Molecular Brain Research, 16 (1992) 119-127
119
© 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00 BRESM 70509
Glucocorticoid regulation of preproenkephalin gene expression in the rat forebrain Rexford S. Ahima a, Meredith M. Garcia a and Richard E. Harlan a,b a Department of Anatomy and b Neuroscience TrainingProgram, Tulane UniversityMedical School, New Orleans, LA 70112 (USA)
(Accepted 23 June 1992)
Key words: Hippocampus;Caudate-putamen; Accumbens;Amygdala;Hypothalamus
The effects of glucocorticoidson the levels of preproenkephalin (PPE) mRNA in the rat forebrain were analyzed with in situ hybridization and dot blots. In adrenally-intact rats, high levels of PPE mRNA, as assessed by in sitn hybridization, were localized in the eaudate-putamen, nucleus accumbens, central amygdala, and ventrolateral ventromedial hypothalamus(VMHVL), and low levels in the hippocampus. After adrenalectomy, the density of PPE mRNA-positive cells and the level of PPE mRNA/cell were decreased in all regions except the hippocampus. Acute treatment with corticosterone (CORT) in adrenalectomized rats increased the level of PPE mRNA/cell in the caudate-putamen and VMHVL. In intact rats, chronic treatment with CORT increased the density of PPE mRNA-positivecells in the caudate-putamen and hippocampus, and the level of PPE mRNA/cell in the caudate-putamen and nucleus accumbens. The effect of chronic CORT treatment on PPE mRNA in the striatum, amygdala, hippocampus and mediobasal hypothalamuswas assessed with dot blots. Chronic CORT treatment increased PPE mRNA levels in the caudate-putamen and hippocampus. There was a good correlation between results on the effect of chronic CORT treatment on PPE mRNA levels in intact rats, obtained from dot blots and in situ hybridization. Results from this study suggest that glucocorticoidsare required for the maintenance of basal PPE mRNA levels in most regions of the rat forebrain. There is, however, considerable regional heterogeneity in the effect of glucocorticoidtreatment on PPE mRNA levels in adrenalectomized and intact rats. Increased PPE mRNA levels in response to high circulating levels of glucocorticoids,e.g. in stress, may have important pathophysiologicalconsequences.
INTRODUCTION Glucocorticoids regulate the expression of various genes in the central nervous system 5'12'29. The translated products of these genes serve neuroendocrine and neuromodulatory functions 12. Glucocorticoid regulation of neuropeptide gene expression may occur indirectly via regulation of other neuronal products which in turn regulate specific neuropeptides 5'15. Alternatively, because some neuropeptide genes, e.g. preproenkephalin (PPE), have a glucocorticoid response element in their regulatory regions 5'16, it has been suggested that glucocorticoids can regulate their expression directly by activating glucocorticoid receptors, which then bind to specific D N A sequences to regulate transcription 3. High levels of P P E m R N A have been localized in the rat forebrain, notably, caudate-putamen, nucleus accumbens, olfactory tubercle, central amygdala and ventrolateral-ventromedial hypothalamus (VMHVL) 13. The PPE gene codes for the opioid
peptides, Met- and Leu-enkephalin 14. The presence of neurons expressing PPE m R N A in regions of the CNS involved in the regulation of sensory, motor, reproductive and limbic activity la suggests that enkephalins may have diverse actions in the CNS. Interestingly, regions in the rat forebrain which express PPE m R N A , also express moderate to high levels of corticosteroid receptors 1,2,13. Two types of corticosteroid receptors have been described in the rat CNS 7 ' s w a type I receptor with a high affinity and low capacity for endogenous glucocorticoids, and an equivalent affinity for mineralocorticoids, and a type II receptor which has high capacity for endogenous glucocorticoids, and high affinity and capacity for synthetic glucocorticoids, e.g. dexamethasone. Glucocorticoids are reportedly required for basal expression of PPE in the striatum, but not in the hippocampus, hypothalamus, ports-medulla and cerebellum 5. Adrenalectomy (ADX) reduces PPE m R N A levels, which can be restored by treatment with corti-
Correspondence: R.E. Harlan, Department of Anatomy,Tulane University Medical School, 1430 Tulane Avenue, New Orleans, LA 70112, USA.
Fax: (1) (504) 584-1687.
l 2()
costerone (CORT) for 5 days 5. In intact rats chronic treatment with C O R T or chronic elevation in plasma glucocorticoid levels following stress, reportedly do not affect PPE m R N A levels in the striatum 5. To address the question as to whether the CORTinduced increases in PPE m R N A levels are peculiar to the caudate putamen, or apply to other regions of the rat forebrain where PPE m R N A is expressed, we examined the effects of chronic C O R T treatment on PPE mRNA in the striatum, amygdala, hypothalamus and hippocampus using dot blot analysis 26, and in the caudate-putamen, nucleus accumbens, central amygdala, and V M H V L using quantitative in situ hybridization
histochemistry 25,26. MATERIALS
AND METHODS
Adrenally-intact and 2-week A D X adult male Sprague-Dawley rats (Charles River) were kept under standard cage conditions and a 12 h light/12 h dark cycle, and allowed free access to chow. Intact rats were allowed free access to tap water and A D X rats to normal saline.
i. Dot blot analysis o f PPE mRNA Two groups of adrenally-intact rats (n = 6) were treated with either daily subcutaneous injections of 10 mg C O R T (Sigma, St. Louis, MO) in a vehicle of sesame oil, or vehicle only for 5 days. T h e following day, the rats were anaesthesized individually with 50 mg pentobarbital intraperitoneally (i.p.) and decapitated. T h e brain from each rat was removed and placed onto a chilled plate over a fluorescent light box. Pieces of tissue corresponding to caudate-putamen, amygdala, mediobasal hypothalamus and hippocampus were dissected out as follows: A coronal cut was made with a razor blade at the caudal end of the optic chiasm. A second coronal cut was m a d e 2 m m caudal to the first cut. With this brain slab rostral side down on the plate, a horizontal cut was made connecting the rhinal fissures of the two hemispheres. Sagittal cuts at the hypothalamic sulci isolated a 2-mm-thick portion of the hypothalamus. On the two lateral pieces, the cortex lateral to the corpus callosum was removed, leaving the amygdala which was combined from the two sides. T h e major remaining piece of brain (caudal to the second cut) was sliced at the interpeduncular fossa and placed caudal side down. A horizontal cut was made through the dorsal corpus callosum, revealing the dorsal hippocampus, which was dissected out visually. The remaining rostral piece of brain (rostral to the first cut) was sliced coronally at the accessory olfactory bulb, about 5 m m rostral to the first coronal cut. This slab was placed rostral surface down and the cortex was removed dorsally and laterally with horizontal and sagittal cuts. Additional sagittal cuts were made at the hypothalamic sulci, and the two lateral pieces were combined. Although this piece was designated caudate-putamen, it also contained the ventral pallidum, olfactory tubercle and part of the nucleus accumbens. Each tissue sample was frozen rapidly on dry ice and stored at - 7 0 ° until R N A extraction was performed. R N A isolation and dot blot analysis of PPE m R N A was carried out as described previously 26. T h e PPE probe, a 435 base pair (bp) cDNA, complimentary to the m R N A encoding amino acids 56-200 of rat PPE, was provided by R. Howells l°'H. The specificity of this probe for P P E m R N A has been described previously 14'2s'26. Statistical comparisons were made with the unpaired Student's t-test. P < 0.05 was considered significant.
ii. In situ hybridization histochemistry Intact rats were divided into two groups (n = 6 per group). Pellets releasing 10 mg C O R T per day (Innovative Research, Toledo, OH), were implanted subcutaneously, under methoxyflurane anesthesia, in
one group for 2 weeks. Control rats received cilolesterol pcllL't:, A D X rats were also divided into two groups (n =.. t~ per group). ()nc group was treated with 1 m g / 1 0 0 g b.wt. C O R T in 2(1(I ,ut 0.005({ e t h a n o l / p h o s p h a t e - b u f f e r e d saline (PBS) intraperitoneally (i.p.) 2 i~ before sacrifice, and the other, vehicle only. tlandling of the l-ms after treatment was minimal. The rats were anaesthesized with 50 mg sodium pentobarbitat i.p., and perfused transcardially with cold PBS for 5 min, and 3
119
© 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00 BRESM 70509
Glucocorticoid regulation of preproenkephalin gene expression in the rat forebrain Rexford S. Ahima a, Meredith M. Garcia a and Richard E. Harlan a,b a Department of Anatomy and b Neuroscience TrainingProgram, Tulane UniversityMedical School, New Orleans, LA 70112 (USA)
(Accepted 23 June 1992)
Key words: Hippocampus;Caudate-putamen; Accumbens;Amygdala;Hypothalamus
The effects of glucocorticoidson the levels of preproenkephalin (PPE) mRNA in the rat forebrain were analyzed with in situ hybridization and dot blots. In adrenally-intact rats, high levels of PPE mRNA, as assessed by in sitn hybridization, were localized in the eaudate-putamen, nucleus accumbens, central amygdala, and ventrolateral ventromedial hypothalamus(VMHVL), and low levels in the hippocampus. After adrenalectomy, the density of PPE mRNA-positive cells and the level of PPE mRNA/cell were decreased in all regions except the hippocampus. Acute treatment with corticosterone (CORT) in adrenalectomized rats increased the level of PPE mRNA/cell in the caudate-putamen and VMHVL. In intact rats, chronic treatment with CORT increased the density of PPE mRNA-positivecells in the caudate-putamen and hippocampus, and the level of PPE mRNA/cell in the caudate-putamen and nucleus accumbens. The effect of chronic CORT treatment on PPE mRNA in the striatum, amygdala, hippocampus and mediobasal hypothalamuswas assessed with dot blots. Chronic CORT treatment increased PPE mRNA levels in the caudate-putamen and hippocampus. There was a good correlation between results on the effect of chronic CORT treatment on PPE mRNA levels in intact rats, obtained from dot blots and in situ hybridization. Results from this study suggest that glucocorticoidsare required for the maintenance of basal PPE mRNA levels in most regions of the rat forebrain. There is, however, considerable regional heterogeneity in the effect of glucocorticoidtreatment on PPE mRNA levels in adrenalectomized and intact rats. Increased PPE mRNA levels in response to high circulating levels of glucocorticoids,e.g. in stress, may have important pathophysiologicalconsequences.
INTRODUCTION Glucocorticoids regulate the expression of various genes in the central nervous system 5'12'29. The translated products of these genes serve neuroendocrine and neuromodulatory functions 12. Glucocorticoid regulation of neuropeptide gene expression may occur indirectly via regulation of other neuronal products which in turn regulate specific neuropeptides 5'15. Alternatively, because some neuropeptide genes, e.g. preproenkephalin (PPE), have a glucocorticoid response element in their regulatory regions 5'16, it has been suggested that glucocorticoids can regulate their expression directly by activating glucocorticoid receptors, which then bind to specific D N A sequences to regulate transcription 3. High levels of P P E m R N A have been localized in the rat forebrain, notably, caudate-putamen, nucleus accumbens, olfactory tubercle, central amygdala and ventrolateral-ventromedial hypothalamus (VMHVL) 13. The PPE gene codes for the opioid
peptides, Met- and Leu-enkephalin 14. The presence of neurons expressing PPE m R N A in regions of the CNS involved in the regulation of sensory, motor, reproductive and limbic activity la suggests that enkephalins may have diverse actions in the CNS. Interestingly, regions in the rat forebrain which express PPE m R N A , also express moderate to high levels of corticosteroid receptors 1,2,13. Two types of corticosteroid receptors have been described in the rat CNS 7 ' s w a type I receptor with a high affinity and low capacity for endogenous glucocorticoids, and an equivalent affinity for mineralocorticoids, and a type II receptor which has high capacity for endogenous glucocorticoids, and high affinity and capacity for synthetic glucocorticoids, e.g. dexamethasone. Glucocorticoids are reportedly required for basal expression of PPE in the striatum, but not in the hippocampus, hypothalamus, ports-medulla and cerebellum 5. Adrenalectomy (ADX) reduces PPE m R N A levels, which can be restored by treatment with corti-
Correspondence: R.E. Harlan, Department of Anatomy,Tulane University Medical School, 1430 Tulane Avenue, New Orleans, LA 70112, USA.
Fax: (1) (504) 584-1687.
l 2()
costerone (CORT) for 5 days 5. In intact rats chronic treatment with C O R T or chronic elevation in plasma glucocorticoid levels following stress, reportedly do not affect PPE m R N A levels in the striatum 5. To address the question as to whether the CORTinduced increases in PPE m R N A levels are peculiar to the caudate putamen, or apply to other regions of the rat forebrain where PPE m R N A is expressed, we examined the effects of chronic C O R T treatment on PPE mRNA in the striatum, amygdala, hypothalamus and hippocampus using dot blot analysis 26, and in the caudate-putamen, nucleus accumbens, central amygdala, and V M H V L using quantitative in situ hybridization
histochemistry 25,26. MATERIALS
AND METHODS
Adrenally-intact and 2-week A D X adult male Sprague-Dawley rats (Charles River) were kept under standard cage conditions and a 12 h light/12 h dark cycle, and allowed free access to chow. Intact rats were allowed free access to tap water and A D X rats to normal saline.
i. Dot blot analysis o f PPE mRNA Two groups of adrenally-intact rats (n = 6) were treated with either daily subcutaneous injections of 10 mg C O R T (Sigma, St. Louis, MO) in a vehicle of sesame oil, or vehicle only for 5 days. T h e following day, the rats were anaesthesized individually with 50 mg pentobarbital intraperitoneally (i.p.) and decapitated. T h e brain from each rat was removed and placed onto a chilled plate over a fluorescent light box. Pieces of tissue corresponding to caudate-putamen, amygdala, mediobasal hypothalamus and hippocampus were dissected out as follows: A coronal cut was made with a razor blade at the caudal end of the optic chiasm. A second coronal cut was m a d e 2 m m caudal to the first cut. With this brain slab rostral side down on the plate, a horizontal cut was made connecting the rhinal fissures of the two hemispheres. Sagittal cuts at the hypothalamic sulci isolated a 2-mm-thick portion of the hypothalamus. On the two lateral pieces, the cortex lateral to the corpus callosum was removed, leaving the amygdala which was combined from the two sides. T h e major remaining piece of brain (caudal to the second cut) was sliced at the interpeduncular fossa and placed caudal side down. A horizontal cut was made through the dorsal corpus callosum, revealing the dorsal hippocampus, which was dissected out visually. The remaining rostral piece of brain (rostral to the first cut) was sliced coronally at the accessory olfactory bulb, about 5 m m rostral to the first coronal cut. This slab was placed rostral surface down and the cortex was removed dorsally and laterally with horizontal and sagittal cuts. Additional sagittal cuts were made at the hypothalamic sulci, and the two lateral pieces were combined. Although this piece was designated caudate-putamen, it also contained the ventral pallidum, olfactory tubercle and part of the nucleus accumbens. Each tissue sample was frozen rapidly on dry ice and stored at - 7 0 ° until R N A extraction was performed. R N A isolation and dot blot analysis of PPE m R N A was carried out as described previously 26. T h e PPE probe, a 435 base pair (bp) cDNA, complimentary to the m R N A encoding amino acids 56-200 of rat PPE, was provided by R. Howells l°'H. The specificity of this probe for P P E m R N A has been described previously 14'2s'26. Statistical comparisons were made with the unpaired Student's t-test. P < 0.05 was considered significant.
ii. In situ hybridization histochemistry Intact rats were divided into two groups (n = 6 per group). Pellets releasing 10 mg C O R T per day (Innovative Research, Toledo, OH), were implanted subcutaneously, under methoxyflurane anesthesia, in
one group for 2 weeks. Control rats received cilolesterol pcllL't:, A D X rats were also divided into two groups (n =.. t~ per group). ()nc group was treated with 1 m g / 1 0 0 g b.wt. C O R T in 2(1(I ,ut 0.005({ e t h a n o l / p h o s p h a t e - b u f f e r e d saline (PBS) intraperitoneally (i.p.) 2 i~ before sacrifice, and the other, vehicle only. tlandling of the l-ms after treatment was minimal. The rats were anaesthesized with 50 mg sodium pentobarbitat i.p., and perfused transcardially with cold PBS for 5 min, and 3
