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Chronic subanesthetic halothane exposure causes selective alterations in neurotransmitter systems in discrete brain regions
EXPERIMENTAL
NEUROLOGY
76, 168- 180 ( 1982)
Chronic Subanesthetic Halothane Exposure Causes Selective Alterations in Neurotransmitter Systems in Discrete Brain Regions GOTTESFELD, PHILIP N. PATSALOS, BENJAMIN AND RICHARD C. WIGGINS’
ZEHAVA
Department
M.
RIGOR,
of Neurobiology and Anatomy and Department of Anesthesiology, University of Texas Medical School, Houston, Texas 77025
Received
September
21, 1981; revision
received
November
25, 1981
The effects of prolonged exposure to subanesthetic concentrations of halothane on dopaminergic, noradrenergic, cholinergic, and GABAergic neuronal systemswere studied in discrete regions of the rat brain. Exposures to 0.25% halothane in air were for 8 h/day, 5 days/week during an I-week period; control rats received only air at an equivalent flow rate. Twenty-four hours after the last exposure the animals were killed and brain, liver, muscle, and serum samples were taken for analyses. Discrete brain regions were removed by microdissection and assayed for norepinephrine, dopamine, glutamate decarboxylase, choline acetyltransferase, and proteins. The remaining tissues and serum were analyzed for halothane content; however, halothane was not detected in any sample tested. Rats which were exposed to halothane exhibited a marked reduction in body weight gain and their urine volume was significantly greater than in the controls. Significant changes were observed in regions associated with the limbic system, for example, dopamine was increased in the ventral tegmental area, choline acetyltransferase was increased in the nucleus accumbens, caudate, and globus pallidus and its activity decreased in the central gray. The activity of glutamate decarboxylase was decreased in the accumbens and in the medial preoptic nuclei but increased in the lateral preoptic nucleus. There was no apparent effect on norepinephrine concentrations. We suggest that these selective changes in limbic regions may cause behavioral deficits consequent to chronic subanesthetic halothane exposure.
’ The authors thank Mrs. Malka Gottesfeld for her excellent technical assitance and Ms. Diana Parker for her invaluable secretarial assistance. A preliminary report was presented at the Annual Meeting of the American Society of Anesthesiologists (35). Address reprint requests to Dr. Zehava Gottesfeld, Department of Neurobiology and Anatomy, P.O. Box 20708, The University of Texas Medical School, Houston, TX 77025. 168 OOl4-4886/82/040168-13$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
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INTRODUCTION Chronic exposure to halothane (2-bromo-2-chloro- 1, 1,l -trifluoroethane), a widely used inhalation anesthetic agent has been implicated in pathologic changes of several organs including kidney, liver, and brain (2, 6, 11, 22). With respect to the brain, changes in cellular ultrastructure, synaptogenesis, myelination, and behavioral deficits including psychomotor activity, mood, and memory, were reported consequent to chronic halothane exposure (1, 3-5, 31, 33). Because behavioral changes may be associated with alterations in brain neurotransmitter systems, we assessed the effect of chronic exposure to subanesthetic concentrations of halothane on noradrenergic, dopaminergic, cholinergic, and GABAergic systems in discrete brain regions. The neuronal markers used were, respectively, norepinephrine (NE), dopamine (DA), glutamic acid decarboxylase (GAD), and choline acetyltransferase (ChAT). We observed that changes occurred in the dopaminergic, yaminobutyric acid-(GABA)ergic, and cholinergic systems in discrete brain regions implicated in limbic functions. Prolonged halothane exposure seemed to have no effect on NE concentrations in the regions under investigation. We suggest that selective chemical alterations in limbic system structures may underlie behavioral changes which result from chronic halothane exposure. MATERIALS
AND
METHODS
Animals. Adult male Sprague-Dawley rats (TIMCO, Houston, Tex.) weighing about 180 g were used. All rats were housed in covered plastic cages (22 X 26 X 46 cm; five rats per cage), with free access to normal laboratory diet (Formulab Chow 5008; Ralston Purina Co.) and water, and were acclimatized to their new environment for 7 days. A 12-h 1ight:dark cycle was maintained and each rat was weighed weekly throughout the experimental period. Halothane. Thirty rats were randomly divided into two groups ( 15 rats/ group; five rats/cage) and designated for halothane or air (control) exposure. Halothane (Halocarbon Laboratories, Hackensack, N.J.) was delivered at 0.25% (2500 ppm in air) from a Fluotec MK2 fluothane vaporizer (Frazer Sweatman, Buffalo, N.Y.), at a flow rate of 2 liters air/min, using a gas flow proportioner. Exposures were for 8 h/day, 5 days/week during an 8-week period. Control rats received only air at an equivalent flow rate. Abbreviations: ChAT-holine tyric acid, GAD-glutamic nephrine, and VTA-ventral
acetyltransferase, acid decarboxylase, tegmental area.
DA-dopamine, NA-nucleus
GABA--y-aminobuaccumbens, NE-norepi-
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ET AL.
In a second experiment a group of six rats was placed singly in metabolic cages, 24 h prior to start of exposure with free access to food and water, and randomly designated for halothane (three rats) or air (three rats) exposure. Method and duration of exposure were exactly as described above. Urine was separated mechanically from the feces over a 24-h period. Urine volume was determined daily. Tissue Collection and Microdissection. At 0800, 24 h after the end of the last exposure regime, the animals were killed by decapitation. Brain, liver, muscle, and serum were taken from each rat, quickly frozen in dry ice, and stored at -70°C until further analyses. Adrenal glands and testes were dissected and weighed as an index of a potential halothane-related stress. Liver, muscle, and serum were analyzed for halothane content as described by Divakaran et al. (14) The lower limit of halothane detection by this method is 1 ng. The frozen brains were mounted on tissue holders and cut into sections (300 pm thick) using a microtome-cryostat set at -8°C. Tissue slices were placed on chilled glass slides, briefly thawed to promote adhesion, and subsequently refrozen and stored at -70°C. Microdissection and chemical analyses were carried out usually within 2 to 4 days. Discrete brain regions were removed from homologous sites of each side of the brain, using stainless-steel cannulae of various internal diameters [( 30) and Table 11. Catecholamines Assay. Brain samples were homogenized in 100 ~1 icecold 0.1 N perchloric acid by sonication. After removing samples ( 10 ~1) for protein determination, the homogenates were centrifuged 5 min at 8000g. Subsequently NE and DA were determined using 25-~1 samples of the supernatant, by the method of Coyle and Henry (12). This method is based on the enzymatic conversion of catecholamines to their O-methylated derivatives in the presence of the tritiated methyl donor [3H-methyl]Sadenosylmethione (S.A. 66 Ci/mmol, New England Nuclear Corp., Boston, Mass.). Glutamic Acid Decarboxylase (GAD) Assay. Tissue pellets were placed in 25 ~1 of a solution containing 10 mM ethylenediaminetetraacetate (EDTA) and 0.2% Triton X-100 at pH 7.4, then homogenized by sonication. GAD was assayed (in duplicate) in 2-~1 samples of the homogenate by the method of Fonnum et al. (17). Choline Acetyltransferase (ChAT) Assay. Tissue samples were prepared as in the GAD assay. Samples consisting of 2-~1 homogenate (in duplicate) were used for the determination of ChAT activity according to Fonnum (16). Proteins Assay. Determination of proteins was made according to Lowry et al. (27) using bovine serum albumin standards. Data Analysis. For neurotransmitters the one-way analysis of variance
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AND NEUROTRANSMITTERS TABLE
1
Microdissection of Discrete Brain Regions
Region Caudate nucleus N. accumbens Frontal cortex N. septum lateralis N. septum medialis N. tractus diagonalis N. tuberculum olfactorium N. preopticus medialis N. preopticus lateralis N. interstitialis stria terminalis Clobus pallidus N. hypothalamic anterior N. amygdaloid medialis N. anterior ventralis thalami N. habenula lateralis N. habenula medialis Habenula Hippocampus N. paraventricularis N. interpeduncularis Ventral tegmental area Substantia nigra pars compacta pars reticulata N. rubrum Central gray Superior colliculus
No. of punches per brain
2 2 2 2 2 2 6 2 2 2 2 2
Size of cannula (mm)
Approximate coordinates” (pm)
1.0 0.5 1.0 0.5 0.5 0.5 0.3 0.5 0.5
A8620 A8620 A8620 A7890 A7890 A7470 A7470 A7190 A7190
0.5 0.5 0.5 0.5 0.5 0.5 0.3 1.0 1.0 0.5 0.7 0.5
A6860 A6280 A6280 A4890 A4620 A41 10 A4110 A3750 A3750 A3430 A1800 A1800
0.3 0.5 0.5 0.5 0.5
A1800 A1800 Al800 A1800 A1800
a According to (23).
was used to assess the significance of differences between groups. P values < 0.05 were considered significant. Differences in weekly body weights were analyzed according to t-statistic of two means. RESULTS Body Weight, Urine Volume, and Halothane Content in Tissues. There was no significant difference in mean body weight between halothane-exposed and control rats during the first 3 weeks of exposure (Table 2). On
172
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ET AL.
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AND
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m
174
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ET AL.
the 4th week of treatment, however, the halothane-exposed rats exhibited a significant (P c 0.01) reduction in body weight gain compared with airexposed (control) rats. This differential rate of weight gain persisted throughout the subsequent 4-weeks exposure period. Gonad and adrenal weights of halothane-exposed animals were not significantly different from control rats. Gonad weights (mean grams + SE, N = 18): halothane exposed 3.72 + 0.04, control 3.85 + 0.01. Adrenal weights (grams f SE; N = 18): halothane exposed 0.45 + 0.01, control 0.45 zk 0.02. Liver, muscle, and serum of each halothane-exposed rat were sampled for halothane content. No halothane was detected 24 h postexposure. Urinary volumes of experimental and control rats were determined daily and were (mean milliliters _+SE, 38 samples): 17.87 +- 0.67 and 14.91 + 0.62, respectively. Urine volume was significantly greater (P < 0.05) in the halothane-exposed rats. Catecholamines Concentration in Discrete Brain Regions. Table 3 shows NE and DA concentrations in 17 discrete brain regions of experimental and control rats. Halothane-exposed rats exhibited a significant reduction (41%, P < 0.01) in DA concentration in the ventral tegmental area (VTA) compared with control rats. The DA concentrations of the other brain regions were not significantly altered. In contrast, no significant changes in NE concentrations were observed after halothane exposure. Glutamic Acid Decarboxylase Activity. The GAD activity in 21 brain regions of halothane-treated and control rats was determined (Table 4) and was observed to be altered in only three brain regions of halothaneexposed rats. Activity was significantly decreased in the nucleus accumbens (NA) (60%, P < 0.002) and preoptic medial nucleus (35%, P < 0.05) and significantly increased in the lateral preoptic nucleus (39%, P < 0.005). Choline Acetyftransferase Activity. Twenty brain regions were assayed for ChAT activity (Table 5). Halothane exposure caused a significant increase in ChAT activity in the globus pallidus (125%, P < 0.003) caudate nucleus (43%, P < 0.003) and NA (41%, P < 0.002) and a significant decrease in the central gray (43%, P < 0.02) compared with control rats. The activity of ChAT was unaffected in the other 16 regions assayed. DISCUSSION The major findings which emerge from this work demonstrate that in consequence to prolonged exposure to subanesthetic concentrations of halothane, there was a marked decrease in body weight gain, a significant increase in urine volume and dopaminergic, GABAergic, and cholinergic neurotransmitter systems were altered selectively in regions associated with the limbic system.
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AND NEUROTRANSMITTERS TABLE
4
Effect of Prolonged Exposure to Halothane on Glutamic Acid Decarboxylase Activity in Discrete Brain Regions” Glutamic
Region Caudate nucleus N. accumbens COrteX N. septum lateralis N. septum medialis N. tractus diagonalis N. preopticus medialis N. preopticus lateralis N. interstitialis stria terminalis Globus pallidus N. hypothalamic anterior N. amygdaloideus medialis N. habenula lateralis Hippocampus N. ventromedialis thalami N. interpeduncularis Ventral tegmental area Substantia nigra pars recticulata N. rubrum Superior colliculus Central gray ‘Results
CO”tIOl 104* 433 + 112r 1002 136 e 164 k 235 + 328 + 396 + 380 k 404 k 240 + 317 + 19? 179 + 133+ 91 *
acid decarboxylase
0.25% Halothane 5 39 8 13 12 12 25 24 18 36 32 16 18 4 17 6 12
557 + 37 112+ 6 3005 13 260 f 18
932 171 + 1022 89+ 108 + 164 + 153 ? 455 + 349 k 365 + 446 + 269 + 280 + 69-+ 174 r 147f 116%
3 14 9 6 IO 12 24 28 30 24 28 17 20 5 I5 II 8
(pmol/g
protein/h)
W Change from control
P
-60
<0.0002
-35 +39
518 + 20 104+ 5 264 k 12 282 ? I3
are mean k SE of 8 to 10 rats.
Reports on changes in body weight which result from chronic halothane exposure have been inconsistent. Investigators observed an increased (38), decreased (38), or unaltered (20, 24, 26) body weight of animals during prolonged halothane exposure compared with control. The conflicting data may be attributed to the use of different animal species, age, and inconsistent conditions (concentration and duration) of halothane exposure. The present findings of increased urine volume in halothane-exposed animals corroborate observations by other investigators on renal malfunctions. It was reported, for instance, that the most consistent histopathologic changes seen in animals treated with halothane occurred in kidney [(6) for review]. Whether or not these observations bear any relevance, however, to the reported high incidence of renal disease among anesthetists (4) remains an open question. The role of neurotransmitters in halothane anesthetic mechanisms was the focus of a number of investigations. It was demonstrated, for instance, that acute halothane anesthesia induced a marked increase of GABA concentrations in cortical slices (7, 9) which, presumably, accounted for the
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TABLE
5
Effect of Prolonged Exposure to Halothane on Choline Acetyltransferase Activity in Discrete Brain Regions” Choline
Region Caudate nucleus N. accumbens Cortex N. septum lateralis N. septum medialis N. tractus diagonalis N. preopticus medialis N. preopticus lateralis N. preopticus stria terminalis Globus pallidus N. hypothalamus anterior N. amygdaloid medialis N. habenula lateralis N. habenula medialis Hippocampus N. interpeduncularis Ventral tegmental area N. rubrum Superior colliculus CentKd
@a,’
-Results
COIltrOl 138 f IO 292 k 16 55* 4 25+ 3 57+ 8 408 k 32 II? 2 53+ 8 35r 9 3lf 3 20+ 3 26+ 3 105 * 12 850 + 19 47f 7 1874 + 132 86+ 9 40t 7 682 II 35+ 5
acetyltransferase
0.25% Halothane 194* 8 412+ 22 57* 4 28+ 4 48+ 8 399 k 28 Is+ 3 53t 4 36k 5 70+ 8 23k 2 31* 4 107 + 13 875 f 21 4lk 4 2092 + 108 so* 7 45k 7 66* 4 20+ 2
(pmolfg
protein)
96 Chan8c from control
P
+43 +41
<0.003 co.002
+12s
<0.003
-43
<0.02
are mean k SE of 8 to 10 rats.
anesthesia-induced inhibition of synaptic transmission (19, 34). The effect of halothane anesthesia on the cholinergic system still is controversial. Thus, it was reported that halothane reduced acetylcholine concentrations (7) and substantially inhibited its synthesis and turnover rate in brain tissue (8, 25, 29). In contrast, it was found that halothane caused a differential increase in acetylcholine in several brain regions, apparently due to decreased utilization (37). Other investigators, however, observed that halothane-induced acetylcholine alterations could be prevented if the degradation enzyme acetylcholine esterase was inactivated by microwave irradiation (29). Acute halothane anesthesia was found also to cause differential changes in the catecholamines NE and DA in discrete brain regions (36). In the present work, we observed that chronic treatment with subanesthetic concentrations of halothane caused heterogeneous changes in GABAergic, cholinergic, and dopaminergic systems in discrete brain regions. To our knowledge, this is the first report which describes alterations in several neurotransmitter systems concurrently in response to prolonged subanesthetic concentrations of halothane. As the half-life of brain halothane is <3h (14) its detection would not be expected at the time of sample
CHRONIC
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analysis as indeed we verified in the present work. Thus, we believe that the observed chemical alterations are a metabolic change consequent’to chronic halothane exposure. It is of interest to note that the brain regions where chemical changes have occurred are either part of or directly connected with the limbic system. At present, no simple explanation can be offered to assess the significance of these changes and one can only speculate on these results. In this study, the regions in which neurotransmitter changes occurred following chronic halothane exposure include the nucleus accumbens, caudate nucleus, globus pallidus, ventral tegmental area, central gray, and the hypothalamic preoptic nuclei medialis and lateralis. The neuronal circuitry which interconnects the NA and the VTA has been extensively investigated. Thus, for instance, it was well documented that the NA receives its dopaminergic innervation via the mesolimbic pathway from the A10 cells in the VTA (13, 40). There is also ample evidence to suggest that fibers project to the VTA from GABAergic cell bodies in the NA (41-45). We observed that DA concentrations decreased in the VTA and that GAD activity decreased in the NA after prolonged exposure to halothane. Whether or not these changes were caused by the interaction between dopaminergic axon terminals and GABAergic cell bodies in the NA remains an open question. Our data do not corroborate the results by Roizen et al. (36) that DA increased in the NA after acute halothane anesthesia; the discrepancy may be attributed to the difference in the experimental conditions, e.g., concentration and duration of halothane exposure. The NA also interconnects with the caudate nucleus (28) and the globus pallidus (21, 28, 32). Although the chemical identity of these connections is not known, it is interesting to note that in this work ChAT activity was altered in the NA as well as in the caudate nucleus and in the globus pallidus. The present data do not reveal whether or not the changes in ChAT are associated with cholinergic interneurons or with long axon neurons (42) which may interconnect between the NA and the basal ganglia. Alternatively, the cholinergic system in these regions may act to modulate (10, 18) other neuronal systems under chronic halothane exposure. Other regions connected with the NA include the central gray, a region considered to be implicated in pain control, and the hypothalamic preoptic region (15). It is interesting to note that we found changes in GAD and ChAT in those regions, respectively, after chronic halothane treatment. Knowledge of the chemical identity of the neuronal connections of the NA with other brain regions is still incomplete and this renders the interpretation of the present results largely speculative. It is tempting, however, to suggest that the NA may play a key role in limbic-system functions,
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and that the chemical alterations of neuronal systems connected with this region may induce behavioral deficits ( 1, 3, 4, 39) consequent to prolonged subanesthetic halothane exposure. REFERENCES 1. ADAM, N. 1973. Effect of general anesthetics on memory function in man. J. Camp. Physiol. Psychol. 83: 294-305. 2. AD Hoc COMMITTEE. 1974. A report of trace anesthetics on the health of operating room personnel. American Society of Anesthesiologists. Occupational disease among operating room personnel: a national study. Anesthesiology 41: 321-369. 3. BRUCE, D. L. 1974. Trace anesthetic effects of perceptual and cognitive skills. Anesth. Rev. 1: 24-25. 4. BRUCE, D. L., K. A. EIDE, H. W. LINDE, AND J. E. ECKENHOFF. 1968. Causes of death among anesthesiologists, a 20 year survey. Anesthesiology 29: 565-569. 5. CHANG, L. W., A. W. DUDLEY, JR., Y. K. LEE, AND J. KATZ. 1974. Ultrastructural changes in the nervous system after chronic exposure to halothane. Ex. Neural. 45: 209-219. 6. CHANG, L. W., AND J. KATZ. 1976. Pathologic effects of chronic halothane inhalation. An overview. Anesthesiology 15: 640-653. 7. CHENG, S.-C., AND E. A. BRUNNER. 1975. A neurochemical hypothesis for halothane anesthesia. Anesth. Anal. 54: 242-246. 8. CHENG, S.-C., AND E. A. BRUNNER. 1978. Alterations of tricarboxyhc acid cycle metabolism in rat brain slices by halothane. .I. Neurochem. 30: 1421-1430. 9. CHENG, S.-C., AND E. A. BRUNNER. 1980. Does GABA have a role in anesthesia? Brain Res. Bull. 5: 909-912. 10. CONSOLO, S., H. LADINSKY, S. BIANCHI, AND D. GHEZZI. 1977. Apparent lack of a dopaminergic-cholinergic link in the rat nucleus accumbens septi-tuberculum olfactorium. Bruin Res. 135: 255-263. 11. CORBETT, T. H., R. G. CORNELL, K. LIEDING, AND J. L. ENDRES. 1973. Incidence of cancer among Michigan nurse anesthetists. Anesfhesiology 38: 250-263. 12. COYLE, J. T., AND D. HENRY. 1973. Catecholamines in fetal and newborn rat brain. J. Neurochem. 21: 6 I-67. 13. DAHLSTROM, A., AND K. FUXE. 1964. Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brainstem neurons. Acta Physiol. Stand. 62: 1094. 14. DIVAKARAN, P., F. JOINER, B. M. RIGOR, AND R. C. WIGGINS. 1980. Accumulation and persistence of halothane in adult and fetal rat brain as a result of subanesthetic exposure. J Neurochem. 34: 1543-I 546. 15. DOMESICK, V. B. 198 1. Further observations on the anatomy of nucleus accumbens and caudate-putamen in the rat: similarities and contrasts. Pages 7-40 R. B. CHRONISTER AND J. F. DEFRANCE, Eds., The Neurobiology of the Nucleus Accumbens. Haer Instit. Electrophysiol. Res., Brunswick, Me. 16. FONNUM, F. 1975. A rapid radiochemical method for the determination of choline acetyltransferase. J. Neurochem. 24: 407-409. 17. FONNUM, F., J. STORM-MATHISEN, AND F.’ WALLBERG. 1970. Glutamate decarboxylase in the inhibitory neurons. A study of the enzyme in Purkinje cell axons and boutons in the cat. Bruin Res. 20: 259-275. 18. FUXE,
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NEUROLOGY
76, 168- 180 ( 1982)
Chronic Subanesthetic Halothane Exposure Causes Selective Alterations in Neurotransmitter Systems in Discrete Brain Regions GOTTESFELD, PHILIP N. PATSALOS, BENJAMIN AND RICHARD C. WIGGINS’
ZEHAVA
Department
M.
RIGOR,
of Neurobiology and Anatomy and Department of Anesthesiology, University of Texas Medical School, Houston, Texas 77025
Received
September
21, 1981; revision
received
November
25, 1981
The effects of prolonged exposure to subanesthetic concentrations of halothane on dopaminergic, noradrenergic, cholinergic, and GABAergic neuronal systemswere studied in discrete regions of the rat brain. Exposures to 0.25% halothane in air were for 8 h/day, 5 days/week during an I-week period; control rats received only air at an equivalent flow rate. Twenty-four hours after the last exposure the animals were killed and brain, liver, muscle, and serum samples were taken for analyses. Discrete brain regions were removed by microdissection and assayed for norepinephrine, dopamine, glutamate decarboxylase, choline acetyltransferase, and proteins. The remaining tissues and serum were analyzed for halothane content; however, halothane was not detected in any sample tested. Rats which were exposed to halothane exhibited a marked reduction in body weight gain and their urine volume was significantly greater than in the controls. Significant changes were observed in regions associated with the limbic system, for example, dopamine was increased in the ventral tegmental area, choline acetyltransferase was increased in the nucleus accumbens, caudate, and globus pallidus and its activity decreased in the central gray. The activity of glutamate decarboxylase was decreased in the accumbens and in the medial preoptic nuclei but increased in the lateral preoptic nucleus. There was no apparent effect on norepinephrine concentrations. We suggest that these selective changes in limbic regions may cause behavioral deficits consequent to chronic subanesthetic halothane exposure.
’ The authors thank Mrs. Malka Gottesfeld for her excellent technical assitance and Ms. Diana Parker for her invaluable secretarial assistance. A preliminary report was presented at the Annual Meeting of the American Society of Anesthesiologists (35). Address reprint requests to Dr. Zehava Gottesfeld, Department of Neurobiology and Anatomy, P.O. Box 20708, The University of Texas Medical School, Houston, TX 77025. 168 OOl4-4886/82/040168-13$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
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INTRODUCTION Chronic exposure to halothane (2-bromo-2-chloro- 1, 1,l -trifluoroethane), a widely used inhalation anesthetic agent has been implicated in pathologic changes of several organs including kidney, liver, and brain (2, 6, 11, 22). With respect to the brain, changes in cellular ultrastructure, synaptogenesis, myelination, and behavioral deficits including psychomotor activity, mood, and memory, were reported consequent to chronic halothane exposure (1, 3-5, 31, 33). Because behavioral changes may be associated with alterations in brain neurotransmitter systems, we assessed the effect of chronic exposure to subanesthetic concentrations of halothane on noradrenergic, dopaminergic, cholinergic, and GABAergic systems in discrete brain regions. The neuronal markers used were, respectively, norepinephrine (NE), dopamine (DA), glutamic acid decarboxylase (GAD), and choline acetyltransferase (ChAT). We observed that changes occurred in the dopaminergic, yaminobutyric acid-(GABA)ergic, and cholinergic systems in discrete brain regions implicated in limbic functions. Prolonged halothane exposure seemed to have no effect on NE concentrations in the regions under investigation. We suggest that selective chemical alterations in limbic system structures may underlie behavioral changes which result from chronic halothane exposure. MATERIALS
AND
METHODS
Animals. Adult male Sprague-Dawley rats (TIMCO, Houston, Tex.) weighing about 180 g were used. All rats were housed in covered plastic cages (22 X 26 X 46 cm; five rats per cage), with free access to normal laboratory diet (Formulab Chow 5008; Ralston Purina Co.) and water, and were acclimatized to their new environment for 7 days. A 12-h 1ight:dark cycle was maintained and each rat was weighed weekly throughout the experimental period. Halothane. Thirty rats were randomly divided into two groups ( 15 rats/ group; five rats/cage) and designated for halothane or air (control) exposure. Halothane (Halocarbon Laboratories, Hackensack, N.J.) was delivered at 0.25% (2500 ppm in air) from a Fluotec MK2 fluothane vaporizer (Frazer Sweatman, Buffalo, N.Y.), at a flow rate of 2 liters air/min, using a gas flow proportioner. Exposures were for 8 h/day, 5 days/week during an 8-week period. Control rats received only air at an equivalent flow rate. Abbreviations: ChAT-holine tyric acid, GAD-glutamic nephrine, and VTA-ventral
acetyltransferase, acid decarboxylase, tegmental area.
DA-dopamine, NA-nucleus
GABA--y-aminobuaccumbens, NE-norepi-
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In a second experiment a group of six rats was placed singly in metabolic cages, 24 h prior to start of exposure with free access to food and water, and randomly designated for halothane (three rats) or air (three rats) exposure. Method and duration of exposure were exactly as described above. Urine was separated mechanically from the feces over a 24-h period. Urine volume was determined daily. Tissue Collection and Microdissection. At 0800, 24 h after the end of the last exposure regime, the animals were killed by decapitation. Brain, liver, muscle, and serum were taken from each rat, quickly frozen in dry ice, and stored at -70°C until further analyses. Adrenal glands and testes were dissected and weighed as an index of a potential halothane-related stress. Liver, muscle, and serum were analyzed for halothane content as described by Divakaran et al. (14) The lower limit of halothane detection by this method is 1 ng. The frozen brains were mounted on tissue holders and cut into sections (300 pm thick) using a microtome-cryostat set at -8°C. Tissue slices were placed on chilled glass slides, briefly thawed to promote adhesion, and subsequently refrozen and stored at -70°C. Microdissection and chemical analyses were carried out usually within 2 to 4 days. Discrete brain regions were removed from homologous sites of each side of the brain, using stainless-steel cannulae of various internal diameters [( 30) and Table 11. Catecholamines Assay. Brain samples were homogenized in 100 ~1 icecold 0.1 N perchloric acid by sonication. After removing samples ( 10 ~1) for protein determination, the homogenates were centrifuged 5 min at 8000g. Subsequently NE and DA were determined using 25-~1 samples of the supernatant, by the method of Coyle and Henry (12). This method is based on the enzymatic conversion of catecholamines to their O-methylated derivatives in the presence of the tritiated methyl donor [3H-methyl]Sadenosylmethione (S.A. 66 Ci/mmol, New England Nuclear Corp., Boston, Mass.). Glutamic Acid Decarboxylase (GAD) Assay. Tissue pellets were placed in 25 ~1 of a solution containing 10 mM ethylenediaminetetraacetate (EDTA) and 0.2% Triton X-100 at pH 7.4, then homogenized by sonication. GAD was assayed (in duplicate) in 2-~1 samples of the homogenate by the method of Fonnum et al. (17). Choline Acetyltransferase (ChAT) Assay. Tissue samples were prepared as in the GAD assay. Samples consisting of 2-~1 homogenate (in duplicate) were used for the determination of ChAT activity according to Fonnum (16). Proteins Assay. Determination of proteins was made according to Lowry et al. (27) using bovine serum albumin standards. Data Analysis. For neurotransmitters the one-way analysis of variance
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AND NEUROTRANSMITTERS TABLE
1
Microdissection of Discrete Brain Regions
Region Caudate nucleus N. accumbens Frontal cortex N. septum lateralis N. septum medialis N. tractus diagonalis N. tuberculum olfactorium N. preopticus medialis N. preopticus lateralis N. interstitialis stria terminalis Clobus pallidus N. hypothalamic anterior N. amygdaloid medialis N. anterior ventralis thalami N. habenula lateralis N. habenula medialis Habenula Hippocampus N. paraventricularis N. interpeduncularis Ventral tegmental area Substantia nigra pars compacta pars reticulata N. rubrum Central gray Superior colliculus
No. of punches per brain
2 2 2 2 2 2 6 2 2 2 2 2
Size of cannula (mm)
Approximate coordinates” (pm)
1.0 0.5 1.0 0.5 0.5 0.5 0.3 0.5 0.5
A8620 A8620 A8620 A7890 A7890 A7470 A7470 A7190 A7190
0.5 0.5 0.5 0.5 0.5 0.5 0.3 1.0 1.0 0.5 0.7 0.5
A6860 A6280 A6280 A4890 A4620 A41 10 A4110 A3750 A3750 A3430 A1800 A1800
0.3 0.5 0.5 0.5 0.5
A1800 A1800 Al800 A1800 A1800
a According to (23).
was used to assess the significance of differences between groups. P values < 0.05 were considered significant. Differences in weekly body weights were analyzed according to t-statistic of two means. RESULTS Body Weight, Urine Volume, and Halothane Content in Tissues. There was no significant difference in mean body weight between halothane-exposed and control rats during the first 3 weeks of exposure (Table 2). On
172
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ET AL.
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m
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ET AL.
the 4th week of treatment, however, the halothane-exposed rats exhibited a significant (P c 0.01) reduction in body weight gain compared with airexposed (control) rats. This differential rate of weight gain persisted throughout the subsequent 4-weeks exposure period. Gonad and adrenal weights of halothane-exposed animals were not significantly different from control rats. Gonad weights (mean grams + SE, N = 18): halothane exposed 3.72 + 0.04, control 3.85 + 0.01. Adrenal weights (grams f SE; N = 18): halothane exposed 0.45 + 0.01, control 0.45 zk 0.02. Liver, muscle, and serum of each halothane-exposed rat were sampled for halothane content. No halothane was detected 24 h postexposure. Urinary volumes of experimental and control rats were determined daily and were (mean milliliters _+SE, 38 samples): 17.87 +- 0.67 and 14.91 + 0.62, respectively. Urine volume was significantly greater (P < 0.05) in the halothane-exposed rats. Catecholamines Concentration in Discrete Brain Regions. Table 3 shows NE and DA concentrations in 17 discrete brain regions of experimental and control rats. Halothane-exposed rats exhibited a significant reduction (41%, P < 0.01) in DA concentration in the ventral tegmental area (VTA) compared with control rats. The DA concentrations of the other brain regions were not significantly altered. In contrast, no significant changes in NE concentrations were observed after halothane exposure. Glutamic Acid Decarboxylase Activity. The GAD activity in 21 brain regions of halothane-treated and control rats was determined (Table 4) and was observed to be altered in only three brain regions of halothaneexposed rats. Activity was significantly decreased in the nucleus accumbens (NA) (60%, P < 0.002) and preoptic medial nucleus (35%, P < 0.05) and significantly increased in the lateral preoptic nucleus (39%, P < 0.005). Choline Acetyftransferase Activity. Twenty brain regions were assayed for ChAT activity (Table 5). Halothane exposure caused a significant increase in ChAT activity in the globus pallidus (125%, P < 0.003) caudate nucleus (43%, P < 0.003) and NA (41%, P < 0.002) and a significant decrease in the central gray (43%, P < 0.02) compared with control rats. The activity of ChAT was unaffected in the other 16 regions assayed. DISCUSSION The major findings which emerge from this work demonstrate that in consequence to prolonged exposure to subanesthetic concentrations of halothane, there was a marked decrease in body weight gain, a significant increase in urine volume and dopaminergic, GABAergic, and cholinergic neurotransmitter systems were altered selectively in regions associated with the limbic system.
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4
Effect of Prolonged Exposure to Halothane on Glutamic Acid Decarboxylase Activity in Discrete Brain Regions” Glutamic
Region Caudate nucleus N. accumbens COrteX N. septum lateralis N. septum medialis N. tractus diagonalis N. preopticus medialis N. preopticus lateralis N. interstitialis stria terminalis Globus pallidus N. hypothalamic anterior N. amygdaloideus medialis N. habenula lateralis Hippocampus N. ventromedialis thalami N. interpeduncularis Ventral tegmental area Substantia nigra pars recticulata N. rubrum Superior colliculus Central gray ‘Results
CO”tIOl 104* 433 + 112r 1002 136 e 164 k 235 + 328 + 396 + 380 k 404 k 240 + 317 + 19? 179 + 133+ 91 *
acid decarboxylase
0.25% Halothane 5 39 8 13 12 12 25 24 18 36 32 16 18 4 17 6 12
557 + 37 112+ 6 3005 13 260 f 18
932 171 + 1022 89+ 108 + 164 + 153 ? 455 + 349 k 365 + 446 + 269 + 280 + 69-+ 174 r 147f 116%
3 14 9 6 IO 12 24 28 30 24 28 17 20 5 I5 II 8
(pmol/g
protein/h)
W Change from control
P
-60
<0.0002
-35 +39
518 + 20 104+ 5 264 k 12 282 ? I3
are mean k SE of 8 to 10 rats.
Reports on changes in body weight which result from chronic halothane exposure have been inconsistent. Investigators observed an increased (38), decreased (38), or unaltered (20, 24, 26) body weight of animals during prolonged halothane exposure compared with control. The conflicting data may be attributed to the use of different animal species, age, and inconsistent conditions (concentration and duration) of halothane exposure. The present findings of increased urine volume in halothane-exposed animals corroborate observations by other investigators on renal malfunctions. It was reported, for instance, that the most consistent histopathologic changes seen in animals treated with halothane occurred in kidney [(6) for review]. Whether or not these observations bear any relevance, however, to the reported high incidence of renal disease among anesthetists (4) remains an open question. The role of neurotransmitters in halothane anesthetic mechanisms was the focus of a number of investigations. It was demonstrated, for instance, that acute halothane anesthesia induced a marked increase of GABA concentrations in cortical slices (7, 9) which, presumably, accounted for the
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TABLE
5
Effect of Prolonged Exposure to Halothane on Choline Acetyltransferase Activity in Discrete Brain Regions” Choline
Region Caudate nucleus N. accumbens Cortex N. septum lateralis N. septum medialis N. tractus diagonalis N. preopticus medialis N. preopticus lateralis N. preopticus stria terminalis Globus pallidus N. hypothalamus anterior N. amygdaloid medialis N. habenula lateralis N. habenula medialis Hippocampus N. interpeduncularis Ventral tegmental area N. rubrum Superior colliculus CentKd
@a,’
-Results
COIltrOl 138 f IO 292 k 16 55* 4 25+ 3 57+ 8 408 k 32 II? 2 53+ 8 35r 9 3lf 3 20+ 3 26+ 3 105 * 12 850 + 19 47f 7 1874 + 132 86+ 9 40t 7 682 II 35+ 5
acetyltransferase
0.25% Halothane 194* 8 412+ 22 57* 4 28+ 4 48+ 8 399 k 28 Is+ 3 53t 4 36k 5 70+ 8 23k 2 31* 4 107 + 13 875 f 21 4lk 4 2092 + 108 so* 7 45k 7 66* 4 20+ 2
(pmolfg
protein)
96 Chan8c from control
P
+43 +41
<0.003 co.002
+12s
<0.003
-43
<0.02
are mean k SE of 8 to 10 rats.
anesthesia-induced inhibition of synaptic transmission (19, 34). The effect of halothane anesthesia on the cholinergic system still is controversial. Thus, it was reported that halothane reduced acetylcholine concentrations (7) and substantially inhibited its synthesis and turnover rate in brain tissue (8, 25, 29). In contrast, it was found that halothane caused a differential increase in acetylcholine in several brain regions, apparently due to decreased utilization (37). Other investigators, however, observed that halothane-induced acetylcholine alterations could be prevented if the degradation enzyme acetylcholine esterase was inactivated by microwave irradiation (29). Acute halothane anesthesia was found also to cause differential changes in the catecholamines NE and DA in discrete brain regions (36). In the present work, we observed that chronic treatment with subanesthetic concentrations of halothane caused heterogeneous changes in GABAergic, cholinergic, and dopaminergic systems in discrete brain regions. To our knowledge, this is the first report which describes alterations in several neurotransmitter systems concurrently in response to prolonged subanesthetic concentrations of halothane. As the half-life of brain halothane is <3h (14) its detection would not be expected at the time of sample
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analysis as indeed we verified in the present work. Thus, we believe that the observed chemical alterations are a metabolic change consequent’to chronic halothane exposure. It is of interest to note that the brain regions where chemical changes have occurred are either part of or directly connected with the limbic system. At present, no simple explanation can be offered to assess the significance of these changes and one can only speculate on these results. In this study, the regions in which neurotransmitter changes occurred following chronic halothane exposure include the nucleus accumbens, caudate nucleus, globus pallidus, ventral tegmental area, central gray, and the hypothalamic preoptic nuclei medialis and lateralis. The neuronal circuitry which interconnects the NA and the VTA has been extensively investigated. Thus, for instance, it was well documented that the NA receives its dopaminergic innervation via the mesolimbic pathway from the A10 cells in the VTA (13, 40). There is also ample evidence to suggest that fibers project to the VTA from GABAergic cell bodies in the NA (41-45). We observed that DA concentrations decreased in the VTA and that GAD activity decreased in the NA after prolonged exposure to halothane. Whether or not these changes were caused by the interaction between dopaminergic axon terminals and GABAergic cell bodies in the NA remains an open question. Our data do not corroborate the results by Roizen et al. (36) that DA increased in the NA after acute halothane anesthesia; the discrepancy may be attributed to the difference in the experimental conditions, e.g., concentration and duration of halothane exposure. The NA also interconnects with the caudate nucleus (28) and the globus pallidus (21, 28, 32). Although the chemical identity of these connections is not known, it is interesting to note that in this work ChAT activity was altered in the NA as well as in the caudate nucleus and in the globus pallidus. The present data do not reveal whether or not the changes in ChAT are associated with cholinergic interneurons or with long axon neurons (42) which may interconnect between the NA and the basal ganglia. Alternatively, the cholinergic system in these regions may act to modulate (10, 18) other neuronal systems under chronic halothane exposure. Other regions connected with the NA include the central gray, a region considered to be implicated in pain control, and the hypothalamic preoptic region (15). It is interesting to note that we found changes in GAD and ChAT in those regions, respectively, after chronic halothane treatment. Knowledge of the chemical identity of the neuronal connections of the NA with other brain regions is still incomplete and this renders the interpretation of the present results largely speculative. It is tempting, however, to suggest that the NA may play a key role in limbic-system functions,
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and that the chemical alterations of neuronal systems connected with this region may induce behavioral deficits ( 1, 3, 4, 39) consequent to prolonged subanesthetic halothane exposure. REFERENCES 1. ADAM, N. 1973. Effect of general anesthetics on memory function in man. J. Camp. Physiol. Psychol. 83: 294-305. 2. AD Hoc COMMITTEE. 1974. A report of trace anesthetics on the health of operating room personnel. American Society of Anesthesiologists. Occupational disease among operating room personnel: a national study. Anesthesiology 41: 321-369. 3. BRUCE, D. L. 1974. Trace anesthetic effects of perceptual and cognitive skills. Anesth. Rev. 1: 24-25. 4. BRUCE, D. L., K. A. EIDE, H. W. LINDE, AND J. E. ECKENHOFF. 1968. Causes of death among anesthesiologists, a 20 year survey. Anesthesiology 29: 565-569. 5. CHANG, L. W., A. W. DUDLEY, JR., Y. K. LEE, AND J. KATZ. 1974. Ultrastructural changes in the nervous system after chronic exposure to halothane. Ex. Neural. 45: 209-219. 6. CHANG, L. W., AND J. KATZ. 1976. Pathologic effects of chronic halothane inhalation. An overview. Anesthesiology 15: 640-653. 7. CHENG, S.-C., AND E. A. BRUNNER. 1975. A neurochemical hypothesis for halothane anesthesia. Anesth. Anal. 54: 242-246. 8. CHENG, S.-C., AND E. A. BRUNNER. 1978. Alterations of tricarboxyhc acid cycle metabolism in rat brain slices by halothane. .I. Neurochem. 30: 1421-1430. 9. CHENG, S.-C., AND E. A. BRUNNER. 1980. Does GABA have a role in anesthesia? Brain Res. Bull. 5: 909-912. 10. CONSOLO, S., H. LADINSKY, S. BIANCHI, AND D. GHEZZI. 1977. Apparent lack of a dopaminergic-cholinergic link in the rat nucleus accumbens septi-tuberculum olfactorium. Bruin Res. 135: 255-263. 11. CORBETT, T. H., R. G. CORNELL, K. LIEDING, AND J. L. ENDRES. 1973. Incidence of cancer among Michigan nurse anesthetists. Anesfhesiology 38: 250-263. 12. COYLE, J. T., AND D. HENRY. 1973. Catecholamines in fetal and newborn rat brain. J. Neurochem. 21: 6 I-67. 13. DAHLSTROM, A., AND K. FUXE. 1964. Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brainstem neurons. Acta Physiol. Stand. 62: 1094. 14. DIVAKARAN, P., F. JOINER, B. M. RIGOR, AND R. C. WIGGINS. 1980. Accumulation and persistence of halothane in adult and fetal rat brain as a result of subanesthetic exposure. J Neurochem. 34: 1543-I 546. 15. DOMESICK, V. B. 198 1. Further observations on the anatomy of nucleus accumbens and caudate-putamen in the rat: similarities and contrasts. Pages 7-40 R. B. CHRONISTER AND J. F. DEFRANCE, Eds., The Neurobiology of the Nucleus Accumbens. Haer Instit. Electrophysiol. Res., Brunswick, Me. 16. FONNUM, F. 1975. A rapid radiochemical method for the determination of choline acetyltransferase. J. Neurochem. 24: 407-409. 17. FONNUM, F., J. STORM-MATHISEN, AND F.’ WALLBERG. 1970. Glutamate decarboxylase in the inhibitory neurons. A study of the enzyme in Purkinje cell axons and boutons in the cat. Bruin Res. 20: 259-275. 18. FUXE,
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