- Email: [email protected]
Studies on noradrenergic alterations in relation to early phenobarbital-induced behavioral changes
In:. 1. Dwl. Neuroscience, Vol. 5, No. 4. pp. 337-344, 1987. Printed in Great Britain.
u736-574ws7 so3.0o+o.aI Pergamon Jourmtls Ltd. @ 1987 ISDN
STUDIES ON NORADRENERGIC ALTERATIONS IN RELATION TO EARLY PHENOBARBITAL-INDUCED BEHAVIORAL CHANGES JOSEPH YANAI and CHAIM G. PICK The Melvin A. and Eleanor Ross Laboratory for Studies in Neural Birth Defects, Department of Anatomy and Embryology, The Hebrew University-Hadassah Medical School, Box 1172, 91010 Jerusalem, Israel (Received
8 January
1987; in revised form
1 July 1987; accepted 3 July 1987)
Abstract-Mice were exposed to phenobarbital prenatally (B offspring) by feeding their mother 3 g/kg phenobarbital in milled food on gestation days 9-18; control dams received unadulterated milled food. At age 50 days, B offspring had fewer fluorescing noradrenergic (NE) cells in the locus coeruleus than control (P
Mice,
Neuron
transplantation,
Early (pre- and neonatal) exposure to phenobarbital induced long-term behavioral and neural changes in experimental animals. ***zJTo gain an understanding of the mechanisms by which the barbiturate exerts its effect on behavior, our research effort focused on behaviors which may be related, to a specific brain area and to particular biochemical processes. Thus, in mice, early exposure to phenobarbital induced deficits in eight-arm maze performance and spontaneous alterations.‘9.20 Both of these behaviors are primarily associated with a specific brain area, the hippocampus. ‘J’**~ We are investigating early phenobarbital-induced alterations in the major innervations to the hippocampus as the possible mechanism mediating hippocampal behavioral deficits. Noradrenergic innervations have an important role in the hippocampus; they originate exclusively from the locus coeruleus where the cell bodies resideI and were shown to have a regulatory role on various behaviors. ‘**L*~In the present experiment, mice were exposed to phenobarbital during their prenatal development. At an older age their locus coeruleushippocampal noradrenegic innervations were studied at morphological and biochemical levels. Since the studies revealed alterations in these innervations, an attempt was made to correct the phenobarbital-induced deficits by hippocampal behaviors by transplantation of normal noradrenergic cells into the impaired hippocampus. METHODS The genetically heterogeneous HS/Ibg mice were employed.” The method of phenobarbital administration to the developing fetuses have been previously described.28 Adult mice were housed in mating groups of 1 male and 4 females. Their offspring (the subjects of the experiments) received phenobarbital prenatally via the placenta. Female parent mice were checked daily at 08.00 hr. Those that had conceived, as verified by the existence of vaginal plug, were separated from the males and housed with other pregnant females. On gestation day 9 (GD 9; evidence of vaginal plug is considered GD l), the females were housed in individual cages. Treated females then received milled mouse food containing 3 g/kg phenobarbital in acid form (their only food source) and water, both available ad libitum. Control females received milled food and water. Drug administration continued until GD 18, when phenobarbital and control diets were replaced with regular mouse pellets. Blood phenobarbital levels were monitored in the en 5:4-n
337
338
J. Yanai and C. G. Pick
females and fetuses as described by Yanai et ~1.~~The blood phenobarbital level averaged 113 rt 12 p&ml during most of the phenobarbital feeding period. The phenobarbital levels of the fetuses were similar to those of the mothers. *’ Following delivery, the barbiturate-exposed offspring (B offspring) and control offspring were maintained with their mothers under standard laboratory conditions and received no further drug treatment. They were weaned on day 25, segregation and maintenance according to gender followed subsequently. Female and male offspring were employed in approximately equal numbers. The data were pooled across gender, as there were no differences between the sexes. Control and B offspring were divided into groups according to the experiments and tested for the following: (a) the number of fluorescing NE cells in the locus coeruleus, (b) levels of NE in the hippocampus and cerebellum, (c) behavior in the eight-arm maze and the effect of NE transplantation on the deficits in this behavior. Catecholamine histojluorescence A detailed description of the method is found in Furness et aL6 Mice were perfused transcardically with 50 ml of 4% parafo~aldehyde and 0.5% glutaraldehyde in a phosphate buffer (pH 7.4, 0.1 M). After dissection, the brain stems were postfixed overnight. Locus coeruleus serial coronal sections (20 pm) from treatment and control mice were obtained on a vibratome. Every 3rd section was picked up, dried on a slide and mounted in paraffin oil. The sections were observed with a Zeiss microscope using a fluorescence attachment, Cell counts of the locus coeruleus were made and the Abercrombie correction factor was applied as follows: N=nt/ t+d) in which N = actual number of cells, n = counted number of cells, t = mean thickness of the section and d = mean diameter of counted units.2.” Norepinephrine assay Hip~campi and cerebella were dissected on an ice-cold plate, weighed, and frozen at -70°C. Catecholamines were assayed as described by Felice et af.5 and Hefti et aA with a few modifications. The tissues were homogenized by a sonicator in ice-cold distilled water containing 0.1 perchloric acid and 0.2 mg/ml sodium metabisulfite. The homogenate was centrifuged (12,000g for 5 min) and 100 t~l of the supematant fluid were taken for analysis. Norepinephrine was isolated using miniature alumina columns as described by Gauchy et al.’ Small pipet tips were stopped with glass wool and filled with 20-25 mg alumina (Woelm, activity grade I, activated by boiling in 2 M HCl). The pipet tips were placed in reagent tubes (12 x 75 mm) with adaptors (cutoff larger pipet tips) and run in a table centrifuge (TJ-6; Beckman Instruments, Palo Alto, CA). The alumina columns were equilibrated with 200 ~10.05 M Tris-HCl buffer (pH 8.6) (100 g for 2 min). One hundred and ninety ~1 of a mixture ~ntaining the sample (100 ~1) and 100 ~1 of 1 M Tris-HCl buffer (pH 8.6 containing 1 mg/ml dithiothreitol) and 10 ~1 of 0.1 M perchloric acid with 10 ng 3,4-dihydroxybenzylamine (internal standard) were pipetted into the columns and centrifuged at 100 g for 3 min. The columns were then washed with 200 t.~lHz0 (100 g for 2 min), transferred into new test tubes and the NE (as well as other catecholamines and DOPAC) subsequently eluted with 150 t.tJof 0.1 M phosphoric acid (400 g for 5 min). The eluate was analysed by reverse-phase high-performance liquid chromatography with electrochemical detection, based on the method described by Felice et aLs The column used was Cl8 reverse-phase (3.9 x 300 mm uBondapak; Waters) and the electrode was glassy carbon (Bioanalytical Systems). The potential was set at +0.7 V.A. Sodium phosphate buffer (0.1 M, pH 3.0), ~ntaining 0.6 mM octyl sodium sulfate as ion-pair reagent, served as mobile phase. Protein dete~ination was made using the method of Lowry et al. ‘* Eight-arm maze test The maze used for this experiment is a scaled down version of the radial maze for rats. lH Briefly, it is constructed on a Plexiglas circular base 76 cm in diameter. Eight arms are attached to this base; the internal dimensions of each arm are 32 x 4 x 4 cm. At the end of each arm, 1.5 cm from its end, is a depression for water reinforcement. All arms are linked with the central platform which is 13.5 cm across. The maze is covered with a circular transparent cover of Plexiglas.
Studies on noradrenergic
alterations
339
Over the center platform in the middle of the cover there is an opening, with a cover through which the mouse is introduced into the maze. Mice were tested in the maze at age 90 days. Control and B offspring transplanted with NE cells or sham-operated were put on a regimen of water deprivation for a week. This included restricting daily administration of water for 30 min. After a week, the mice were introduced individually into the apparatus for 10 min of habituation without reinforcement of water. The first 16 entries were observed. The mice received reinforcement with water drops of 50 ~1 during the subsequent 5 day testing period unlike the habituation day, when animals were left in the maze until they had either entered all eight arms, or had made 16 entries, whichever occurred first. Animals were considered to have reached criterion if there were eight correct entries out of the possible eight for two consecutive days. Thus it was possible to calculate the number of days it took to reach criterion. Animals that reached criterion after day 1 (eight correct responses out of eight trials on habituation day+the following day) received the score 0. Animals that reached criterion on the 2 days after habituation day received the score 1, etc. Animals not reaching criterion during the entire testing period received the score 6. Transplantation
of noradrenergic
cells
Brains of normal mouse embryos were dissected on gestation day 15 under an operating microscope. The locus coeruleus was selected as a source of noradrenergic cells. It was dissected at the level of the pontine flexure and the rhombic lip. Two mm tissue sections from the same brain region were pooled (20 embryos) and transferred to a conical centrifuge tube containing 5 ml of versene (1:5000). Tissues were gently dissociated by aspiration pipetting 15-20 times, and the cell suspension was centrifuged at 1000 g for 10 min. The pellet was washed once with Hank’s Balanced Salt Solution. The washed cells were resuspended in 200-300 p.1 of Hank’s BSS.4*‘5 Transplantation was applied to 30-day-old mice; control and B offspring were divided into groups receiving NE cells and sham controls. Accordingly, the following groups were defined: controlanimals which received control diet prenatally and at adulthood were either sham-operated or given NE transplants. Sham-operated and NE groups were pooled due to similarities in eight-arm maze behavior; B-sham-operated animals with prenatal exposure to phenobarbital; B/NE-B animals receiving NE transplants. All mice were anesthetized and placed in a stereotaxic apparatus. The cell suspension (2-5 1t.1)was injected 1.8 mm posterior to bregma, 1.5 mm lateral from the midline and 1.7 mm below the calvarium. ‘” Sham-operated offspring were injected with medium only. After behavioral testing, the existence of transplants in the hippocampus was verified with the histofluorescence technique described above. The coronal sections (50 pm) were made throughout the entire hippocampus. Adjacent sections were stained in Cresyl Violet. RESULTS For the purpose of studying the NE cells in the locus coeruleus, two animals, one control and one B were perfused each day, their brains cut and the cells counted on the same day. This resulted in a high within-pairs correlation (r = 0.743). Because of the large variations between pairs, statistical analysis was applied on the mean difference within pairs. In all the nine brain pairs studied, control locus coeruleus had more NE cells than B locus coeruleus (P
J. Yanai
and
C. 6. Pick
Table 1. Number of fluorescing NE cells in the locus coeruleus and NE levels in the hippocampus and cerebellum of control mice offspring and offspring with prenatal exposure to phenoba~tal (B offspring) NE levels
(ng/mgprotein) Treatment
NE cells
Control
1367~ 107 (9)
B offspring Mean difference between tested pairs
1064+81 (9) 3031t71’”
Hippocampus
Cerebellum
4.92kO.23
3.01 2 0.29
(8) 3.97 t 0.479 (8) -
(7) 3.42 ItO.25 (8) -
Numbers are mean + S.E.M. Numbers in parentheses represent sample sizes. ‘PcO.05 for the reduction from control level (treatment by brain intera~ion). ***f
Fig. I. The number of days to reach criterion in the eight-arm maze. Sample sizes are: control= 17, B = 12, B/NE = 10. **P
Fluorescing NE cells could be identified in the hippocampus of the transplanted while no similar phenomenon could be detected in the sham-operated mice.
mice (Fig. 2)
DISCUSSION Prenatal exposure to phenobarbital induced long-term alterations in the locus coeruleushippocampal noradrenergic pathway of mice. This was shown on a morphofogical level in the number of locus coeruleus NE cells which are known to innervate the hippocampus. The reduction of NE cells in the hippocampus further supports this finding on the biochemical level. Both the hip~ampus and the cerebellum receive their noradrenergic input from the locus coeruleus.*4 Yet, only the hippocampal NE was affected, suggesting that the effect of phenobarbital on NE level was, at least to some extent, specific. The changes induced by early barbiturate exposure are relatively small. However, even small long-term developmental changes may have extensive consequences on behavior. Previous studies have demonstrated short-term alteration in whole brain NE” and long-term changes in hypothalamic but not striatal NE levels” after early exposure to phenobarbital.
Studies on noradrenergic
alterations
Fig. 2. Fluorescing transplanted NE cell in the hippocampus (arrows). Cells were viewed in a Zeiss microscope with fluorescence attachment.
341
Studies on noradrenergic
alterations
343
Under regular microscopic examination, the locus coeruleus sections of the treated offspring appeared normal and did not differ from control. However, previous studies which applied fine quantitative histological techniques made it possible to elucidate relatively small differences between inbred mouse strains for locus coeruleus cells. *SagApplying these techniques, the present study revealed deficits induced by prenatal exposure to phenobarbital in the number of the fluorescing noradrenergic cells in the locus coeruleus. Eight-arm maze behavior is known to be related to the hippocampus. w*” Therefore it may be used as an indicator for early phenobarbital-induced impai~ent of the hip~campus. The present experiment confirmed previous findingP that early phenobradital administration impairs hippocampus-related behavior. An attempt was made in the present experiment to ascertain whether the mechanism by which phenobarbital-induced behavioral effects are mediated rests in changes in the noradrenergic innervations of the hippocampus. This hypothesis appeared feasible since manipulating the locus coeruleus-hippocampal pathway had an effect on several behaviors.7*2’*24Neuron transplantation was applied in the present experiment as a method of ascertaining a possible causal relationship between phenobarbital-induced alterations in NE and behavioral deficits. Experience accumulated in transplantation studies suggests that the specific type of transplant reversing behavioral deficits also indicates the neural impairment underlying the behavioral deficit.3 Transplantation NE cells survived in the impaired hippocampus. but did not reverse behavioral deficits. On the other hand, in previous studies, transplantation of cholinergic cells into the hip~mpus reversed most of the early phenoba~ital-indu~d deficits in eight-arm maze behavior. 30It appears, therefore, that the mechanism by which phenobarbitalinduced behavioral deficits are mediated may rest in alterations in septohippocampal cholinergic pathways, but not in alterations in locus coeruleus-hippocampal pathways. Alterations in noradrenergic innervations may have a role in other behavioral deficits induced by early phenobarbital administration.** The noradrenergic innervations in the hip~ampus originate from the locus coeruleus, where the cell bodies reside.14 Consequently, no fluorescing noradrenergic cell bodies were observed in the hippocampus of nontransplanting animals. Therefore, the fluorescing noradrenergic cells shown in Fig. 2 could only be transplanted cells. The histofluorescence studies confirmed the viability of the transplant, as no dead cells can be observed using this particular histofluore~nce procedure. This technique was implemented several weeks following transplantation. Although in the present experiment, the transplantation does exert an effect on behavior in a parallel study, transplantation of embryonic noradrenergic cells from the locus coeruleus into the hippocampus of normal mice markedly prolonged their barbital-induced narcosis.” Yet, further studies are necessary to ascertain that the noradrenergic transplantation indeed formed proper synaptic connections with the host brain rather than simply acting as a ‘neurotransmitter pump’ releasing NE into the neigh~~ng CNS tissues.
REFERENCES 1. Becker J. T., Walker J. A. and Oiton D. S. (1980) Neuroanatomicai basis of spatial memory. Brain Res. 2&l, 307-
320. 2. Berger B., Hewe D., Dolphin A., Bartheiemy C., Gay M. and Tassin J. P. (1979) Genetically determined differences in noradrenergic input to the brain cortex: A histochemicai and biochemical study in two inbred strains of mice. Neuroscience 4, 877-888. 3. Bjorklund A. and Stenevi U. (1984) Intracerebrai neural implants: neuronai replacement and reconstruction of damaged circuitries. Ann. Rev. Neurosci. 7, 279-308. 4. Bjorklund A., Stenevi U., Schmidt, Dunnett S. B. and Gage F. H. (1983) Intracerebrai grafting of neuronai cell suspensions I. Introduction and general method of preparation. ACM physiof. sand. Suppi., 522, l-7. 5. Felice L. J., Felice J. D. and Kinnin~er P. T. (1978) Dete~ination of catecholamines in rat brain parts by reversephase ion-pair liquid chromatographi. 3. Ne&che& 31, 1461-1466. 6. Fumess J. B.. Costa M. and Wilson A. J. (1977) Water-stable fluorophores, produced by reaction with aidehyde solutions, for the histochemicai localization bf caiechol- and indoiethyiamines.‘H~roc/re&&y 52, 159-170. _ 7. Flicker C. and Geyer M. A. (1982) Behavior during hippocampai microinfusions. I. Norepinephrine and diversive exploration. Brain Res. Rev. 4, 79-103. 8. Gauchy C., Tassin J. P., Giowinski J. and Cheramy A. (1976) Isolation and radioenzymatic estimation of picogram quantities of dopamine and norepinephrine in biological samples. 1. Neurocirern. 26,471-4&o. 9. H&i F. (1979) A simple, sensitive method for measuring 3,~ihyd~x~~nyla~t~ acid and ~vanil1~ acid in rat brain tissue using high performance liquid ch~mat~raphy with electrochemical detection. Life Sci. 25, 77%781.
J. Yanai and C. G. Pick
344
10. Jaffard R., Ebel A., Destrade C., Dunkint I., Mandel P. and Cardo B. (1977) Effects of hippocampal electrical stimulation on long-term memory and on cholinergic mechanism in three inbred strains of mice. Brain Res. 133,277298. 11. Konigsmark B. W. (1970) Methods for the counting of neurons. In Contemporary Research Methods in Neuroanatomy (eds Nalta W. J. H. and Ebbison S. 0. E.), pp. 315-340. Springer, New York. 12. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-272. 13. McCleam G. E., Wilson J. R. and Meredith W. (1970) The use of isogenic and heterogenic mouse stocks in behavioral research. In Contributions to Behavior-Genetic Analysis: The Mouse as a Prototype (eds Lindzey G. and Thiessen D. D.), pp. 3-22, Appleton-Century Crofts, New York. 14. Moore R. Y. and Bloom F. E. (1979) Central catecholamine neuron systems: Anatomy and physiology of the nonepinephrine and epinephrine systems. Ann. Rev. Neurosci. 2, 113. 15. McRae-Degueurce A., Bellin S. I., Serrano A., Landas S., Wilkin L., Scatton B. and Johnson A. K. (1984) Behavioral and neurochemical models to investigate functional recovery with transplants. Proceedings of Femstrom Symposium on Transplantation in the Mammalian CNS. 16. Middaugh L. D., Thomas T. N., Simpson L. W. and Zemp J. W. (1981) Effect of prenatal maternal injections of phenobarbital on brain neurotransmitters and behavior of young C57 mice. Neurobehav. Toxicol. Terutol. 3, 271275.
17. Olton D. S. and Papas B. C. (1979) Spatial memory and hippocampal function. Neuropsychologiu 17, 669482. 18. Olton D. S. and Samuelson R. J. (1976) Remembrance of places passed: spatial memory in rats. J. exp. Psychol. Animal
Eehav.
Processes
2, 97-l 16.
19. Pick C. G. and Yanai J. (1984) Long-term reduction in spontaneous alternations after early exposure to phenobarbital. Int. 1. devl Neurosci. 2, 223-228. 20. Pick C. G. and Yanai J. (1985) Long-term reduction in eight arm maze performance after early exposure to phenobarbital. Int. 1. devl Neurosci. 3, 223-227. 21. Plaznik A. W., Danysz W. and Kostowsky W. (1983) Some behavioral effects of microinjections of NA and serotonin, into the hippocampus of the rat. fhysiol. Behttv. 31, 625-631. 22. Reinisch J. M. and Sanders S. A. (1982) Early barbiturate exposure: The brain, sexually dimorphic behavior and learning. Neurosci. Biobehov. Rev. 67, 311-319. 23. Roberts W. W., Dember W. N. and Brodwick M. (1962) Alternation and exploration in rats with hippocampal lesions. 1. camp. Physiol. Phychol. 55, 695-700. 24. Segal M. and Edelson A. (1978) Effects of priming stimulation of catecholamine containing nuclei in rat brain on runway performance. Brain Res. Bull. 3, 203-206. 25. Siegel S. (1956) Non-Purametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. 26. Touret M., Valatx J. L. and Jouvet M. (1982) The locus coeruleus: a quantitative and genetic study in mice. Bruin Res. 250, 353-357.
27. Winer B. J. (1971) Statistical Principles in Experimental Design. McGraw-Hill, New York. 28. Yanai J. (1984) An animal model for barbiturate effect on CNS development. In Neurobehoviorul Temtology (ed. Yanai J.), pp. 111-132. Elsevier, Amsterdam. 29. Yanai J., Rosselli-Austin L. and Tabakoff B. (1979) Neuronal deficits in mice following prenatal exposure to phenobarbital. Expl Neurol. 64, 237-244. 30. Yanai J., Sze P. Y., lser C. and Melamed E. (1985) Studies on brain monoamine neurotransmitters in mice after prenatal exposure to barbiturate. Pharmac. Biochem. Behav. 23, 215-219. 31. Yanai J., Pick C. G. and McRae-Degueurce A. (1986) The use of brain tissue transplantation in neuroteratological studies. Neurotoxicology 7, 143-150.
u736-574ws7 so3.0o+o.aI Pergamon Jourmtls Ltd. @ 1987 ISDN
STUDIES ON NORADRENERGIC ALTERATIONS IN RELATION TO EARLY PHENOBARBITAL-INDUCED BEHAVIORAL CHANGES JOSEPH YANAI and CHAIM G. PICK The Melvin A. and Eleanor Ross Laboratory for Studies in Neural Birth Defects, Department of Anatomy and Embryology, The Hebrew University-Hadassah Medical School, Box 1172, 91010 Jerusalem, Israel (Received
8 January
1987; in revised form
1 July 1987; accepted 3 July 1987)
Abstract-Mice were exposed to phenobarbital prenatally (B offspring) by feeding their mother 3 g/kg phenobarbital in milled food on gestation days 9-18; control dams received unadulterated milled food. At age 50 days, B offspring had fewer fluorescing noradrenergic (NE) cells in the locus coeruleus than control (P
Mice,
Neuron
transplantation,
Early (pre- and neonatal) exposure to phenobarbital induced long-term behavioral and neural changes in experimental animals. ***zJTo gain an understanding of the mechanisms by which the barbiturate exerts its effect on behavior, our research effort focused on behaviors which may be related, to a specific brain area and to particular biochemical processes. Thus, in mice, early exposure to phenobarbital induced deficits in eight-arm maze performance and spontaneous alterations.‘9.20 Both of these behaviors are primarily associated with a specific brain area, the hippocampus. ‘J’**~ We are investigating early phenobarbital-induced alterations in the major innervations to the hippocampus as the possible mechanism mediating hippocampal behavioral deficits. Noradrenergic innervations have an important role in the hippocampus; they originate exclusively from the locus coeruleus where the cell bodies resideI and were shown to have a regulatory role on various behaviors. ‘**L*~In the present experiment, mice were exposed to phenobarbital during their prenatal development. At an older age their locus coeruleushippocampal noradrenegic innervations were studied at morphological and biochemical levels. Since the studies revealed alterations in these innervations, an attempt was made to correct the phenobarbital-induced deficits by hippocampal behaviors by transplantation of normal noradrenergic cells into the impaired hippocampus. METHODS The genetically heterogeneous HS/Ibg mice were employed.” The method of phenobarbital administration to the developing fetuses have been previously described.28 Adult mice were housed in mating groups of 1 male and 4 females. Their offspring (the subjects of the experiments) received phenobarbital prenatally via the placenta. Female parent mice were checked daily at 08.00 hr. Those that had conceived, as verified by the existence of vaginal plug, were separated from the males and housed with other pregnant females. On gestation day 9 (GD 9; evidence of vaginal plug is considered GD l), the females were housed in individual cages. Treated females then received milled mouse food containing 3 g/kg phenobarbital in acid form (their only food source) and water, both available ad libitum. Control females received milled food and water. Drug administration continued until GD 18, when phenobarbital and control diets were replaced with regular mouse pellets. Blood phenobarbital levels were monitored in the en 5:4-n
337
338
J. Yanai and C. G. Pick
females and fetuses as described by Yanai et ~1.~~The blood phenobarbital level averaged 113 rt 12 p&ml during most of the phenobarbital feeding period. The phenobarbital levels of the fetuses were similar to those of the mothers. *’ Following delivery, the barbiturate-exposed offspring (B offspring) and control offspring were maintained with their mothers under standard laboratory conditions and received no further drug treatment. They were weaned on day 25, segregation and maintenance according to gender followed subsequently. Female and male offspring were employed in approximately equal numbers. The data were pooled across gender, as there were no differences between the sexes. Control and B offspring were divided into groups according to the experiments and tested for the following: (a) the number of fluorescing NE cells in the locus coeruleus, (b) levels of NE in the hippocampus and cerebellum, (c) behavior in the eight-arm maze and the effect of NE transplantation on the deficits in this behavior. Catecholamine histojluorescence A detailed description of the method is found in Furness et aL6 Mice were perfused transcardically with 50 ml of 4% parafo~aldehyde and 0.5% glutaraldehyde in a phosphate buffer (pH 7.4, 0.1 M). After dissection, the brain stems were postfixed overnight. Locus coeruleus serial coronal sections (20 pm) from treatment and control mice were obtained on a vibratome. Every 3rd section was picked up, dried on a slide and mounted in paraffin oil. The sections were observed with a Zeiss microscope using a fluorescence attachment, Cell counts of the locus coeruleus were made and the Abercrombie correction factor was applied as follows: N=nt/ t+d) in which N = actual number of cells, n = counted number of cells, t = mean thickness of the section and d = mean diameter of counted units.2.” Norepinephrine assay Hip~campi and cerebella were dissected on an ice-cold plate, weighed, and frozen at -70°C. Catecholamines were assayed as described by Felice et af.5 and Hefti et aA with a few modifications. The tissues were homogenized by a sonicator in ice-cold distilled water containing 0.1 perchloric acid and 0.2 mg/ml sodium metabisulfite. The homogenate was centrifuged (12,000g for 5 min) and 100 t~l of the supematant fluid were taken for analysis. Norepinephrine was isolated using miniature alumina columns as described by Gauchy et al.’ Small pipet tips were stopped with glass wool and filled with 20-25 mg alumina (Woelm, activity grade I, activated by boiling in 2 M HCl). The pipet tips were placed in reagent tubes (12 x 75 mm) with adaptors (cutoff larger pipet tips) and run in a table centrifuge (TJ-6; Beckman Instruments, Palo Alto, CA). The alumina columns were equilibrated with 200 ~10.05 M Tris-HCl buffer (pH 8.6) (100 g for 2 min). One hundred and ninety ~1 of a mixture ~ntaining the sample (100 ~1) and 100 ~1 of 1 M Tris-HCl buffer (pH 8.6 containing 1 mg/ml dithiothreitol) and 10 ~1 of 0.1 M perchloric acid with 10 ng 3,4-dihydroxybenzylamine (internal standard) were pipetted into the columns and centrifuged at 100 g for 3 min. The columns were then washed with 200 t.~lHz0 (100 g for 2 min), transferred into new test tubes and the NE (as well as other catecholamines and DOPAC) subsequently eluted with 150 t.tJof 0.1 M phosphoric acid (400 g for 5 min). The eluate was analysed by reverse-phase high-performance liquid chromatography with electrochemical detection, based on the method described by Felice et aLs The column used was Cl8 reverse-phase (3.9 x 300 mm uBondapak; Waters) and the electrode was glassy carbon (Bioanalytical Systems). The potential was set at +0.7 V.A. Sodium phosphate buffer (0.1 M, pH 3.0), ~ntaining 0.6 mM octyl sodium sulfate as ion-pair reagent, served as mobile phase. Protein dete~ination was made using the method of Lowry et al. ‘* Eight-arm maze test The maze used for this experiment is a scaled down version of the radial maze for rats. lH Briefly, it is constructed on a Plexiglas circular base 76 cm in diameter. Eight arms are attached to this base; the internal dimensions of each arm are 32 x 4 x 4 cm. At the end of each arm, 1.5 cm from its end, is a depression for water reinforcement. All arms are linked with the central platform which is 13.5 cm across. The maze is covered with a circular transparent cover of Plexiglas.
Studies on noradrenergic
alterations
339
Over the center platform in the middle of the cover there is an opening, with a cover through which the mouse is introduced into the maze. Mice were tested in the maze at age 90 days. Control and B offspring transplanted with NE cells or sham-operated were put on a regimen of water deprivation for a week. This included restricting daily administration of water for 30 min. After a week, the mice were introduced individually into the apparatus for 10 min of habituation without reinforcement of water. The first 16 entries were observed. The mice received reinforcement with water drops of 50 ~1 during the subsequent 5 day testing period unlike the habituation day, when animals were left in the maze until they had either entered all eight arms, or had made 16 entries, whichever occurred first. Animals were considered to have reached criterion if there were eight correct entries out of the possible eight for two consecutive days. Thus it was possible to calculate the number of days it took to reach criterion. Animals that reached criterion after day 1 (eight correct responses out of eight trials on habituation day+the following day) received the score 0. Animals that reached criterion on the 2 days after habituation day received the score 1, etc. Animals not reaching criterion during the entire testing period received the score 6. Transplantation
of noradrenergic
cells
Brains of normal mouse embryos were dissected on gestation day 15 under an operating microscope. The locus coeruleus was selected as a source of noradrenergic cells. It was dissected at the level of the pontine flexure and the rhombic lip. Two mm tissue sections from the same brain region were pooled (20 embryos) and transferred to a conical centrifuge tube containing 5 ml of versene (1:5000). Tissues were gently dissociated by aspiration pipetting 15-20 times, and the cell suspension was centrifuged at 1000 g for 10 min. The pellet was washed once with Hank’s Balanced Salt Solution. The washed cells were resuspended in 200-300 p.1 of Hank’s BSS.4*‘5 Transplantation was applied to 30-day-old mice; control and B offspring were divided into groups receiving NE cells and sham controls. Accordingly, the following groups were defined: controlanimals which received control diet prenatally and at adulthood were either sham-operated or given NE transplants. Sham-operated and NE groups were pooled due to similarities in eight-arm maze behavior; B-sham-operated animals with prenatal exposure to phenobarbital; B/NE-B animals receiving NE transplants. All mice were anesthetized and placed in a stereotaxic apparatus. The cell suspension (2-5 1t.1)was injected 1.8 mm posterior to bregma, 1.5 mm lateral from the midline and 1.7 mm below the calvarium. ‘” Sham-operated offspring were injected with medium only. After behavioral testing, the existence of transplants in the hippocampus was verified with the histofluorescence technique described above. The coronal sections (50 pm) were made throughout the entire hippocampus. Adjacent sections were stained in Cresyl Violet. RESULTS For the purpose of studying the NE cells in the locus coeruleus, two animals, one control and one B were perfused each day, their brains cut and the cells counted on the same day. This resulted in a high within-pairs correlation (r = 0.743). Because of the large variations between pairs, statistical analysis was applied on the mean difference within pairs. In all the nine brain pairs studied, control locus coeruleus had more NE cells than B locus coeruleus (P
J. Yanai
and
C. 6. Pick
Table 1. Number of fluorescing NE cells in the locus coeruleus and NE levels in the hippocampus and cerebellum of control mice offspring and offspring with prenatal exposure to phenoba~tal (B offspring) NE levels
(ng/mgprotein) Treatment
NE cells
Control
1367~ 107 (9)
B offspring Mean difference between tested pairs
1064+81 (9) 3031t71’”
Hippocampus
Cerebellum
4.92kO.23
3.01 2 0.29
(8) 3.97 t 0.479 (8) -
(7) 3.42 ItO.25 (8) -
Numbers are mean + S.E.M. Numbers in parentheses represent sample sizes. ‘PcO.05 for the reduction from control level (treatment by brain intera~ion). ***f
Fig. I. The number of days to reach criterion in the eight-arm maze. Sample sizes are: control= 17, B = 12, B/NE = 10. **P
Fluorescing NE cells could be identified in the hippocampus of the transplanted while no similar phenomenon could be detected in the sham-operated mice.
mice (Fig. 2)
DISCUSSION Prenatal exposure to phenobarbital induced long-term alterations in the locus coeruleushippocampal noradrenergic pathway of mice. This was shown on a morphofogical level in the number of locus coeruleus NE cells which are known to innervate the hippocampus. The reduction of NE cells in the hippocampus further supports this finding on the biochemical level. Both the hip~ampus and the cerebellum receive their noradrenergic input from the locus coeruleus.*4 Yet, only the hippocampal NE was affected, suggesting that the effect of phenobarbital on NE level was, at least to some extent, specific. The changes induced by early barbiturate exposure are relatively small. However, even small long-term developmental changes may have extensive consequences on behavior. Previous studies have demonstrated short-term alteration in whole brain NE” and long-term changes in hypothalamic but not striatal NE levels” after early exposure to phenobarbital.
Studies on noradrenergic
alterations
Fig. 2. Fluorescing transplanted NE cell in the hippocampus (arrows). Cells were viewed in a Zeiss microscope with fluorescence attachment.
341
Studies on noradrenergic
alterations
343
Under regular microscopic examination, the locus coeruleus sections of the treated offspring appeared normal and did not differ from control. However, previous studies which applied fine quantitative histological techniques made it possible to elucidate relatively small differences between inbred mouse strains for locus coeruleus cells. *SagApplying these techniques, the present study revealed deficits induced by prenatal exposure to phenobarbital in the number of the fluorescing noradrenergic cells in the locus coeruleus. Eight-arm maze behavior is known to be related to the hippocampus. w*” Therefore it may be used as an indicator for early phenobarbital-induced impai~ent of the hip~campus. The present experiment confirmed previous findingP that early phenobradital administration impairs hippocampus-related behavior. An attempt was made in the present experiment to ascertain whether the mechanism by which phenobarbital-induced behavioral effects are mediated rests in changes in the noradrenergic innervations of the hippocampus. This hypothesis appeared feasible since manipulating the locus coeruleus-hippocampal pathway had an effect on several behaviors.7*2’*24Neuron transplantation was applied in the present experiment as a method of ascertaining a possible causal relationship between phenobarbital-induced alterations in NE and behavioral deficits. Experience accumulated in transplantation studies suggests that the specific type of transplant reversing behavioral deficits also indicates the neural impairment underlying the behavioral deficit.3 Transplantation NE cells survived in the impaired hippocampus. but did not reverse behavioral deficits. On the other hand, in previous studies, transplantation of cholinergic cells into the hip~mpus reversed most of the early phenoba~ital-indu~d deficits in eight-arm maze behavior. 30It appears, therefore, that the mechanism by which phenobarbitalinduced behavioral deficits are mediated may rest in alterations in septohippocampal cholinergic pathways, but not in alterations in locus coeruleus-hippocampal pathways. Alterations in noradrenergic innervations may have a role in other behavioral deficits induced by early phenobarbital administration.** The noradrenergic innervations in the hip~ampus originate from the locus coeruleus, where the cell bodies reside.14 Consequently, no fluorescing noradrenergic cell bodies were observed in the hippocampus of nontransplanting animals. Therefore, the fluorescing noradrenergic cells shown in Fig. 2 could only be transplanted cells. The histofluorescence studies confirmed the viability of the transplant, as no dead cells can be observed using this particular histofluore~nce procedure. This technique was implemented several weeks following transplantation. Although in the present experiment, the transplantation does exert an effect on behavior in a parallel study, transplantation of embryonic noradrenergic cells from the locus coeruleus into the hippocampus of normal mice markedly prolonged their barbital-induced narcosis.” Yet, further studies are necessary to ascertain that the noradrenergic transplantation indeed formed proper synaptic connections with the host brain rather than simply acting as a ‘neurotransmitter pump’ releasing NE into the neigh~~ng CNS tissues.
REFERENCES 1. Becker J. T., Walker J. A. and Oiton D. S. (1980) Neuroanatomicai basis of spatial memory. Brain Res. 2&l, 307-
320. 2. Berger B., Hewe D., Dolphin A., Bartheiemy C., Gay M. and Tassin J. P. (1979) Genetically determined differences in noradrenergic input to the brain cortex: A histochemicai and biochemical study in two inbred strains of mice. Neuroscience 4, 877-888. 3. Bjorklund A. and Stenevi U. (1984) Intracerebrai neural implants: neuronai replacement and reconstruction of damaged circuitries. Ann. Rev. Neurosci. 7, 279-308. 4. Bjorklund A., Stenevi U., Schmidt, Dunnett S. B. and Gage F. H. (1983) Intracerebrai grafting of neuronai cell suspensions I. Introduction and general method of preparation. ACM physiof. sand. Suppi., 522, l-7. 5. Felice L. J., Felice J. D. and Kinnin~er P. T. (1978) Dete~ination of catecholamines in rat brain parts by reversephase ion-pair liquid chromatographi. 3. Ne&che& 31, 1461-1466. 6. Fumess J. B.. Costa M. and Wilson A. J. (1977) Water-stable fluorophores, produced by reaction with aidehyde solutions, for the histochemicai localization bf caiechol- and indoiethyiamines.‘H~roc/re&&y 52, 159-170. _ 7. Flicker C. and Geyer M. A. (1982) Behavior during hippocampai microinfusions. I. Norepinephrine and diversive exploration. Brain Res. Rev. 4, 79-103. 8. Gauchy C., Tassin J. P., Giowinski J. and Cheramy A. (1976) Isolation and radioenzymatic estimation of picogram quantities of dopamine and norepinephrine in biological samples. 1. Neurocirern. 26,471-4&o. 9. H&i F. (1979) A simple, sensitive method for measuring 3,~ihyd~x~~nyla~t~ acid and ~vanil1~ acid in rat brain tissue using high performance liquid ch~mat~raphy with electrochemical detection. Life Sci. 25, 77%781.
J. Yanai and C. G. Pick
344
10. Jaffard R., Ebel A., Destrade C., Dunkint I., Mandel P. and Cardo B. (1977) Effects of hippocampal electrical stimulation on long-term memory and on cholinergic mechanism in three inbred strains of mice. Brain Res. 133,277298. 11. Konigsmark B. W. (1970) Methods for the counting of neurons. In Contemporary Research Methods in Neuroanatomy (eds Nalta W. J. H. and Ebbison S. 0. E.), pp. 315-340. Springer, New York. 12. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-272. 13. McCleam G. E., Wilson J. R. and Meredith W. (1970) The use of isogenic and heterogenic mouse stocks in behavioral research. In Contributions to Behavior-Genetic Analysis: The Mouse as a Prototype (eds Lindzey G. and Thiessen D. D.), pp. 3-22, Appleton-Century Crofts, New York. 14. Moore R. Y. and Bloom F. E. (1979) Central catecholamine neuron systems: Anatomy and physiology of the nonepinephrine and epinephrine systems. Ann. Rev. Neurosci. 2, 113. 15. McRae-Degueurce A., Bellin S. I., Serrano A., Landas S., Wilkin L., Scatton B. and Johnson A. K. (1984) Behavioral and neurochemical models to investigate functional recovery with transplants. Proceedings of Femstrom Symposium on Transplantation in the Mammalian CNS. 16. Middaugh L. D., Thomas T. N., Simpson L. W. and Zemp J. W. (1981) Effect of prenatal maternal injections of phenobarbital on brain neurotransmitters and behavior of young C57 mice. Neurobehav. Toxicol. Terutol. 3, 271275.
17. Olton D. S. and Papas B. C. (1979) Spatial memory and hippocampal function. Neuropsychologiu 17, 669482. 18. Olton D. S. and Samuelson R. J. (1976) Remembrance of places passed: spatial memory in rats. J. exp. Psychol. Animal
Eehav.
Processes
2, 97-l 16.
19. Pick C. G. and Yanai J. (1984) Long-term reduction in spontaneous alternations after early exposure to phenobarbital. Int. 1. devl Neurosci. 2, 223-228. 20. Pick C. G. and Yanai J. (1985) Long-term reduction in eight arm maze performance after early exposure to phenobarbital. Int. 1. devl Neurosci. 3, 223-227. 21. Plaznik A. W., Danysz W. and Kostowsky W. (1983) Some behavioral effects of microinjections of NA and serotonin, into the hippocampus of the rat. fhysiol. Behttv. 31, 625-631. 22. Reinisch J. M. and Sanders S. A. (1982) Early barbiturate exposure: The brain, sexually dimorphic behavior and learning. Neurosci. Biobehov. Rev. 67, 311-319. 23. Roberts W. W., Dember W. N. and Brodwick M. (1962) Alternation and exploration in rats with hippocampal lesions. 1. camp. Physiol. Phychol. 55, 695-700. 24. Segal M. and Edelson A. (1978) Effects of priming stimulation of catecholamine containing nuclei in rat brain on runway performance. Brain Res. Bull. 3, 203-206. 25. Siegel S. (1956) Non-Purametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. 26. Touret M., Valatx J. L. and Jouvet M. (1982) The locus coeruleus: a quantitative and genetic study in mice. Bruin Res. 250, 353-357.
27. Winer B. J. (1971) Statistical Principles in Experimental Design. McGraw-Hill, New York. 28. Yanai J. (1984) An animal model for barbiturate effect on CNS development. In Neurobehoviorul Temtology (ed. Yanai J.), pp. 111-132. Elsevier, Amsterdam. 29. Yanai J., Rosselli-Austin L. and Tabakoff B. (1979) Neuronal deficits in mice following prenatal exposure to phenobarbital. Expl Neurol. 64, 237-244. 30. Yanai J., Sze P. Y., lser C. and Melamed E. (1985) Studies on brain monoamine neurotransmitters in mice after prenatal exposure to barbiturate. Pharmac. Biochem. Behav. 23, 215-219. 31. Yanai J., Pick C. G. and McRae-Degueurce A. (1986) The use of brain tissue transplantation in neuroteratological studies. Neurotoxicology 7, 143-150.