 1 hr before learned heIpiessness training. All groups (7--g animals each) are identi~ed by the number of days of imipramine availability at the time of learned helplessness training. Initially, the animals were exposed to an 0.7 mA shock on a 10 set on/50 set off schedule for 1 hr in the apparatus used for later testing. The apparatus consisted of a 12 x 18 x 12” box with a grid floor. After this learned helplessness training, the animals were allowed 2 hr in their home cage with tap (drug-free) water to drink. After 2 hr, the animals were returned to the same apparatus for Ien trials of escape testing. For escape testing, a 3 x 3” platform 9” above the grid floor was inserted through the side of the box and shock (0.4 mA) initiated. If. after 10 see, the animal had not escaped onto the platform. the shock was terminated. Animals escaping onto the platform were allowed to stay there for the remaining time until 10 set had elapsed and were returned to the grid floor. After an intertrial interval varying around 20 set, the trial was repeated. After 10 trials, the animals were sacrificed by cervical fracture and brains removed on a cold plate mainThe foll~)w~ng sections were tained at -6-C. obtained: hippocampus, frontal neocortex (anterior to the posterior limit of the septum), temporal (remaining) neocortex, and remainder of brain exclusive of the cerebellum. Each section was homogenized in 4 vol of 5?/, trichloroacetic acid, amj~rip~y~ene*H~~ (Merck, Sharp & Dohme) equivalent to I ,ug base was imipramine
added, and the sample centrifuged at 16,OOOg for 10min at 4°C. The supernatant was passed over a 4 x 0.5 cm Amberiite XAD-2 column which had been washed with 10 vo1 of water. chloroform, and water successively. The column was washed with 0.7 ml of water, then imipramine, desipramine and the internal standard amitriptylene were eluted with 2 ml of chIaroform. The chloroform was concentrated to less than 50 ~1 and a 5 ~1 aliquot was injected onto a I m x 2 mm column of OV-17 in a Bendix 2500 gas chromatograph operating at 210°C. The injector and detector temperatures were 22W and 245” respectively. Amitriptytene, imipramine and desipramine eluted at 3.4, 4.1 and 4.9 min respectively, and peak areas were determined on a lT1 digital integrator. Recovery as determined for 3H-imipramine was 84 i 8%.
RESULTS
Free drug levels (Table 1) were essentially constant from the first day onward, possibly affected by seIfregulation of intake by the animals since the total amount of drug received would depend on total water consumption. On each day, animals were classified into two groups; “responders” to imipramine intervention had 0 or 1 escape failures (1Osec) on the final five trials of escape testing, while “non-responders” had 4 or 5 failures. No animals had 2 or 3 escape failures.
Table 2. Regional drug levels (imipramine plus desipramine) in two areas of brains of “responders” and “non-responders” Region -“-__ Frontal neocortex
._.--~
Day
Responders
Non-responders
1
717 * 387 (3) 586 + 315 (6) 508 t 61 (4) 513 I: 126(X) 2133 + 453 (4) 1712 rt 321 (6) 2184 k 288 (4) 2238 & 344(8)
448 It. 123 (4) 1394 & 8 (2)$ 1032 f 71 (4)*
2 3 4 Hippocampus
to imipramine intervention in learned helplessness
1 2 3 4
1165 1131 (3)t 1294 ir 28 (1) 1194 ) 169 (4)t
* Significantly higher than responders; P -c 0.05 by Mann-Whitney U-test. t Significantly lower than responders; P < 0.05 by Mann-Whitney U-test. Data are mean -fr SD ngig. Number of animals in parentheses. Conditions as in Table 1.
Imipramine Table
3. Hippocampal
Day
Responders
1 2 3 4 All
ND 49 * 147 + 531 *
161
distribution
drug levels in responders and non-responders intervention in learned helplessness Imipramine Non-responders
(3) 30(5) 90(6) 104(8)
244 + 102 (22)
Responders
to imipramine
Desipramine Non-responders
146 f 97* (4) 325 k 129* (3) 1I2 k 56 (2)
2133 1663 2037 1707
453 (3) 261(5) 316(6) 441 (8)
1019 + l96* (4) 969 + 489* (3) 1081 f 20* (2)
198 f 72(9)
1833 f 405 (22)
1036 k 205* (9)
+ + k *
Data are mean ngig k SD. Number of animals in parentheses. * P < 0.05 vs responders by Mann-Whitney U-test. Other conditions
Based on this classification, no reliable differences in temporal neocortical levels or levels in the “remainder” of the brain were observed in comparing responders and non-responders. In the hippocampus and frontal neocortex (Table 2) however, differences reached statistical significance. Beginning with the second day, lower drug levels in the frontal neocortex were associated with response to drug. From the first day, higher hippocampal levels were seen in responders as opposed to non-responders. The presence of unmetabolized imipramine (Table 3) at higher levels in the hippocampi of “non-responders” as opposed to “responders” supports earlier suggestions (Dingell, er al., 1964) that the demethylated metabolite desipramine might be the active agent following imipramine administration. Accordingly, for all days, the desipramine level in hippocampi of responders was significantly higher than levels for nonresponders to imipramine intervention in learned helplessness. A hippocampal concentration of 5 x 10m6 M (I 335 ng desipramine/g) divided responders and nonresponders exactly for the first 3 days, with responders exceeding this level and non-responders failing to reach this level, e.g. Day 1: 4/4 responders and O/3 non-responders (P = 0.029); Day 2: 616 vs O/2 (P = 0.036); Day 3: 4/4 vs O/4 (P = 0.014).
DISCUSSION
The typical delayed response to TCA administration can be seen when all animals are taken together. On Day 1, 4/8 were classified as “responders” (i.e. 0 or 1 escape failures on the last five trials) resulting in a probability of 0.50. On day 4, however, all animals acquired the task and had 0 or 1 escape failures on the final five trials (P = 0.04). This 4-day delay in response to TCAs is in contrast to the 2 weeks normally considered necessary before full therapeutic response is established in humans. There are several possible explanations for this difference. First, the present data are based on a model system which may, in fact, be based on variables quite different from those related to depression in humans. While the model system parallels human depression
as in Table
I.
in several aspects, there may be important differences as yet untested. Second, the lack of a “placebo effect” may be very relevant, Clinically, those patients which appear to respond to TCAs within the first few days may be classified as actually having had no response since many patients improve without therapy during this time. In the model system, it is most unusual for an undrugged control not to acquire the learned helplessness and thus not performed well on the escape responding task which follows it. Thus, it may not be possible to differentiate those patients who actually respond to TCA therapy within a few days from while this represents a minor “placebo responders,” problem in the model system. Thirdly, the average dose of imipramine (7.5 mg,ikg per day) is substantially higher than that given clinically, which only rarely exceeds 4 mg/kg per day. Finally. differences in hepatic metabolism rates or transfer rates from the CSF may be different enough to produce a difference such as this. Reference to Table 1 and autoradiographic data (Cassano and Hansson. 1966) reveals a reasonably good overlap considering the differences in methodology. In both cases, the ratio of drug in the frontal neocortex to temporal and occipital neocortex exceeds I.0 and averages 1.25 & 0.39 in the present study. This difference appears to be due to a regionally high concentration in the most frontal areas (Cassano and Hansson. 1966). The higher level in hippocampus was also confirmed with hippocampal levels 224 times higher than other areas measured, This confirmation (in a different species) tends to support this hypothesis that the increased level and retention of TCAs in the hippocampal formation may be related to the slow diffusion of drug into the hippocampus from the CSF. The large hippocampal surface exposed to the lateral ventricle makes this an attractive explanation. Further support comes from studies involving acetazolamide in which high hippocampal concentrations of drug were attributed to this mechanism. The finding of statistically-reliable differences in drug level between responders and non-responders appears unlikely to represent a random difference in membrane permeability to TCAs. For example. no
162
A. D. SHEKMAN and G. L. ALLERS
reliable differences were observed in the temporal neocortex or the “rest of brain” sections where drug levels were found to be lower than the other two sections. In the frontal neocortex, the lower level in responders appears to be due to the accumulation of TCA in non-responders. In the hippocampal formation. where the highest TCA levels &e found, the situation appears quite different. From the first day. higher levels of desipramine were present in the hippocampi of responders than in non-responders. In those cases in which either reduced demethylation of the parent compound by liver microsomes or decreased uptake of the secondary amine metabolite resulted in a lower level of desipramine, the animals failed to respond to imipramine administration. In considering these distributional studies, it should be stressed that the animals tested on the first day received drugs via intraperitoneal injection while all other animals received drug orally in drinking water. The presence of unmetabolized imipramine in the hippocampus followed a time-dependent course in those animals which responded to TCA administration. Beginning with the second day, levels in the hippocampus increase in a logarithmic manner and represent a substantial proportion of the total drug present by the fourth day. In non-responders. levels are essentially constant. As related to TCA therapy of depression in humans, these data suggest that hippocampal drug levels may be a critical variable in establishing the efficacy of these agents in a clinical situation. Furthermore. the extremely high ratio of its secondary amine metabolite to imipramine supports the hypothesis of
Dingeil et rtl. (1964) that desipramine may be the effective antidepressant following imipramine administration.
REFEREILCES
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