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Reduced metabolic response of the rat brain to haloperidol after chronic treatment
Brain Research, 337 (1985) 1-9 Elsevier
1
BRE 10795
Research Reports
Reduced Metabolic Response of the Rat Brain to Haloperidol After Chronic Treatment GILBERTO PIZZOLATO*, TIMOTHY T. SONCRANT, DENISE M. LARSON and STANLEY I. RAPOPORT Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, MD 20205 (U.S.A.)
(Accepted September llth, 1984) Key words: haloperidol - - tolerance - - chronic treatment - - cerebral metabolism - - deoxyglucose - - dopamine antagonist
Local cerebral glucose utilization (LCGU) was determined, using the quantitative autoradiographic [14C]2-deoxy-D-glucosetechnique, in 47 brain regions of awake rats, after acute and chronic haloperidol (HAL) administration (1 mg/kg or 1 mg/kg/day). LCGU was reduced in fewer regions after chronic HAL (19%) than after acute HAL (72%); the average reduction for all regions was smaller (8% and 25 %, respectively). The reduced metabolic effect of chronic HAL is not due to a lower brain concentration of the drug, since similar effects on LCGU were found in rats which received an acute i.p. injection of HAL (as in the acutely treated animals) after chronic administration of HAL for 3 weeks. Furthermore, continuous infusion of HAL for 3 weeks or 1 day resulted in similar tolerance to the metabolic effect of HAL. Tolerance was not observed in the mesocortical dopamine (DA) system. The present findings show that tolerance develops to the effect of HAL on cerebral metabolism, even after 1 day of HAL treatment. Lack of tolerance in the mesocortical pathway may implicate this system in the neuroleptic effect of chronic HAL. INTRODUCTION Clinical actions of neuroleptic drugs such as haloperidol ( H A L ) result from blockade of dopamine (DA) neurotransmission in the central nervous system 14. Much experimental data regarding the effects of neuroleptics on brain D A refer to the effects produced by a single dose of drug; however, as antipsychotics, they are administered chronically. H A L alters neurochemical, electrophysiological and behavioral parameters after acute administrationS,33. After repeated or chronic treatment, however, tolerance develops. Increases in D A turnover 30,37, enhancement of the firing rate of the nigrostriatal D A system 8, activation of striatal tyrosine hydroxylase18, 28 and cataleptic behaviorlAS, all produced by acute H A L administration, are less prominent after chronic treatment. Other effects are produced by repeated H A L administration, and are thought to be responsible for behavioral supersensitivity to D A agonists 35. Chronic blockade of D A re-
ceptors results in a compensatory increase in the number of D A receptors17, 34, and in changes in other central neurotransmitter systems (i.e. GABA22, 40, serotonin 34, acetylcholine aS, substance p21). Though the time-course of the development of behavioral and biochemical changes varies during chronic neuroleptic treatment in the rat, the changes usually are fully developed after 1 week of repeated treatment 35. In our laboratory, we previously determined peripheral pharmacokinetics and time-dependent brain concentrations of H A L in Fischer-344 rats after acute i.p. administration of H A L 0.5 mg/kg, or continuous i.p. infusion of the same daily dose of drug for 4 days 24. Regional concentrations of H A L were found to be homogeneous throughout the brain. The concentration of H A L in frontal cortex after H A L 0.5 mg/kg i.p. peaked 30-60 min later (470 ng/g). Thereafter, brain concentration fell in proportion to plasma concentration; after 90 rain, the brain concentration was about half of the peak level (263 ng/g). After establishment of a steady-state brain
Present address: Clinica delle Malattie Nervose e Mentali, University of Padua, Italy. Correspondence: T. T. Soncrant, Laboratory of Neurosciences, NIA NIH, Clinical Center 10/6C103, Bethesda, MD 20205, U.S.A.
*
0006-8993/85/$03.30 (~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)
2 concentration of H A L by continuous infusion of HAL 0.5 mg/kg/day, the frontal cortex concentration of HAL was 120 ng/g, or 25% of the peak level achieved after acute administration. Plasma clearance of H A L was reduced after chronic treatment, suggesting that continuous H A L administration inhibits its own metabolism. We also determined the time-course of the effect of HAL 0.5 and 1 mg/kg i.p. in awake Fischer-344 rats 39. A peak metabolic effect was observed 60 min after i.p. administration of H A L 1 mg/kg. In the present study local cerebral glucose utilization (LCGU) was determined in the awake rat, using the [14C]2-deoxy-D-glucose ([14C]DG) method 45, after acute or chronic administration of H A L 1 mg/kg. The results demonstrate that tolerance to the effects of H A L on LCGU develops within 1 day of continuous administration. MATERIALS AND METHODS Experiments were performed in awake, 3-monthold, male Fischer-344 rats (Charles River Breeding Laboratories, Wilmington, MA). [14C]DG, spec. act. 50-55 mCi/mmol, was purchased from New England Nuclear (Boston, MA), and was rechromatographed to ascertain purity. H A L was a gift from McNeil Pharmaceuticals (Spring House, PA). Drug treatments Acute HAL. Acute Control and Acute H A L rats received the vehicle solution for H A L (1.8 mg methylparaben and 0.2 mg propylparaben in each ml of distilled water, 1 ml/kg), or H A L 1 mg/kg i.p., respectively, 60 min before [14C]DG injection. In Chronic Control rats, subcutaneously-implanted Alzet osmotic pumps (model 2ML4; Alza, Palo Alto, CA) dispensed the vehicle solution continuously at a rate of 0.055 ml/day for 3 weeks prior to [14C]DG injection. Chronic HAL: H A L 1 mg/kg/day was delivered continuously throughout the experiment for 3 weeks by the osmotic pump at a rate of 0.055 ml/day. LCGU was measured on the 21st day of treatment. Chronic + Acute HAL. Rats received H A L 1 mg/kg daily by single subcutaneous injection for 20 days. On the 21st day, 24 h after the last s.c. injection, H A L 1 mg/kg was administered i.p. LCGU was
measured 60 min after this last dose of drug (on the 21st treatment day). Short-term HAL. To establish the time-course of the chronic effects of HAL on LCGU, osmotic pumps, which dispensed H A L 1 mg/kg/day, were implanted for 30 h prior to measurement of LCGU. This time period permitted pumps to achieve a steady-state pumping rate (4 h) and the establishment of a constant plasma concentration of HAL (about 10 half-lives24). Preparation of animals Animals had free access to food and water until the morning of the experiment. After insertion of femoral vein and artery catheters under ether anesthesia, the rats were restrained with a plaster cast that enabled them to move their forequarters only, and were allowed to recover from anesthesia for 6 h in a temperature-controlled enclosure before i.v. injection of [14C]DG. Body temperature was maintained at 35-36 °C with a rectal thermoprobe connected to a feedback device that could activate an external heating element. Arterial blood pressure, heart rate and body temperature were monitored throughout the experiment. Behavioral observations Catalepsy was evaluated in Acute H A L and Chronic + Acute H A L animals at 15-min intervals after final H A L or vehicle injection, and was scored on a four-point scale from 0 (no effect, animal normal) to 4 (animal completely cataleptic). Measures of catalepsy were: presence and intensity of reduced spontaneous movements of the head and forelimbs; hypertonic-akinetic posture with kyphotic trunk, extended head, broad-based support, up-turned tail; resistance and bracing to imposed horizontal displacement of the forequarters; and ptosis. All catalepsy determinations were made by a single rater (G.P.). Determination of local cerebral glucose utilization LCGU was determined after a bolus injection of [14C]DG (125 /~Ci/kg body weight, i.v.). Fourteen timed arterial blood samples were collected during the subsequent 50 min and were centrifuged. Aliquots of plasma were taken for assessment of glucose (Glucose Analyzer II, Beckman Instruments, Irvine,
CA) and of [14C]DG (Model LS9000 Liquid Scintilla-
spread reductions in LCGU. Significant declines
tion Spectrometer,Beckman) concentrations. Rats were killed at 50 min by an i.v. overdose of sodium pentobarbital. The brains were removed rapidly and frozen in 2-methylbutane cooled to -40 °C. Coronal sections (20/~m thick) were obtained with a cryostat/microtome (Bright Model 5030, Hacker Instruments, Fairfield, N J) maintained at -20 °C. XRay film (SB-5, Kodak Rochester, NY) was exposed to the sections, as well as methylmethacrylate standards of known 14C concentration, for 7 days. Radioactivity in brain regions was determined by quantitative autoradiography. Optical densities of autoradiograms were measured with a microdensitometer (Model 700-10-90, Gamma Scientific, San Diego, CA). Six separate determinations were made at each region in both left and right sides of the brain, and the means for the two sides were averaged. Anatomical regions were identified by comparing autoradiograms with atlases of the rat brain27, 38 and, where necessary, with cresyl violet-stained sections taken adjacent to those used for autoradiography. LCGU was calculated from brain and plasma radioactivities and from plasma glucose concentrations, using equations and constants given by Sokoloff et al.45.
were observed in 72% of 47 brain regions examined;
Statistical analysis Metabolic data were analyzed for statistical significance by one-way analysis of variance and Bonferroni's multiple comparison test 32 which produced significance values for the following 5 pairwise comparisons: Acute Control to Chronic Control; Acute H A L to Acute Control; Chronic HAL, Chronic + Acute HAL, Short-term H A L to Chronic Control. Statistical significance was taken as P < 0.05. RESULTS Mean rates of LCGU in 47 examined regions of the rat brain are presented in Table I. No significant differences were found between acute and chronic vehicle-treated animals. Values in both control groups were similar to rates of LCGU previously reported for untreated, awake rats 45.
Metabolic effect of acute HAL HAL 1 mg/kg, administered 60 min prior to [14C]DG injection (Acute HAL), produced wide-
average LCGU for all examined regions (except the lateral habenula) was reduced by 25%, as compared to control. Only in the lateral habenula was LCGU increased after HAL. Acute H A L significantly reduced LCGU in several regions with known DA receptors or terminals9, 26 (caudate-putamen, substantia nigra, ventral tegmental area, precentral frontal cortex). However, significant declines were found in many brain regions which are not known to contain DA terminals (e.g. cortical, cerebellar and brainstem regions).
Metabolic effect of chronic HAL After 3 weeks of continuous infusion of HAL 1 mg/kg/day (Chronic HAL), fewer regions were significantly affected than in the Acute HAL group (19% vs 72% of those examined). The mean reduction in LCGU for all regions, excluding the lateral habenula, was 8%. After chronic treatment, therefore, fewer regions were affected significantly than after acute HAL, and the decreases in LCGU were generally less. Again, LCGU was elevated only in the lateral habenula. Many brain regions which were affected after acute H A L did not show significant changes after chronic treatment (e.g. frontal cortex areas 8 and 10, auditory cortex, thalamus ventromedial n., posterior hypothalamus, locus coeruleus, superior olive, cerebellar hemispheres and nuclei). In two regions, however, LCGU declined significantly only after chronic but not acute H A L (lateral amygdala and preoptic magnocellular nucleus). Extrapolating from our previous pharmacokinetic study24, the steady-state brain concentration of HAL at the time of [14C]DG injection in Chronic HAL rats was about one-quarter of the brain level of the drug in Acute H A L animals. However, the smaller metabolic effect of chronic H A L is not related to lower brain concentrations of the drug. In fact, rates of LCGU in animals which received a bolus injection of HAL 1 mg/kg i.p. after 20 days of HAL 1 mg/kg s.c. (Chronic + Acute HAL) were similar to those found after chronic continuous infusion alone (Chronic HAL) (Table I). The mean decline in LCGU for all examined regions (except the lateral habenula) in Chronic + Acute H A L animals was 8%; 17% of the regions showed significant changes. Because chronic
4 TABLE I
L CG U after acute and chronic HA L in Fischer-344 ratsa Mean + S.E.M. for the n u m b e r of animals shown in parentheses.
L C G U (~mol/lO0 g/min) Acute Control
Acute HAL
Chronic Control
Chronic HAL
Chronic + Acute H A L
Short-term HAL
(5)
(5)
(5)
(5)
(4)
(5)
Cortical regions Precentral medial Precentral agranular Frontal area 8 Frontal area 10 Somatosensory Auditory Visual Pyriform Entorh inal
114 112 104 99 108 137 103 86 68
+ + + + + + + + +
5 4 5 4 7 3 3 4 2
84 82 79 73 80 94 77 71 55
+ + + + + + + + +
3* 6* 2* 4* 3* 6* 4* 2 2*
129 128 118 110 119 145 111 97 73
+ 3 + 4 + 2 + 2 + 3 + 3 + 3 + 3 + 5 ___4 + 4
38 45 41 62 39 59 70 57 64 44 23
+ + + + + + + + + + +
2* 2* 2* 3* 4* 4 4 3 3* 3* 2*
59 75 58 94 59 79 70 75 93 62 46
69 + 64 + 154 + 58 + 62 + 32 + 54 + 70 + 62 +
2* 3* 9* 3* 3* 2 3* 4* 2
+ + + + + + + + +
4 6 5 7 5 5 4 5 2
106 109 116 100 115 145 97 94 64
+ 2 _+ 4 + 1 + 3 + 2 + 3 + 5 + 4 + 4 + 2 + 2
48 64 50 74 53 71 69 69 78 56 40
+ 6* + 7 + 8 + 5 _+ 10 + 6 + 4* + 6 + 5
108 101 112 110 114 160 100 94 65
+ 4* _+ 3* + 2 + 6 + 2 + 3 + 2 + 1 + 1
101 98 108 106 104 137 96 83 64
4* 4 3* 3* 3 5 5 5 5* 4 2
50 62 51 80 49 75 74 70 77 53 35
+ 1 + 3 + 1 + 3 _+ 2 + 4 + 2 + 3 + 2* + 3 + 1"
48 60 49 81 46 76 62 69 82 52 39
3* 7 13" 5* 5 5 4 6 5
88 + 89 + 209 + 72 + 82 + 40 + 65 + 117 + 73 +
___3* + 4* + 4 + 6 + 4 + 5 + 4 + 3 + 1
Limbic regions Dorsal hippocampus CA1 CA3 D e n t a t e gyrus Lateral amygdala Lateral septum Diagonal band n. Olfactory tubercle Lateral preoptic area Magnocellular preoptic area Medial forebrain bundle A n t e r i o r commissure
52 66 55 82 52 71 82 68 84 60 37
+ + + + + + + + + + +
+ + + + + + + + + + +
1" 1" 2* 2 1" 2 2 2 3 1 2
87 + 93 + 241 + 72 + 82 + 34 + 69 + 103 + 72 +
1" 3 6* 2 3 1 4 3 3
Diencephalon Thalamus med iodorsal n. Thalamus v e n t r o m e d i a l n. Lateral habenula Subthalamic n. Hypothal. posterior Hypothal. arcuate n. Hypothal. lateral n. Medial geniculate Lateral geniculate
101 + 88 + 119 + 82 + 83 + 38 + 70 + 107 + 76 +
3 4 6 3 3 2 4 3 2
116 + 2 101 + 3 123 __+3 86 + 3 91 + 4 37 + 1 68 + 2 123 + 4 77 + 2
93 + 95 + 184 + 69 + 81 + 44 + 66 + 104 + 69 +
2* 2 5* 1" 3 2 3 2 3
Basal ganglia Globus pallidus Caudate-putamen mediolateral mediomed ial Accumb ens n.
46 + 4
35 ___2
52 + 4
63 + 4
56 + 3
47 + 3
97 + 3 82 + 4 72 + 5
77 + 3 62 + 3* 69 + 3
98 + 4 84 + 4 72 + 3
103 + 9 86 + 7 73 + 4
95 + 2 79 + 3 91 + 4*
90 + 6 68 + 3 61 + 2
Brainstem Substantia nigra pars compacta pars reticulata Ventral teg men tal area Dorsal raphe Median raphe Superior colliculus Inferior colliculus Locus coeruleus
70 49 73 78 92 80 173 75
+ + + + + + + +
1 2 3 4 4 3 9 3
56 37 56 66 74 56 151 51
_+ 3* + 3* + 3* + 4 + 3* + 3* + 6 + 2*
72 53 77 88 97 85 180 72
+ + + + + + + +
2 2 3 4 3 1 3 1
67 51 77 82 93 72 165 69
+ + + + + + + +
3 3 5 3 4 5 7 5
81 49 79 81 94 72 184 63
+ + + + + + + +
2 1 1 2 2 2 6 2
75 51 76 83 104 70 185 64
+ + + + + + + +
3 1 2 1 5 2* 14 3
TABLE I continued L CG U (tzmol/lO0 g/min) Acute Control (5) Superior olive Inferior olive
Acute HAL (5)
Chronic Control (5)
Chronic HAL (5)
Chronic + Acute H A L (4)
Short-term HAL (5)
115 + 7 74 + 3
123 + 5 75 + 5
119 + 3 71 ___3
127 + 2 84 + 5
93 + 7* 68 + 3*
121 + 5 71 + 2
94 59 100 96
64 43 72 71
89 57 100 92
Cerebellum Vermis Hemispheres Interpositus n. Dentate n.
+ + + +
5 3 3 4
+ + + +
3* 2* 2* 2*
+ + + +
3 3 3 3
93 55 89 87
+ + + +
6 4 5 6
93 53 90 89
+ + + +
3 2 3 3
81 49 97 93
+ + + +
3 1 3 3
* Significantly different from control (P < 0.05). Acute H A L and Chronic Control groups were compared to Acute Control. Other groups were compared to Chronic Control. a Refer to text for details of drug treatments (Acute HAL: 1 mg/kg; Chronic HAL: 1 mg/kg/day).
exposure to H A L decreases plasma clearance of the drug 24, brain levels in Chronic + Acute H A L animals should have been similar to, or greater than, those in Acute H A L animals at the time of [14C]DG injection. The pattern of metabolic response in Chronic H A L and Chronic + Acute H A L also was similar. Paradoxically, L C G U increased in the nucleus accumbens and inferior olive in Chronic + Acute H A L rats. These increases were not seen in the acutely treated animals. Overall, the smaller metabolic response after chronic H A L indicates that tolerance develops to the drug. 47
Catarepsy after HA1
3-
o
~2C~
a
1-
o Acute HAL Chronic. (60 rnin) Acute HAL Fig. 1. Catalepsy after HAL. Catalepsy was determined at 60 rain after i.p. administration of H A L 1 mg/kg in previously untreated rats (Acute H A L ) , and in rats which had previously received H A L 1 mg/kg by daily s.c. injection for 3 weeks (Chronic + Acute HAL). Scoring was on a scale of 0 (no effect) to 4 (maximal catalepsy). N u m b e r of rats in each group = 5. Vertical bars indicate S.E.M.; * = significant difference from mean score after Acute H A L by two-tailed t-test (P < 0.05).
As shown in Fig. 1, animals which received an acute i.p. administration of H A L 1 mg/kg after 3 weeks of chronic treatment (Chronic + Acute H A L ) were tolerant also to the cataleptic effect of HAL. The mean catalepsy score 60 min after H A L 1 mg/kg i.p. in Chronic + Acute H A L rats was significantly less than in the Acute H A L animals (1.87 + 0.1 and 3.25 + 0.3, respectively, P < 0.05).
Time-course of development of metabolic tolerance to HAL Remarkably, a similar reduction of the metabolic effects of H A L which was observed after treatment for 3 weeks (Chronic HAL) also was produced by constant infusion of the same daily dose of drug for only about 1 day. In animals which received H A L 1 mg/kg/day by pump for 30 h (Short-term H A L ) , the mean decline in L C G U for all regions was 10% and 19% of examined regions were significantly affected (Fig. 2). The topography of the metabolic effect of H A L after 30 h of treatment was similar to that found after 3 weeks of continuous H A L administration (Table I). Brain concentrations of H A L in these rats should have been at steady-state levels (after 10 halflives), and similar to those of Chronic H A L animals. However, they were lower than in the Acute H A L rats z4. The smaller effect of Short-term H A L on L C G U could, therefore, represent an.effect of lower brain levels of the drug. On the other hand, the similar magnitude and pattern of metabolic alterations after 1 day as after 3 weeks of continuous H A L infusion may indicate that tolerance develops during the first day of treatment.
80-
LCGU
after
Acute
and
Chronic
Haloperidol
fJJ
60-
8 L_ 40-
f~J f~J J~J
I
a_
l
°/o o f r e g i o n s
i
f~J f~J f~J f~J
affected
A v e r a g e °Io decline in LCGU
l
20-
Acute HAL (60 min)
Chronic HAL Short-term HAL
Acute HAL (go min)
Fig. 2. Effect of HAL on LCGU in 47 brain regions. The percentage of regions in which LCGU was significantly reduced from control (hatched bars) and the average reduction in LCGU (solid bars) are shown. Vertical bars indicate S.E.M. Refer to text for details of drug treatment.
DISCUSSION
These results show that the effect of long-term H A L treatment on cerebral metabolism is smaller than that of acute HAL. The behavioral effect (catalepsy) of H A L is also smaller after chronic administration, as previously reported by others1,15. Therefore, our findings indicate that, after repeated H A L treatment, tolerance develops to the action of H A L on cerebral metabolism, as it does to its behavioral and neurochemical effects.
Patterns of metabolic tolerance H A L affected metabolism in some D A regions, as well as in other areas not known to be directly connected with the D A system. We have previously discussed the regional pattern of change in L C G U after acute H A L , and the mechanisms by which non-DA regions might be influenced by H A L 39. Most of the D A innervation in the brain arises from cell groups in the brainstem. D A neurons in substantia nigra heavily innervate the striatum, which returns direct projections to nigral neurons 5. The ventral tegmental area, the other major D A region in the brain, gives rise to widespread projections to mesolimbic (mainly nucleus accumbens and olfactory tubercle) and cortical (anterior cingulate and frontal cortex) structures 43. Numerous studies have indicated that different
D A systems in the brain may not respond identically to chronic neuroleptic treatment. Whereas development of tolerance in the nigrostriatal system has been reported for several parameters2S,30,37, disparate results have been reported for mesolimbic regions 6,20,41, and tolerance does not seem to develop in the mesocortical system3,29,42. Both structural components of the nigrostriatal system were significantly affected after acute H A L , and returned to control levels after chronic treatment. Among mesolimbic regions, L C G U in the ventral tegmental area was significantly reduced after acute H A L , but not after chronic treatment. In the nucleus accumbens, L C G U was unaffected by acute or chronic HAL, but when the two treatments were combined (Chronic + Acute HAL), it was increased by 27%. In precentral medial and agranular cortex (mesocortical receptive areas), L C G U was reduced by acute H A L ; the reductions persisted after chronic H A L , indicating that tolerance to metabolic effects of H A L d o e s not develop in mesocortical D A regions. In short, our findings agree with previous reports that tolerance develops after chronic H A L in nigrostriatal and some mesolimbic D A structures. On the other hand, these and previous biochemical studies indicate that the effects of acute H A L are maintained in the mesocortical system during chronic treatment. The three major D A systems in the brain are believed to mediate different effects of neuroleptics. Clinically, tolerance develops to the extrapyramidal side-effects of neuroleptics (thought to be nigrostriatal in origin 46) but not to their antipsychotic effect 2, which may result from-their persistent action on the mesocortical areas4, 31.
Mechanisms of development of tolerance to H A L Tolerance to H A L , achieved by prolonged daily treatment, prohibited further reductions in L C G U by an additional, acute dose of drug. H A L 1 mg/kg administered to Untreated rats caused widespread reductions in LCGU. After chronic treatment, changes in L C G U were less evident, and subsequent administration of H A L 1 mg/kg produced no further discernable change, despite brain levels which should have beeia four times those of animals receiving continuous daily infusion of the same dose of drug by osmotic pump 24. If prolonged H A L administration leads to satu-
ration of available D A receptors, additional H A L would not further increase receptor occupancy, and therefore, would have no additional effect on metabolism. Dose independence has been reported for neurochemical changes after chronic administration of various doses of H A L , ranging from 0.25 to 10 mg/kg/day 35, and for the incidence of tardive dyskinesia, which develops in man after chronic neuroleptic treatment 13. Time-course o f development o f metabolic tolerance to HAL Similar steady-state brain levels should have been achieved after 30 h as after 3 weeks of continuous infusion of H A L via an osmotic pump, because the plasma elimination half-life for H A L in 3-month-old Fischer-344 rats is 2.62 h, and a fixed brain:plasma ratio of H A L (37:1) is maintained 24. In our experiments, similar tolerance to the metabolic effect of H A L was observed 30 h and 3 weeks after osmotic pump implantation. Because glucose utilization is a measure of ftinctional activity in each brain region 44, our findings indicate that adaptative changes in the brain to persistent D A receptor saturation 16 by H A L may occur within 2 days after sub-acute treatment. Other adaptive changes which occur after chronic H A L do not develop as rapidly. Previous studies of development of tolerance to H A L have focused on adaptations which develop over several days to weeks. For example, catalepsy normalizes gradually over 5 days of H A L administration in the rat 15. Clinically, therapeutic effects of H A L develop gradually over 2-3 weeks 23. However, more rapid adaptations of the brain to H A L have been described. Behavioral and biochemical supersensitivity occurs in mice within a few hours after acute interruption of D A transmission 12, and an increased behavioral effect of apomorphine was shown 1 day after a single dose ol
REFERENCES 1 Asper, H., Baggiolini, M., B/irki, H. R., Launer, H., Ruch, W. and Stille, G., Tolerance phenomena with neuroleptics. Catalepsy, apomorphine stereotypies and striatal dopamine metabolism in the rat after single and repeated administration of loxapine and haloperidol, Europ. J. Pharrnacol., 22 (1973) 287-294. 2 Ayd, F. G., Jr., Neuroleptics and antiparkinsonian drugs, Int. Drug Ther. News Lett., 6 (1973) 33-45. 3 Bacopoulos, N. G., Biochemical mechanism of tolerance to
H A L ~0. Further, we have previously demonstrated that the effect of H A L 1 mg/kg on cerebral metabolism is maximal at 60 min after i.p. administration, and returns to pre-drug levels in most affected regions at 90 min39. The overall metabolic effect at 90 min after H A L resembles closely the response after 1 day or 3 weeks of treatment (Fig. 2), and suggests that some tolerance may occur within hours. The mechanism for tolerance is not clear. Whereas adaptation to long-term H A L administration has been attributed to new receptor formation and to changes in D A metabolism and turnover36, 41, an additional homeostatic mechanism, of very rapid onset, may be responsible for such an early adaptation as we describe here. Neuronal feedback circuits could play a role, as such circuits have been shown to be activated by acute H A L treatment7A 9. The early development of supersensitivity to D A agonists suggests that rapid adaptive changes occur at D A receptor sites. Such changes could represent new receptor formation 12, or modifications of D A receptor-effector mechanismslk This study demonstrates that tolerance, as measured by L C G U , develops after chronic H A L treatment in rats. Saturation of D A receptors appears to be responsible for the tolerance phenomenon. Tolerance appears within 1-2 days, and perhaps earlier. Tolerance does not develop equally in all D A systems. Persistence of the metabolic effect of H A L after chronic treatment in some D A regions may identify these areas as sites at which chronic H A L produces therapeutic and side effects. ACKNOWLEDGEMENT The authors thank H. Holloway and S. Carlson for their excellent technical assistance.
neuroleptic drugs; regional differences in rat brain, Europ. J. Pharmacol., 70 (1981) 585-586. 4 Bacopoulos, N. G., Spokes, E. G., Bird, E. D. and Roth, R. H., Antipsychotic drug action in schizophrenic patients: effect on cortical dopamine metabolism after long-term treatment, Science, 205 (1979) 1405-1407. 5 Beckstead, R. M., Domesik, V. B. and Nauta, W. J. H., Efferent connections of the substantia nigra and ventral tegmental area in the rat, Brain Research, 175 (1979) 191-217. 6 Bowers, M. B., Jr. and Rozitis, A., Brain homovanillic
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24
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the treatment of acute schizophrenia, Lancet, 1 (1978) 848-851. Kapetanovic, I. M., Sweeney, D. J. and Rapoport, S. I., Age effects on haloperidol pharmacokinetics in male, Fischer-344 rats, J. Pharmacol. exp. Ther., 221 (1982) 434-438. Kerwin, R. W., Rupniak, N. M. J., Jenner, P. and Marsden, C. D., A comparison of acute and one year's continuous neuroleptic treatment on the release of [3H]glutamate and [3H]acetylcholine from rat striatal slices, Neuroscience, 11 (1984) 205-210. Klemm, N., Murrin, L. C. and Kuhar, M. J., Neuroleptic and dopamine receptors: autoradiographic localization of [3H]spiperone in rat brain, Brain Research, 169 (1979) 1-9. Kt3nig, J. F. R. and Klippel, R. A., The Rat B r a i n - A Ster-
eotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Robert E. Krieger Publishing, Huntington, NY, 1967. 28 Lerner, P., Nose, P., Gordon, E. K. and Lovenberg, W., Haloperidol: effect of long-term treatment on rat striatal dopamine synthesis and turnover, Science, 197 (1977) 181-183. 29 Matsumoto, T., Uchimura, H., Hirano, M., Kim, J. S., Yokoo, H., Shimomura, M., Nakahara, T., Inoue, K. and Oomagari, K., Differential effects of acute and chronic administration of haloperidol on homovanillic acid levels in discrete dopaminergic areas of rat brain, Europ. J. Pharmacol., 89 (1983) 27-33. 30 Meller, E., Friedhoff, A. J. and Friedman, E., Differential effects of acute and chronic haloperidol treatment on striatal and nigral 3,4-dihydroxyphenylacetic acid (DOPAC) levels, Life Sci., 26 (1980) 541-547. 31 Meltzer, H. Y. and Stahl, S. M., The dopamine hypothesis of schizophrenia: a review, Schizophrenia Bull., 2 (1976) 19-76. 32 Miller, R. G., Jr., Simultaneous Statistical Inference, McGraw-Hill, New York, 1966, pp. 76-81. 33 Moleman, P., Bruinvels, J. and van Valkenburg, C. F. M., On the relation between haloperidol-induced alterations in dopamine release and dopamine metabolism in rat striaturn, Life Sci., 23 (1978) 611-616. 34 Muller, P. and Seeman, P., Brain neurotransmitter receptors after long-term haloperidol: dopamine, acetylcholine, serotonin, a-noradrenergic and naloxone receptors, Life Sci., 21 (1977) 1751-1758. 35 Muller, P. and Seeman, P., Dopaminergic supersensitivity after neuroleptics: time-course and specificity, Psychopharmacology, 60 (1978) 1-11. 36 Muller, P., Svensson, T. H. and Carlsson, A., Pre- and postsynaptic mechanisms in haloperidol-induced sensitization to dopamine agonists, Advanc. Biochem. Psychopharmacol., 24 (1980) 69-74. 37 Nicolaou, N. M., Acute and chronic effects of neuroleptics and acute effects of apomorphine and amphetamine on dopamine turnover in corpus striatum and substantia nigra of the rat brain, Europ. J. Pharmacol., 64 (1980) 123-132. 38 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, 1982. 39 Pizzolato, G., Soncrant, T. T. and Rapoport, S. I., Haloperidol and cerebral metabolism in the conscious rat: relation to pharmacokinetics, J. Neurochem., 43 (1984) 724-732. 40 Rastogi, S. K., Rastogi, R. B., Singhal, R. L. and Lapierre, Y. D., Behavioral and biochemical alterations following haloperidol treatment and withdrawal: the animal model of
tardive dyskinesia reexamined, Progr. Neuro-Psychopharmacol. Biol. Psychiatr., 7 (1983) 153-164 41 Rupniak, N. M. J,, Jenner, P. and Marsden, C. D., The effect of chronic neuroleptic administration on cerebral dopamine receptor function, Life Sci., 32 (1983) 2289-2311. 42 Scatton, B., Differential regional development of tolerance to increase in dopamine turnover upon repeated neuroleptic administration, Europ. J. Pharmacol., 46 (1977) 363-369. 43 Simon, H., LeMoal, M. and Calas, A., Efferents and afferents of the ventral tegmental-A10 region studied after local injection of [3H]leucine and horseradish peroxidase, Brain Research, 178 (1979) 17-40. 44 Sokoloff, L., Relation between physiological function and
energy metabolism in the central nervous system, J. Neurochem., 29 (1977) 13-26. 45 Sokoloff, L., Reivich, M., Kennedy, C., Des Rosiers, M. H., Patlak, C. S., Pettigrew, K. D., Sakurada, O. and Shinohara, M., The [14C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat, J. Neurochem., 28 (1977) 897-916. 46 Souner, R. and Di Mascio, A., Extrapyramidal syndromes and other neurological side effects of psychotropic drugs, In M. A. Lipton, A. Di Mascio and K. F. Killian (Eds.), Psychopharmacology: a Generation of Progress, Raven Press, New York, 1978, pp. 1021-1032.
1
BRE 10795
Research Reports
Reduced Metabolic Response of the Rat Brain to Haloperidol After Chronic Treatment GILBERTO PIZZOLATO*, TIMOTHY T. SONCRANT, DENISE M. LARSON and STANLEY I. RAPOPORT Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, MD 20205 (U.S.A.)
(Accepted September llth, 1984) Key words: haloperidol - - tolerance - - chronic treatment - - cerebral metabolism - - deoxyglucose - - dopamine antagonist
Local cerebral glucose utilization (LCGU) was determined, using the quantitative autoradiographic [14C]2-deoxy-D-glucosetechnique, in 47 brain regions of awake rats, after acute and chronic haloperidol (HAL) administration (1 mg/kg or 1 mg/kg/day). LCGU was reduced in fewer regions after chronic HAL (19%) than after acute HAL (72%); the average reduction for all regions was smaller (8% and 25 %, respectively). The reduced metabolic effect of chronic HAL is not due to a lower brain concentration of the drug, since similar effects on LCGU were found in rats which received an acute i.p. injection of HAL (as in the acutely treated animals) after chronic administration of HAL for 3 weeks. Furthermore, continuous infusion of HAL for 3 weeks or 1 day resulted in similar tolerance to the metabolic effect of HAL. Tolerance was not observed in the mesocortical dopamine (DA) system. The present findings show that tolerance develops to the effect of HAL on cerebral metabolism, even after 1 day of HAL treatment. Lack of tolerance in the mesocortical pathway may implicate this system in the neuroleptic effect of chronic HAL. INTRODUCTION Clinical actions of neuroleptic drugs such as haloperidol ( H A L ) result from blockade of dopamine (DA) neurotransmission in the central nervous system 14. Much experimental data regarding the effects of neuroleptics on brain D A refer to the effects produced by a single dose of drug; however, as antipsychotics, they are administered chronically. H A L alters neurochemical, electrophysiological and behavioral parameters after acute administrationS,33. After repeated or chronic treatment, however, tolerance develops. Increases in D A turnover 30,37, enhancement of the firing rate of the nigrostriatal D A system 8, activation of striatal tyrosine hydroxylase18, 28 and cataleptic behaviorlAS, all produced by acute H A L administration, are less prominent after chronic treatment. Other effects are produced by repeated H A L administration, and are thought to be responsible for behavioral supersensitivity to D A agonists 35. Chronic blockade of D A re-
ceptors results in a compensatory increase in the number of D A receptors17, 34, and in changes in other central neurotransmitter systems (i.e. GABA22, 40, serotonin 34, acetylcholine aS, substance p21). Though the time-course of the development of behavioral and biochemical changes varies during chronic neuroleptic treatment in the rat, the changes usually are fully developed after 1 week of repeated treatment 35. In our laboratory, we previously determined peripheral pharmacokinetics and time-dependent brain concentrations of H A L in Fischer-344 rats after acute i.p. administration of H A L 0.5 mg/kg, or continuous i.p. infusion of the same daily dose of drug for 4 days 24. Regional concentrations of H A L were found to be homogeneous throughout the brain. The concentration of H A L in frontal cortex after H A L 0.5 mg/kg i.p. peaked 30-60 min later (470 ng/g). Thereafter, brain concentration fell in proportion to plasma concentration; after 90 rain, the brain concentration was about half of the peak level (263 ng/g). After establishment of a steady-state brain
Present address: Clinica delle Malattie Nervose e Mentali, University of Padua, Italy. Correspondence: T. T. Soncrant, Laboratory of Neurosciences, NIA NIH, Clinical Center 10/6C103, Bethesda, MD 20205, U.S.A.
*
0006-8993/85/$03.30 (~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)
2 concentration of H A L by continuous infusion of HAL 0.5 mg/kg/day, the frontal cortex concentration of HAL was 120 ng/g, or 25% of the peak level achieved after acute administration. Plasma clearance of H A L was reduced after chronic treatment, suggesting that continuous H A L administration inhibits its own metabolism. We also determined the time-course of the effect of HAL 0.5 and 1 mg/kg i.p. in awake Fischer-344 rats 39. A peak metabolic effect was observed 60 min after i.p. administration of H A L 1 mg/kg. In the present study local cerebral glucose utilization (LCGU) was determined in the awake rat, using the [14C]2-deoxy-D-glucose ([14C]DG) method 45, after acute or chronic administration of H A L 1 mg/kg. The results demonstrate that tolerance to the effects of H A L on LCGU develops within 1 day of continuous administration. MATERIALS AND METHODS Experiments were performed in awake, 3-monthold, male Fischer-344 rats (Charles River Breeding Laboratories, Wilmington, MA). [14C]DG, spec. act. 50-55 mCi/mmol, was purchased from New England Nuclear (Boston, MA), and was rechromatographed to ascertain purity. H A L was a gift from McNeil Pharmaceuticals (Spring House, PA). Drug treatments Acute HAL. Acute Control and Acute H A L rats received the vehicle solution for H A L (1.8 mg methylparaben and 0.2 mg propylparaben in each ml of distilled water, 1 ml/kg), or H A L 1 mg/kg i.p., respectively, 60 min before [14C]DG injection. In Chronic Control rats, subcutaneously-implanted Alzet osmotic pumps (model 2ML4; Alza, Palo Alto, CA) dispensed the vehicle solution continuously at a rate of 0.055 ml/day for 3 weeks prior to [14C]DG injection. Chronic HAL: H A L 1 mg/kg/day was delivered continuously throughout the experiment for 3 weeks by the osmotic pump at a rate of 0.055 ml/day. LCGU was measured on the 21st day of treatment. Chronic + Acute HAL. Rats received H A L 1 mg/kg daily by single subcutaneous injection for 20 days. On the 21st day, 24 h after the last s.c. injection, H A L 1 mg/kg was administered i.p. LCGU was
measured 60 min after this last dose of drug (on the 21st treatment day). Short-term HAL. To establish the time-course of the chronic effects of HAL on LCGU, osmotic pumps, which dispensed H A L 1 mg/kg/day, were implanted for 30 h prior to measurement of LCGU. This time period permitted pumps to achieve a steady-state pumping rate (4 h) and the establishment of a constant plasma concentration of HAL (about 10 half-lives24). Preparation of animals Animals had free access to food and water until the morning of the experiment. After insertion of femoral vein and artery catheters under ether anesthesia, the rats were restrained with a plaster cast that enabled them to move their forequarters only, and were allowed to recover from anesthesia for 6 h in a temperature-controlled enclosure before i.v. injection of [14C]DG. Body temperature was maintained at 35-36 °C with a rectal thermoprobe connected to a feedback device that could activate an external heating element. Arterial blood pressure, heart rate and body temperature were monitored throughout the experiment. Behavioral observations Catalepsy was evaluated in Acute H A L and Chronic + Acute H A L animals at 15-min intervals after final H A L or vehicle injection, and was scored on a four-point scale from 0 (no effect, animal normal) to 4 (animal completely cataleptic). Measures of catalepsy were: presence and intensity of reduced spontaneous movements of the head and forelimbs; hypertonic-akinetic posture with kyphotic trunk, extended head, broad-based support, up-turned tail; resistance and bracing to imposed horizontal displacement of the forequarters; and ptosis. All catalepsy determinations were made by a single rater (G.P.). Determination of local cerebral glucose utilization LCGU was determined after a bolus injection of [14C]DG (125 /~Ci/kg body weight, i.v.). Fourteen timed arterial blood samples were collected during the subsequent 50 min and were centrifuged. Aliquots of plasma were taken for assessment of glucose (Glucose Analyzer II, Beckman Instruments, Irvine,
CA) and of [14C]DG (Model LS9000 Liquid Scintilla-
spread reductions in LCGU. Significant declines
tion Spectrometer,Beckman) concentrations. Rats were killed at 50 min by an i.v. overdose of sodium pentobarbital. The brains were removed rapidly and frozen in 2-methylbutane cooled to -40 °C. Coronal sections (20/~m thick) were obtained with a cryostat/microtome (Bright Model 5030, Hacker Instruments, Fairfield, N J) maintained at -20 °C. XRay film (SB-5, Kodak Rochester, NY) was exposed to the sections, as well as methylmethacrylate standards of known 14C concentration, for 7 days. Radioactivity in brain regions was determined by quantitative autoradiography. Optical densities of autoradiograms were measured with a microdensitometer (Model 700-10-90, Gamma Scientific, San Diego, CA). Six separate determinations were made at each region in both left and right sides of the brain, and the means for the two sides were averaged. Anatomical regions were identified by comparing autoradiograms with atlases of the rat brain27, 38 and, where necessary, with cresyl violet-stained sections taken adjacent to those used for autoradiography. LCGU was calculated from brain and plasma radioactivities and from plasma glucose concentrations, using equations and constants given by Sokoloff et al.45.
were observed in 72% of 47 brain regions examined;
Statistical analysis Metabolic data were analyzed for statistical significance by one-way analysis of variance and Bonferroni's multiple comparison test 32 which produced significance values for the following 5 pairwise comparisons: Acute Control to Chronic Control; Acute H A L to Acute Control; Chronic HAL, Chronic + Acute HAL, Short-term H A L to Chronic Control. Statistical significance was taken as P < 0.05. RESULTS Mean rates of LCGU in 47 examined regions of the rat brain are presented in Table I. No significant differences were found between acute and chronic vehicle-treated animals. Values in both control groups were similar to rates of LCGU previously reported for untreated, awake rats 45.
Metabolic effect of acute HAL HAL 1 mg/kg, administered 60 min prior to [14C]DG injection (Acute HAL), produced wide-
average LCGU for all examined regions (except the lateral habenula) was reduced by 25%, as compared to control. Only in the lateral habenula was LCGU increased after HAL. Acute H A L significantly reduced LCGU in several regions with known DA receptors or terminals9, 26 (caudate-putamen, substantia nigra, ventral tegmental area, precentral frontal cortex). However, significant declines were found in many brain regions which are not known to contain DA terminals (e.g. cortical, cerebellar and brainstem regions).
Metabolic effect of chronic HAL After 3 weeks of continuous infusion of HAL 1 mg/kg/day (Chronic HAL), fewer regions were significantly affected than in the Acute HAL group (19% vs 72% of those examined). The mean reduction in LCGU for all regions, excluding the lateral habenula, was 8%. After chronic treatment, therefore, fewer regions were affected significantly than after acute HAL, and the decreases in LCGU were generally less. Again, LCGU was elevated only in the lateral habenula. Many brain regions which were affected after acute H A L did not show significant changes after chronic treatment (e.g. frontal cortex areas 8 and 10, auditory cortex, thalamus ventromedial n., posterior hypothalamus, locus coeruleus, superior olive, cerebellar hemispheres and nuclei). In two regions, however, LCGU declined significantly only after chronic but not acute H A L (lateral amygdala and preoptic magnocellular nucleus). Extrapolating from our previous pharmacokinetic study24, the steady-state brain concentration of HAL at the time of [14C]DG injection in Chronic HAL rats was about one-quarter of the brain level of the drug in Acute H A L animals. However, the smaller metabolic effect of chronic H A L is not related to lower brain concentrations of the drug. In fact, rates of LCGU in animals which received a bolus injection of HAL 1 mg/kg i.p. after 20 days of HAL 1 mg/kg s.c. (Chronic + Acute HAL) were similar to those found after chronic continuous infusion alone (Chronic HAL) (Table I). The mean decline in LCGU for all examined regions (except the lateral habenula) in Chronic + Acute H A L animals was 8%; 17% of the regions showed significant changes. Because chronic
4 TABLE I
L CG U after acute and chronic HA L in Fischer-344 ratsa Mean + S.E.M. for the n u m b e r of animals shown in parentheses.
L C G U (~mol/lO0 g/min) Acute Control
Acute HAL
Chronic Control
Chronic HAL
Chronic + Acute H A L
Short-term HAL
(5)
(5)
(5)
(5)
(4)
(5)
Cortical regions Precentral medial Precentral agranular Frontal area 8 Frontal area 10 Somatosensory Auditory Visual Pyriform Entorh inal
114 112 104 99 108 137 103 86 68
+ + + + + + + + +
5 4 5 4 7 3 3 4 2
84 82 79 73 80 94 77 71 55
+ + + + + + + + +
3* 6* 2* 4* 3* 6* 4* 2 2*
129 128 118 110 119 145 111 97 73
+ 3 + 4 + 2 + 2 + 3 + 3 + 3 + 3 + 5 ___4 + 4
38 45 41 62 39 59 70 57 64 44 23
+ + + + + + + + + + +
2* 2* 2* 3* 4* 4 4 3 3* 3* 2*
59 75 58 94 59 79 70 75 93 62 46
69 + 64 + 154 + 58 + 62 + 32 + 54 + 70 + 62 +
2* 3* 9* 3* 3* 2 3* 4* 2
+ + + + + + + + +
4 6 5 7 5 5 4 5 2
106 109 116 100 115 145 97 94 64
+ 2 _+ 4 + 1 + 3 + 2 + 3 + 5 + 4 + 4 + 2 + 2
48 64 50 74 53 71 69 69 78 56 40
+ 6* + 7 + 8 + 5 _+ 10 + 6 + 4* + 6 + 5
108 101 112 110 114 160 100 94 65
+ 4* _+ 3* + 2 + 6 + 2 + 3 + 2 + 1 + 1
101 98 108 106 104 137 96 83 64
4* 4 3* 3* 3 5 5 5 5* 4 2
50 62 51 80 49 75 74 70 77 53 35
+ 1 + 3 + 1 + 3 _+ 2 + 4 + 2 + 3 + 2* + 3 + 1"
48 60 49 81 46 76 62 69 82 52 39
3* 7 13" 5* 5 5 4 6 5
88 + 89 + 209 + 72 + 82 + 40 + 65 + 117 + 73 +
___3* + 4* + 4 + 6 + 4 + 5 + 4 + 3 + 1
Limbic regions Dorsal hippocampus CA1 CA3 D e n t a t e gyrus Lateral amygdala Lateral septum Diagonal band n. Olfactory tubercle Lateral preoptic area Magnocellular preoptic area Medial forebrain bundle A n t e r i o r commissure
52 66 55 82 52 71 82 68 84 60 37
+ + + + + + + + + + +
+ + + + + + + + + + +
1" 1" 2* 2 1" 2 2 2 3 1 2
87 + 93 + 241 + 72 + 82 + 34 + 69 + 103 + 72 +
1" 3 6* 2 3 1 4 3 3
Diencephalon Thalamus med iodorsal n. Thalamus v e n t r o m e d i a l n. Lateral habenula Subthalamic n. Hypothal. posterior Hypothal. arcuate n. Hypothal. lateral n. Medial geniculate Lateral geniculate
101 + 88 + 119 + 82 + 83 + 38 + 70 + 107 + 76 +
3 4 6 3 3 2 4 3 2
116 + 2 101 + 3 123 __+3 86 + 3 91 + 4 37 + 1 68 + 2 123 + 4 77 + 2
93 + 95 + 184 + 69 + 81 + 44 + 66 + 104 + 69 +
2* 2 5* 1" 3 2 3 2 3
Basal ganglia Globus pallidus Caudate-putamen mediolateral mediomed ial Accumb ens n.
46 + 4
35 ___2
52 + 4
63 + 4
56 + 3
47 + 3
97 + 3 82 + 4 72 + 5
77 + 3 62 + 3* 69 + 3
98 + 4 84 + 4 72 + 3
103 + 9 86 + 7 73 + 4
95 + 2 79 + 3 91 + 4*
90 + 6 68 + 3 61 + 2
Brainstem Substantia nigra pars compacta pars reticulata Ventral teg men tal area Dorsal raphe Median raphe Superior colliculus Inferior colliculus Locus coeruleus
70 49 73 78 92 80 173 75
+ + + + + + + +
1 2 3 4 4 3 9 3
56 37 56 66 74 56 151 51
_+ 3* + 3* + 3* + 4 + 3* + 3* + 6 + 2*
72 53 77 88 97 85 180 72
+ + + + + + + +
2 2 3 4 3 1 3 1
67 51 77 82 93 72 165 69
+ + + + + + + +
3 3 5 3 4 5 7 5
81 49 79 81 94 72 184 63
+ + + + + + + +
2 1 1 2 2 2 6 2
75 51 76 83 104 70 185 64
+ + + + + + + +
3 1 2 1 5 2* 14 3
TABLE I continued L CG U (tzmol/lO0 g/min) Acute Control (5) Superior olive Inferior olive
Acute HAL (5)
Chronic Control (5)
Chronic HAL (5)
Chronic + Acute H A L (4)
Short-term HAL (5)
115 + 7 74 + 3
123 + 5 75 + 5
119 + 3 71 ___3
127 + 2 84 + 5
93 + 7* 68 + 3*
121 + 5 71 + 2
94 59 100 96
64 43 72 71
89 57 100 92
Cerebellum Vermis Hemispheres Interpositus n. Dentate n.
+ + + +
5 3 3 4
+ + + +
3* 2* 2* 2*
+ + + +
3 3 3 3
93 55 89 87
+ + + +
6 4 5 6
93 53 90 89
+ + + +
3 2 3 3
81 49 97 93
+ + + +
3 1 3 3
* Significantly different from control (P < 0.05). Acute H A L and Chronic Control groups were compared to Acute Control. Other groups were compared to Chronic Control. a Refer to text for details of drug treatments (Acute HAL: 1 mg/kg; Chronic HAL: 1 mg/kg/day).
exposure to H A L decreases plasma clearance of the drug 24, brain levels in Chronic + Acute H A L animals should have been similar to, or greater than, those in Acute H A L animals at the time of [14C]DG injection. The pattern of metabolic response in Chronic H A L and Chronic + Acute H A L also was similar. Paradoxically, L C G U increased in the nucleus accumbens and inferior olive in Chronic + Acute H A L rats. These increases were not seen in the acutely treated animals. Overall, the smaller metabolic response after chronic H A L indicates that tolerance develops to the drug. 47
Catarepsy after HA1
3-
o
~2C~
a
1-
o Acute HAL Chronic. (60 rnin) Acute HAL Fig. 1. Catalepsy after HAL. Catalepsy was determined at 60 rain after i.p. administration of H A L 1 mg/kg in previously untreated rats (Acute H A L ) , and in rats which had previously received H A L 1 mg/kg by daily s.c. injection for 3 weeks (Chronic + Acute HAL). Scoring was on a scale of 0 (no effect) to 4 (maximal catalepsy). N u m b e r of rats in each group = 5. Vertical bars indicate S.E.M.; * = significant difference from mean score after Acute H A L by two-tailed t-test (P < 0.05).
As shown in Fig. 1, animals which received an acute i.p. administration of H A L 1 mg/kg after 3 weeks of chronic treatment (Chronic + Acute H A L ) were tolerant also to the cataleptic effect of HAL. The mean catalepsy score 60 min after H A L 1 mg/kg i.p. in Chronic + Acute H A L rats was significantly less than in the Acute H A L animals (1.87 + 0.1 and 3.25 + 0.3, respectively, P < 0.05).
Time-course of development of metabolic tolerance to HAL Remarkably, a similar reduction of the metabolic effects of H A L which was observed after treatment for 3 weeks (Chronic HAL) also was produced by constant infusion of the same daily dose of drug for only about 1 day. In animals which received H A L 1 mg/kg/day by pump for 30 h (Short-term H A L ) , the mean decline in L C G U for all regions was 10% and 19% of examined regions were significantly affected (Fig. 2). The topography of the metabolic effect of H A L after 30 h of treatment was similar to that found after 3 weeks of continuous H A L administration (Table I). Brain concentrations of H A L in these rats should have been at steady-state levels (after 10 halflives), and similar to those of Chronic H A L animals. However, they were lower than in the Acute H A L rats z4. The smaller effect of Short-term H A L on L C G U could, therefore, represent an.effect of lower brain levels of the drug. On the other hand, the similar magnitude and pattern of metabolic alterations after 1 day as after 3 weeks of continuous H A L infusion may indicate that tolerance develops during the first day of treatment.
80-
LCGU
after
Acute
and
Chronic
Haloperidol
fJJ
60-
8 L_ 40-
f~J f~J J~J
I
a_
l
°/o o f r e g i o n s
i
f~J f~J f~J f~J
affected
A v e r a g e °Io decline in LCGU
l
20-
Acute HAL (60 min)
Chronic HAL Short-term HAL
Acute HAL (go min)
Fig. 2. Effect of HAL on LCGU in 47 brain regions. The percentage of regions in which LCGU was significantly reduced from control (hatched bars) and the average reduction in LCGU (solid bars) are shown. Vertical bars indicate S.E.M. Refer to text for details of drug treatment.
DISCUSSION
These results show that the effect of long-term H A L treatment on cerebral metabolism is smaller than that of acute HAL. The behavioral effect (catalepsy) of H A L is also smaller after chronic administration, as previously reported by others1,15. Therefore, our findings indicate that, after repeated H A L treatment, tolerance develops to the action of H A L on cerebral metabolism, as it does to its behavioral and neurochemical effects.
Patterns of metabolic tolerance H A L affected metabolism in some D A regions, as well as in other areas not known to be directly connected with the D A system. We have previously discussed the regional pattern of change in L C G U after acute H A L , and the mechanisms by which non-DA regions might be influenced by H A L 39. Most of the D A innervation in the brain arises from cell groups in the brainstem. D A neurons in substantia nigra heavily innervate the striatum, which returns direct projections to nigral neurons 5. The ventral tegmental area, the other major D A region in the brain, gives rise to widespread projections to mesolimbic (mainly nucleus accumbens and olfactory tubercle) and cortical (anterior cingulate and frontal cortex) structures 43. Numerous studies have indicated that different
D A systems in the brain may not respond identically to chronic neuroleptic treatment. Whereas development of tolerance in the nigrostriatal system has been reported for several parameters2S,30,37, disparate results have been reported for mesolimbic regions 6,20,41, and tolerance does not seem to develop in the mesocortical system3,29,42. Both structural components of the nigrostriatal system were significantly affected after acute H A L , and returned to control levels after chronic treatment. Among mesolimbic regions, L C G U in the ventral tegmental area was significantly reduced after acute H A L , but not after chronic treatment. In the nucleus accumbens, L C G U was unaffected by acute or chronic HAL, but when the two treatments were combined (Chronic + Acute HAL), it was increased by 27%. In precentral medial and agranular cortex (mesocortical receptive areas), L C G U was reduced by acute H A L ; the reductions persisted after chronic H A L , indicating that tolerance to metabolic effects of H A L d o e s not develop in mesocortical D A regions. In short, our findings agree with previous reports that tolerance develops after chronic H A L in nigrostriatal and some mesolimbic D A structures. On the other hand, these and previous biochemical studies indicate that the effects of acute H A L are maintained in the mesocortical system during chronic treatment. The three major D A systems in the brain are believed to mediate different effects of neuroleptics. Clinically, tolerance develops to the extrapyramidal side-effects of neuroleptics (thought to be nigrostriatal in origin 46) but not to their antipsychotic effect 2, which may result from-their persistent action on the mesocortical areas4, 31.
Mechanisms of development of tolerance to H A L Tolerance to H A L , achieved by prolonged daily treatment, prohibited further reductions in L C G U by an additional, acute dose of drug. H A L 1 mg/kg administered to Untreated rats caused widespread reductions in LCGU. After chronic treatment, changes in L C G U were less evident, and subsequent administration of H A L 1 mg/kg produced no further discernable change, despite brain levels which should have beeia four times those of animals receiving continuous daily infusion of the same dose of drug by osmotic pump 24. If prolonged H A L administration leads to satu-
ration of available D A receptors, additional H A L would not further increase receptor occupancy, and therefore, would have no additional effect on metabolism. Dose independence has been reported for neurochemical changes after chronic administration of various doses of H A L , ranging from 0.25 to 10 mg/kg/day 35, and for the incidence of tardive dyskinesia, which develops in man after chronic neuroleptic treatment 13. Time-course o f development o f metabolic tolerance to HAL Similar steady-state brain levels should have been achieved after 30 h as after 3 weeks of continuous infusion of H A L via an osmotic pump, because the plasma elimination half-life for H A L in 3-month-old Fischer-344 rats is 2.62 h, and a fixed brain:plasma ratio of H A L (37:1) is maintained 24. In our experiments, similar tolerance to the metabolic effect of H A L was observed 30 h and 3 weeks after osmotic pump implantation. Because glucose utilization is a measure of ftinctional activity in each brain region 44, our findings indicate that adaptative changes in the brain to persistent D A receptor saturation 16 by H A L may occur within 2 days after sub-acute treatment. Other adaptive changes which occur after chronic H A L do not develop as rapidly. Previous studies of development of tolerance to H A L have focused on adaptations which develop over several days to weeks. For example, catalepsy normalizes gradually over 5 days of H A L administration in the rat 15. Clinically, therapeutic effects of H A L develop gradually over 2-3 weeks 23. However, more rapid adaptations of the brain to H A L have been described. Behavioral and biochemical supersensitivity occurs in mice within a few hours after acute interruption of D A transmission 12, and an increased behavioral effect of apomorphine was shown 1 day after a single dose ol
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H A L ~0. Further, we have previously demonstrated that the effect of H A L 1 mg/kg on cerebral metabolism is maximal at 60 min after i.p. administration, and returns to pre-drug levels in most affected regions at 90 min39. The overall metabolic effect at 90 min after H A L resembles closely the response after 1 day or 3 weeks of treatment (Fig. 2), and suggests that some tolerance may occur within hours. The mechanism for tolerance is not clear. Whereas adaptation to long-term H A L administration has been attributed to new receptor formation and to changes in D A metabolism and turnover36, 41, an additional homeostatic mechanism, of very rapid onset, may be responsible for such an early adaptation as we describe here. Neuronal feedback circuits could play a role, as such circuits have been shown to be activated by acute H A L treatment7A 9. The early development of supersensitivity to D A agonists suggests that rapid adaptive changes occur at D A receptor sites. Such changes could represent new receptor formation 12, or modifications of D A receptor-effector mechanismslk This study demonstrates that tolerance, as measured by L C G U , develops after chronic H A L treatment in rats. Saturation of D A receptors appears to be responsible for the tolerance phenomenon. Tolerance appears within 1-2 days, and perhaps earlier. Tolerance does not develop equally in all D A systems. Persistence of the metabolic effect of H A L after chronic treatment in some D A regions may identify these areas as sites at which chronic H A L produces therapeutic and side effects. ACKNOWLEDGEMENT The authors thank H. Holloway and S. Carlson for their excellent technical assistance.
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