Neurochemical Research, Vol. 17, No. 6, 1992, pp. 565-569
Differential Sensitivity of Cholinergic and GABAergic Neurons in Chick Embryos Treated Intracerebrally with Ethanol at 8 Days of Embryonic Age Kendall Lee 1, Susan KentrotP, and Antonia Vernadakis 1 (Acceptetd October 28, 1991)
We have shown that in embryos treated with ethanol in ovo during days 1-3, a critical period of neuroembryogenesis, cholinergic neuronal phenotypic expression is decreased whereas GABAergic and catecholaminergic neuronal populations are increased as assessed by neuronal markers choline acetyltransferase (CHAT), glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) respectively. In this study, ethanol was administered intracerebrally to embryos at embryonic day 8, embryos were sacrificed at day 9 and ChAT and GAD activities assayed separately in cerebral hemispheres and remaining brain (diencephalon-midbrain and optic lobes). We found that ChAT activity was enhanced in the cerebral hemispheres only, whereas GAD activity was decreased in b.oth cerebral hemispheres and remaining brain. We have concluded that the differential responses of neuronal phenotypes to ethanol may reflect compensatory mechanisms to ethanol insult. Moreover, these findings emphasize the vulnerability of the GABAergie neuronal phenotypes to ethanol neurotoxicity during early brain development in the chick. KEY WORDS: Ethanol; chick embryo; GABAergic neuron; cholinergic neuron.
INTRODUCTION
hanced (4,6,15,16). However, when neuronal cultures derived from 3-day-old chick embryos (E3WE), which consist primarily of neuroblasts, are exposed to ethanol in vitro both cholinergic and GABAergic neuronal phenotypes are retarded (17). In contrast, in neuronal cultures derived from 8-day-old chick embryo cerebral hemispheres (E8CH) consisting of differentiated neurons ethanol exposure results in a decline in GABAergic neuronal phenotypes and an enhancement of cholinergic neuronal expression (5). These findings suggest that the stage of neuronal maturation (neuroblasts versus differentiated neurons) and the local microenvironment (in ovo versus in culture) may play an important role in neuronal sensitivity to ethanol. In the present study we attempted to mimic some components of both in ovo and in vitro paradigms. Ethanol was introduced directly into the brain of developing embryos via intracerebral injection. Embryos were
We have reported that in ovo administration of ethanol to chick embryos via the air sac during embryonic days 1 to 3, a period of active neuronal proliferation (2,3) exerts differential effects on neuronal phenotypic expression as assessed by the enzyme markers, choline acetyltransferase (cholinergic), tyrosine hydroxylase (catecholaminergic) and glutamic acid decarboxylase (GABAergic); cholinergic expression is retarded whereas both catecholaminergic and GABAergic expression are eni Departments of Psychiatry and Pharmacology, University of Colorado School of Medicine, Denver, CO 80262. z Department of Pharmacology, University of Colorado School of Medicine, Denver, CO 80262. To whom to address reprint requests: Professor Antonia Vernadakis, Departments of Psychiatry and Pharmacology, University of Colorado School of Medicine, 4200 East Ninth Avenue, Denver, CO 80262.
565 0364-3190192/0600-0565506,50/09 1992PlenumPublishingCorporation
566 treated at 8 days of development, a stage at which cerebral neurons are almost entirely differentiated. The merit of this experimental paradigm as opposed to administration via the aft sac is that developing neurons are directly exposed to ethanol, thus allowing the test substance (ethanol) to reach the brain versus other developing organs and thus bypassing interaction among other cell populations. The virtue of intracerebral administration as opposed to the culture model system is that neuronal complexity and integration can be maintained. We report that after intracerebral administration of ethanol to 8day-old chick embryo, cholinergic expression, as assessed by choline acetyltransferase (CHAT), was enhanced but only in the cerebral hemispheres whereas GABAergic expression, as assessed by glutamic acid decarboxylase, was decreased in both cerebral hemispheres and remaining brain. These results together with our previous findings reported earlier support our view that differentiated neurons and proliferating neuroblasts respond differentially to ethanol suggesting alternative mechanisms of action.
EXPERIMENTAL PROCEDURE Animals. All experiments were performed on white Leghorn chick embryos (SPAFAS, Norwich, CT) Ethanol treatment. Figure 1 is a schematic representation of intracerebral ethanol administration in chick embryos at 8 days of embryonic age. The egg shell above the air sac was carefully removed. The shell membrane was removed very carefully so as not to damage the underlying blood vessels and capillaries communicating with the embryo. This is of paramount significance since any damage results in the death of the embryo. The embryo within the amniotic cavity was gently lifted with a wire scoop instrument and thus the head of the embryo was visible. Care was taken not to damage the yolk sac in which case the embryo would not survive. A volume of 2 ixl of test substance (ethanol) or vehicle (saline) was injected into the cerebrum using a Hamilton syringe fitted with a 30 gauge needle. The entire procedure was performed under sterile conditions. Embryos were sacrificed, brains removed and cerebral hemispheres and remaining brain were dissected and frozen separately at - 20~ until assayed (not later than one week). Viability results using this method were: Saline control 59.4%; 10 ~g (108 raM) ETOH 52.9%; 40 Ixg (443 raM) ETOH, 50.6%; 100 ~g (1.08 M) ETOH, 62.2%. Thus none of the ethanol doses used produced higher mortality of chick embryo than saline. Choline Acetyltransferase Activity. Choline acetyltransferase (CHAT) activity was measured by the radiometric method of Fonnum (9) as modified by us for embryonic tissue and culture (25). Glutamic Acid Decarboxylase Activity. Glutamic acid decarboxylase (GAD) activity was determined using the method of Wilson et al. (28), as modified by Quinn and Cagan (24) and also by us for embryonic tissue and culture (20). Protein Assay. Protein content was determined in aliquots of the supernatant according to the method of Lowry et al. (19) using bovine serum albumin as a standard.
Lee, Kentroti, and Vernadakis Statistics. All the data are analyzed statistically using the student t-test for comparison of the means.
RESULTS
Choline actyltransferase activity. Ethanol or saline (2 Ixl)were administered intracerebraUyin 8-day-old chick embryos as described in Methods. Twenty-four hours later, embryonic day 9, embryos were sacrificed, brain removed, and cerebral hemispheres and remaining brain (optic lobes, diencephalon-midbrain, brain stem) were dissected and frozen separately at -20~ until ChAT analysis. Administration of either 10 p~g or 40 Ixg of ethanol produced a statistically significant increase in ChAT activity in cerebral hemispheres (P
Differential Sensitivity of Cholinergic and GABAergic Neurons in Chick Embryos Treated Intracerebrally with Ethanol at 8 Days of Embryonic Age Kendall Lee 1, Susan KentrotP, and Antonia Vernadakis 1 (Acceptetd October 28, 1991)
We have shown that in embryos treated with ethanol in ovo during days 1-3, a critical period of neuroembryogenesis, cholinergic neuronal phenotypic expression is decreased whereas GABAergic and catecholaminergic neuronal populations are increased as assessed by neuronal markers choline acetyltransferase (CHAT), glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) respectively. In this study, ethanol was administered intracerebrally to embryos at embryonic day 8, embryos were sacrificed at day 9 and ChAT and GAD activities assayed separately in cerebral hemispheres and remaining brain (diencephalon-midbrain and optic lobes). We found that ChAT activity was enhanced in the cerebral hemispheres only, whereas GAD activity was decreased in b.oth cerebral hemispheres and remaining brain. We have concluded that the differential responses of neuronal phenotypes to ethanol may reflect compensatory mechanisms to ethanol insult. Moreover, these findings emphasize the vulnerability of the GABAergie neuronal phenotypes to ethanol neurotoxicity during early brain development in the chick. KEY WORDS: Ethanol; chick embryo; GABAergic neuron; cholinergic neuron.
INTRODUCTION
hanced (4,6,15,16). However, when neuronal cultures derived from 3-day-old chick embryos (E3WE), which consist primarily of neuroblasts, are exposed to ethanol in vitro both cholinergic and GABAergic neuronal phenotypes are retarded (17). In contrast, in neuronal cultures derived from 8-day-old chick embryo cerebral hemispheres (E8CH) consisting of differentiated neurons ethanol exposure results in a decline in GABAergic neuronal phenotypes and an enhancement of cholinergic neuronal expression (5). These findings suggest that the stage of neuronal maturation (neuroblasts versus differentiated neurons) and the local microenvironment (in ovo versus in culture) may play an important role in neuronal sensitivity to ethanol. In the present study we attempted to mimic some components of both in ovo and in vitro paradigms. Ethanol was introduced directly into the brain of developing embryos via intracerebral injection. Embryos were
We have reported that in ovo administration of ethanol to chick embryos via the air sac during embryonic days 1 to 3, a period of active neuronal proliferation (2,3) exerts differential effects on neuronal phenotypic expression as assessed by the enzyme markers, choline acetyltransferase (cholinergic), tyrosine hydroxylase (catecholaminergic) and glutamic acid decarboxylase (GABAergic); cholinergic expression is retarded whereas both catecholaminergic and GABAergic expression are eni Departments of Psychiatry and Pharmacology, University of Colorado School of Medicine, Denver, CO 80262. z Department of Pharmacology, University of Colorado School of Medicine, Denver, CO 80262. To whom to address reprint requests: Professor Antonia Vernadakis, Departments of Psychiatry and Pharmacology, University of Colorado School of Medicine, 4200 East Ninth Avenue, Denver, CO 80262.
565 0364-3190192/0600-0565506,50/09 1992PlenumPublishingCorporation
566 treated at 8 days of development, a stage at which cerebral neurons are almost entirely differentiated. The merit of this experimental paradigm as opposed to administration via the aft sac is that developing neurons are directly exposed to ethanol, thus allowing the test substance (ethanol) to reach the brain versus other developing organs and thus bypassing interaction among other cell populations. The virtue of intracerebral administration as opposed to the culture model system is that neuronal complexity and integration can be maintained. We report that after intracerebral administration of ethanol to 8day-old chick embryo, cholinergic expression, as assessed by choline acetyltransferase (CHAT), was enhanced but only in the cerebral hemispheres whereas GABAergic expression, as assessed by glutamic acid decarboxylase, was decreased in both cerebral hemispheres and remaining brain. These results together with our previous findings reported earlier support our view that differentiated neurons and proliferating neuroblasts respond differentially to ethanol suggesting alternative mechanisms of action.
EXPERIMENTAL PROCEDURE Animals. All experiments were performed on white Leghorn chick embryos (SPAFAS, Norwich, CT) Ethanol treatment. Figure 1 is a schematic representation of intracerebral ethanol administration in chick embryos at 8 days of embryonic age. The egg shell above the air sac was carefully removed. The shell membrane was removed very carefully so as not to damage the underlying blood vessels and capillaries communicating with the embryo. This is of paramount significance since any damage results in the death of the embryo. The embryo within the amniotic cavity was gently lifted with a wire scoop instrument and thus the head of the embryo was visible. Care was taken not to damage the yolk sac in which case the embryo would not survive. A volume of 2 ixl of test substance (ethanol) or vehicle (saline) was injected into the cerebrum using a Hamilton syringe fitted with a 30 gauge needle. The entire procedure was performed under sterile conditions. Embryos were sacrificed, brains removed and cerebral hemispheres and remaining brain were dissected and frozen separately at - 20~ until assayed (not later than one week). Viability results using this method were: Saline control 59.4%; 10 ~g (108 raM) ETOH 52.9%; 40 Ixg (443 raM) ETOH, 50.6%; 100 ~g (1.08 M) ETOH, 62.2%. Thus none of the ethanol doses used produced higher mortality of chick embryo than saline. Choline Acetyltransferase Activity. Choline acetyltransferase (CHAT) activity was measured by the radiometric method of Fonnum (9) as modified by us for embryonic tissue and culture (25). Glutamic Acid Decarboxylase Activity. Glutamic acid decarboxylase (GAD) activity was determined using the method of Wilson et al. (28), as modified by Quinn and Cagan (24) and also by us for embryonic tissue and culture (20). Protein Assay. Protein content was determined in aliquots of the supernatant according to the method of Lowry et al. (19) using bovine serum albumin as a standard.
Lee, Kentroti, and Vernadakis Statistics. All the data are analyzed statistically using the student t-test for comparison of the means.
RESULTS
Choline actyltransferase activity. Ethanol or saline (2 Ixl)were administered intracerebraUyin 8-day-old chick embryos as described in Methods. Twenty-four hours later, embryonic day 9, embryos were sacrificed, brain removed, and cerebral hemispheres and remaining brain (optic lobes, diencephalon-midbrain, brain stem) were dissected and frozen separately at -20~ until ChAT analysis. Administration of either 10 p~g or 40 Ixg of ethanol produced a statistically significant increase in ChAT activity in cerebral hemispheres (P
