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Developmental and age-related changes in the D2 dopamine receptor mRNA subtypes in rat brain
Neurochera. Int. Vol, 20, Suppl.,pp. 49S-58S, 1992 Printed in Great Britain.All rightsreserved
0197-0186/92 $5.00+0.00 Copyright© 1992PergamonPressplc
DEVELOPMENTAL A N D AGE-RELATED CHANGES IN THE D2 D O P A M I N E RECEPTOR m R N A SUBTYPES IN RAT BRAIN BENJAMIN WEISS, JANG FAN CHEN, SU1POZHANG and LONG-Wu ZHOU Division of Neuropsychopharmacology,Department of Pharmacology, Medical College of Pennsylvania at Eastern Pennsylvania Psychiatric Institute, 3200 Henry Avenue, Philadelphia, PA, U.S.A.
Abstract--The influence of ontogeny and aging on the D2 dopamine receptor mRNA in rat brain were examined using/n situ hybridization histochemistry and Northern analysis utilizing oligonueleotide probes complementary to the different D2 mRNA subtypes. At birth, there was a high level of D2 dopamine receptor mRNA in corpus striatum relative to that found in the cerebral cortex and other brain areas. The hybridization signal of striatum (using a probe that hybridizes to both the D2Aand D2s mRNA) increased during the first two postnatal weeks, reached a peak at day 16, then declined slightly. The D2A mRNA showed a similar distribution and developmental pattern. Intracisternal injection of 6-hydroxydopamine into neonates did not significantly alter the increase of the D 2 dopamine receptor mRNAs, suggesting that neuronal input does not influence the ontogenetic development of this mRNA. In striatum, olfactory tubercule and inferior colliculus, the D2AmRNA declined between 3 and 24 months of age. By contrast, there was an age-related increase in the D2^ mRNA in the anterior and intermediate lobes of the pituitary. The mRNA for the D2a dopamine receptor showed very low but nevertheless detectable levels in striatum, olfactory tubercule and pituitary. Like with the D~ mRNA, in 24-month-old rats the D2BmRNA declined in striatum and olfactory tubercule and increased in pituitary. These results show that there are differential tissue-related changes in the mRNAs for the D 2 dopamine receptor during both development and aging.
An important challenge in neurobiology is to explain at a biochemical and molecular level the mechanisms by which dopaminergically mediated behaviors are modulated. Earlier studies showed that the behavioral supersensitivity induced in mice by unilaterally lesioning the corpus striatum with 6- hydroxydoparnine could be overcome by continuously infusing the mice with dopaminergic agonists selective for the D~ or D2 dopamine receptor (Winkler et al., 1988; Winkler and Weiss, 1989; Weiss et al., 1990). The behavioral downregulation induced by the D2 dopamine agonist quinpirole was accompanied by a reduction in the level of D2 dopamine receptor and a decrease in the m R N A for the D2 dopamine receptor (Weiss et al., 1990). Since very young and very old animals have a reduction in certain dopaminergic responses (Weiss et al., 1984; Morelli et aL, 1990; Feigenbraum & Yanai, 1984), we have begun a series of studies designed to examine the effect of age (from birth to senility) on the level of the D2 dopamine receptor m R N A in the brain of rodents. The original publication of the e D N A sequence for the D2 dopamine receptor reported by Bunzow et al. (1988) was soon followed by evidence that there was more than one subtype of this receptor, two of which are derived by alternative splicing (Chio et al., 1989; Dal Toso et al., 1989; Giros et al., 1989; Monsma et al.,
1989). Indeed, the more recently described receptor subtype, which has a 29 amino acid insert in the third intracellular loop, the region of the receptor thought to be involved in the binding of G-proteins, appears to be the major form of the D2 dopamine receptor. To study the different subtypes of the D2 dopamine receptor m R N A , we have developed oligonucleotide probes selective for these dopamine receptor mRNAs. Using these probes and utilizing the techniques of in situ hybridization histochemistry and Northern analysis, we have examined the ontogenetie development of these D2 dopamine receptor mRNAs in rat brain, the changes that take place during advanced age, and some of the factors that influence the development of these mRNAs. EXPERIMENTALPROCEDURES
Developments of probes We have developed three oligodeoxynucleotide probes: one is a 39 mer sequence complementary to the region on the mRNA that codes for the second extracelluiarloop (common probe); another is a 39 mcr sequence complementary to the region coding for part of the 29 amino acid insert sequence on the purported G-protein binding site of the receptor (insert probe, specific for one D~ mRNA); and the third is a 43 mer sequence complementary to the region coding for that area that spans the site where the insert sequence wouldbe (bridge probe, specific for the D2B mRNA).
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In situ hybridization histochemistry Animals were quickly decapitated and the brains were removed and immediately frozen on dry ice. Ten micron sections were cut on a cryostat and thaw-mounted onto gelain-coated slides. The slides were kept at 70 °C until hybridization. For hybridization, the sections were warmed to room temperature, fixed for 5 min in buffered 4% paraformaldehyde, and briefly rinsed in 0.1 M phosphate buffered saline and 2 X SSC(1 x SSC = 0.15 M sodium chloride/ 0.025 M sodium citrate). Some sections were pretreated with unlabeled probes or RNase A (Sigma Chemical Corp.) to study the probe specificity. The sections were dehydrated in ethanol and then prehybridized at 32°C for 3 hours in the hybridization buffer, consisting of 4 X SSC, 1X Denhardt's solution, 0.5 mg/ml salmon sperm DNA, 0.25 mg/ml yeast tRNA, 50% formamide, 10% dextran sulfate and 10 mM dithiothreitol. This was followed by an overnight hybridization at 32"C with approximately 0.4 nM[~SS]-labelled probe (about 1.5x106 cpm/300 ,ul/slide) in hybridization buffer. After hybridization, the slides were washed in I X SSC at room temperature for 2 hr and then in decreasing salt concentrations to a final stringency of0.5X SSC at 48°C for 1 hour, each wash containing 14 ,uM 2-mercaptoethanol and I% sodium thiosulphate. The slides were then dehydrated and delipidated, first in ethanol, then xylene, then ethanol again. Washed tissue sections were dried and placed in an X-ray cassette with Kodak X-ray film (XAR-5) placed directly over the mounted sections. The X-ray film was developed after approximately 5 days. Subsequent densitometric analyses of the X-ray film were performed on a Drexel University Image Analysis System (DUMAS). Following exposure to X-ray films, the slides were dipped into photographic emulsion (NTB2, Kodak, diluted 1:1 with distilled water), allowed to dry, and stored in light-tight slides boxes at 4"C. After about 40 days exposure, the photographic emulsion was developed. The slides were then counter-stained with eosin-hematoxylin and examined microscopically for the cellular localization of autoradiographic grains.
Northern blot analysis The total RNA was extracted from rat and mouse striata using either a guanidium thiocyanate/CsC12 gradient as described by Davis et al (1986) or by lithium chloride: chloroform extraction as described by Chromczynski and Sacchi (1987). Poly (A)~RNA was isolated with oligo d(T) cellulose columns as described by Davis et al. (1986). RNA content was quantified by measuring the absorbance at 260 nm or from the fluorescence ofethidium bromide (0.1 ,ug/ml) at an excitation wavelength of 250 nm and an emission wavelength 600 nm. Samples of total RNA (6ug) were subjected to electrophoresis on a 1% agarose gel containing 0.66 M formaldehyde. The RNA was then transferred onto nitricellulose membranes by capillary transfer overnight in 10x SCC. The blots were immobilized and prehybridized for 2 hr at room temperature in 4X SCC, 10x Denhardt's solution and 100/tg/ ml salmon sperm DNA, and then hybridized at 65°C overnight with [32P]-dATP-labeled oligonucleotide probe (I,5 XI06 cpm/ng probe; 0.13 nm) in the same buffer. The blots were washed three times at 55"C and once at 65"C (30 min. each) with a solution of 2X SCC and exposed to X-ray film with an intensifying screen at 70°C.
RESULTS AND DIS(T[ SSION
Spec(ficity of probes Figure 1 shows a N o r t h e r n blot o f the D2 m R N A using the three oligonucleotide probes. As can be seen, in each case there was a single b a n d at 2.9 kb c o r r e s p o n d i n g to the molecular weight o f the D, d o p a m i n e receptor m R N A (no measurable differences were seen a m o n g the 3 probes for the two different m R N A s ; this is not surprising since the extra 87 base sequence accounts for too small a portion of the total m R N A sequence to be differentiated). The figure also shows t h a t the radiolabeled insert p r o b e was displaced by a d d i n g 100 fold excess o f the unlabeled insert probe but not by excess bridge probe. Similarly, the radiolabeled bridge p r o b e was displaced by adding 100 fold excess o f the unlabeled bridge probe but not by the insert probe.
Distribution olD: dopamine receptor mRNAs In situ hybridization studies of the distribution of the d o p a m i n e receptor m R N A in m o u s e brain using the c o m m o n p r o b e (Weiss et aL, 1990) yielded results similar to those found by others in studies of the rat. The highest levels of the D2 d o p a m i n e receptor m R N A in mouse brain were in regions c o n t a i n i n g the cells bodies a n d their projection fields of the nigrostriatal, mesolimbic a n d mesocortical dopaminergic systems, with particularly high levels in the corpus striatum a n d olfactory tubercule. Studies o f other brain sections showed high levels in substantia nigra pars compacta, ventral tegmental area, a n d anterior and intermediate lobes of the pituitary gland, and lower levels in the cerebral cortex, dentate gyrus a n d pyramidal cell layer of the h i p p o c a m p u s , certain thalamic nuclei, inferior colliculus, a n d medulla. Little or no signal was seen in the s u b s t a n t i a nigra pars reticulata, cerebellum, or posterior lobe of pituitary. The small but nevertheless detectable levels seen in cerebral cortex a n d h i p p o c a m p u s using in situ hybridization histochemistry was confirmed by N o r t h e r n analysis o f p o l y (A) + m R N A isolated from brain areas by oligo d (T) columns. Large quantities of D2 m R N A were found in striatum, and small but significant quantities in h i p p o c a m p u s and cerebral cortex. Surprisingly, relatively high c o n c e n t r a t i o n s were also found in spinal cord. N o detectable levels, however, were seen in cerebellum or liver (Chen, Qin, Szele; Bai and Weiss, u n p u b l i s h e d observations). Cellular analysis olD: doparnine receptor mRNA A cellular analysis o f the D2 d o p a m i n e receptor m R N A in m o u s e corpus striatum or s u b s t a n t i a nigra
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Fig. I. Northern analysis ofdopamine D2receptor mRNA in mouse brain. Total cellular RNA was extracted from mouse striatum using the method of Chromczynski and Sacchi (1987). Poly (A) + RNA was isolated with oligo d(T) cellulose columns as described by Davis et al. (1986). The yield of RNA was about 2.7% of total RNA. For Northern blots, 3/zg of poly (A)* RNA was fractioned on 1% agarose gel containing 2.2 M formaldehyde and blotted onto nitrocellulose membranes. The mRNA was hybridized with labeled probe in the absence or presence of 100 fold excess unlabeiled oligonucleotide probe (displacer) as described in the section on Experimental Procedures. The autoradiography was carried out after exposing the X-ray film to the hybrid at-70°C for 17 days. The figure demonstrates specificdisplacement of the hybridization signal of D2 dopamine receptor mRNA by the corresponding insert or bridge probe. C = common probe, I = insert probe, B = bridge probe. pars compacta showed that the signal was associated with cell bodies; there were relatively few grains evident in the cellular matrix (Weiss et al. 1990). The cellular location of the m R N A was examined further using selective lesions induced with 6hydroxydopamine. Rats were unilaterally lesioned in the corpus striatum or substantia nigra with 6hydroxydopamine, and at various times after these lesions in situ hybridization studies were performed using the 02 m R N A common probe. The results of the studies showed that lesioning the substantia nigra greatly reduced the signal in the nigra but failed to substantially alter the signal in the striatum (Figure 2). Similarly, lesioning the striatum had little effect on the hybridization signal in striatum but greatly reduced the signal in the nigra and ventral tegmental area on the lesioned side (not shown). Taken together, these results suggest: (1) that the D 2 dopamine receptor m R N A in substantia nigra pars compacta is localized largely in dopaminergic cell bodies whose terminal projections lie in the striatum, and that this m R N A might code for D2 autoreceptors; (2) that the D2 dopamine receptor m R N A of striatum is in non-dopaminergic cell bodies intrinsic to the striatum and probably codes for post-synaptic D2 dopamine receptors.
Relationship between alterations of D: mRNA and behavior To determine the possible relationship between the loss of dopamine cell bodies in substantia nigra, with dopaminergic behavioral supersensitivity, we
administered 6-hydroxydopamine unilaterally into the striatum of mice. We then determined their rotational response to apomorphine and the levels of D2 dopamine receptor m R N A in substantia nigra. As can be seen (Figure 3), there was a significant correlation between these two events. Animals that evidenced the greatest rotational activity showed the greatest reduction in the levels of D2 receptor mRNA. These results show that the reduction of cell bodies containing D 2 receptor m R N A in the substantia nigra correlates well with the development of dopaminergically mediated behavioral supersensitivity.
Ontogenesis olD: dopamine receptor mRNA In studing the ontogenetic development of the D2 dopamine receptor m R N A in brain using in situ hybridization histocbemistry, it is important to note that the levels of D E m R N A are relatively low, particularly in newborn rats, and that there was significant non-specific binding of the probe. Therefore, all the studies were performed in the absence and presence of excess unlabeled oligonucleotide probe to displace the radiolabeled probe from specific binding sites on the D2 mRNA. For example, adding 100 fold excess D2 probe to brain sections of 2-day-old rats abolished the signal in olfactory bulb and striatum but had little influence on the signal in cortex or cerebellum. Similar results were found when adult brain was studied. Using greater stringency conditions, such as higher posthybridization wash temperatures, also reduced the non-specific hybridization.
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Fig. 2. Effect of 6-hydroxydopamine lesions on D~ dopamine receptor mRNA in corpus striatum and substantia nigra.Mice were unilaterally lesioned in the substantia nigra with microinjections of 6hydroxydopamine. The animals were killed 6 days after lesioning,the brains were removed and immediately frozen on dry ice. Ten micron sections were subjected to in situ hybridization histochemistry as described in Experimental Procedures• The results showed that lesioning the substantia nigra almost completely abolished the hybridization signal in substantia nigra pars compacta ipsilateral to the lesion, but had no significant effect on the hybridization signal in the striatum.
Figure 4 shows the ontogenetic development of the D 2 m R N A in striatum of rats 1 to 32 days of age. In these experiments, in situ hybridization studies were performed with the D2 m R N A c o m m o n probe and the results quantified with an image analyzer• As can be see, the D 2 m R N A increased soon after birth, reached a maximum at 16 days, then declined slightly. Studies
of the D2Am R N A using the insert probe showed an identical distribution and developmental pattern (not shown). The time course for the expression of the D2 dopamine receptor m R N A correlates well with the ontogenetic development of the D2 receptor (Feigenbraum & Yanai, 1984; Sales e t al., 1989; Gelbard et al., 1989).
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D, dopamine receptor mRNA in substantia nigra with rotational response to apomorphine. Mice were unilaterally lesioned in the substantia nigra with microinjections of 6hydroxydopamine. After one week the mice were challenged with apomorphine and rotational behavior was determined. The animals were then killed and the D2 mRNA was measured by/n situ hybridization histochemistry. The results show a positive correlation between the decrease of D2 mRNA in substantia nigra with the increased rotational response to apomorphine. 0.06
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striatum, mice were administered 6-hydroxydopamine intracerebroventriculady at 2 and 6 days of age. Animals were then killed at various times and the D2 m R N A determined by in situ hybridization histochemistry and by Northern analysis. The results showed that the administration of 6-hydroxydopamine failed to alter the development of the D2 mRNA, suggesting that this m R N A and, therefore, presumably the D2 dopamine receptor, can develop in the absence of dopaminergic input. Similar results were noted earlier for the beta adrenergic receptor where sympathetically denervating the pineal gland from birth still allowed the development of the beta adrenergic receptor (Weiss et al., 1984). In contrast with the results with the m R N A for the D: dopamine receptor, 6-hydroxydopamine treatment to newborn rats caused an increase in the enkephalin m R N A in striatum (not shown). These results on the development of the D: m R N A are consistent with those of Duncan et al. (1987) who showed that treating neonates with 6-hydroxydopamine produced little changes in D2 receptors of adult striatum. By contrast, Broaddus and Bennett (1990) reported about a 50% reduction in D 2receptors in caudate of adult rats following 6-hydroxydopamine treatment of neonates. Other investigators (Kostrzewa and Seleh 1989) reported that treatment of newborn rats with spiroperidol impaired the development of D 2 receptors.
Cellular analysis of ontogenetic development of D: dopamine receptor mRNA
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Fig. 4. Ontogenetic development of 02 dopamine receptor mRNA in rat striatum. Brains from rats at various postnatal ages were removed and parasagital sections at the level of the corpus striatum were prepared for /n situ hybrLdiTation histochemistry using the [35S]-labelledD~dopamine common probe. After high stringency post-hybridization washings, the hybridization signals were detected by film autoradiography and analyzed with the DUMAS image analysis system. To examine the effect of dopaminergic neuronal input on the development of the D2 m R N A in
Figure 5 shows the cellular distribution of the D2 dopamine receptor m R N A in rat striatum during postnatal development. As may be seen, at postnatal day 2 (P2), the hybridization signal for the D2 m R N A was scattered throughout the field, with only relatively few cellular elements labeled; those that were labeled were of relatively low density. By P32 only a certain subpopulations of cells were labeled, and these cells were considerabily more heavily labeled. These results showing that the increase in D2 m R N A during ontogeny is caused by a selectiveincrease in the density of specific types of cells in the striatum suggests that the development expression of the D2 dopamine receptor m R N A in certain striatal neurons may be associated with the maturation and differentiation of these neurons.
Effects of advancedage on D2 dopamine receptor mRNA During aging, there is a reduced level of D:
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P32 Fig. 5. Cellular analysis of D2 dopamine receptor mRNA in rat striatum during postnatal development. Brains from 2- and 32-day-old rats were removed and prepared as described in the legend to figure 4. Following in situ hybridization, the brain sections were kipped in Kodak NTB-2 emulsion, developed after 25 days of exposure and counterstained with hemotoxylin-eosin. The figure showed high power (1250X) bright field photomicroscopy of the hybridization signal for D 2dopamine receptor mRNA.
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Fig. 6. Northern blot analysis of D 2 dopamine receptor mRNA in the brains of young and old rats (insert probe). Brains of 3- and 24-month-old Fisher-344 rats were dissected, and total RNA was extracted. The RNA was subjected to Northern blot analysis using the D2 dopamine receptor insert probe. IC --~ inferior colliculus; OT = olfactory tubercle; ST = corpus striatum; PIT = pituitary gland.
dopamine receptors in various brain regions of the rat (Weiss et al., 1984; Severson and Randall, 1985; Morelli et al., 1990). To determine whether or not this decrease in receptors might be caused by a reduction in their synthesis, we have studied the effects of advanced age on the D2 dopamine receptor mRNA in several brain regions. In these studies, we have compared the levels of the D2 m R N A in 3- and 24 month-old rats by Northern blot analysis using both the insert and bridge probes. As can be seen (Figure 6), using the insert probe, 24-month-old rats had a significantly lower level of D2 dopamine receptor mRNA in corpus striatum, olfactory tubercle and inferior colliculus. By contrast, in pituitary the levels of D2 dopamine receptor mRNA was higher in 24-month-old rats than in 3-month-old rats. These results of Northern blot analysis in several brain areas of 3- and 24-month-old rats using the 'insert D2 mRNA probe are quantified and summarized in Table 1. The data show that both in 3- and 24-month-old rats, striatum has more than two times the level of D2 dopamine receptor mRNA than olfactory tubercle or pituitary and about 10 to 20 times more than the inferior and superior colliculi. Several other tissues showed non-detectable levels using these analyses. Tissues from aged rats had lower levels of the D2 mRNA in all brain areas examined, with the notable exception of pituitary gland. In striatum and olfactory tubercule, there was about 50% reduction in 24-month-old rats, in inferior colliculus there was about a 34% reduction, and in the superior colliculus the m R N A was the insert probe, the use of the bridge probe indicated that 24-
Table 1. Distributio~of D2mRNA(Insert)in 3-month-oldand 24month-oldrat brain D2mRNA(fmol/Gmtotal RNA) Area Striatum Olfactory Pituitary Inferiorcolliculus Superiorcolliculus Cerebellum Cortex Hippocampus Hypothalamus Olfactorybulb Spinalcord Thalamus
3 Month
24 Month
430 150 140 32 21 ND ND ND ND ND ND ND
220 73 310 21 ND ND ND ND ND ND ND ND
Brains from 3- and 24-month-oldrats were dissected, the RNA extractedand the I)2mRNAanalyzedbyNorthernanalysisusing the D2mRNAinsertprobe.The autoradiogramswerequantified usinga DUMASimaseanalyzer. ND = not detectable.
month-old rats had significantly less of the d2 bridge m R N A in striatum and olfactory tubercle, and more of this subtype of the D, mRNA in pituitary. A calculation of the ratio of the two subtypes of D2 mRNA shows thatthere was from 6 to 9 times more signal using the Dz m R N A insert probe than the D2 mRNA bridge probe in all tissues regardless of the age of the animal. Further examination of the effect of age on the D2 mRNA in pituitary using in situ hybridization histochemistry confirmed that there was a significantly higher density of the D2 mRNA signal in the anterior
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Table 2. Distribution of D2mRNA (Bridge) in 3-month-oldand 24month-old rat brain D:, mRNA (fmol;Gm total RNA)
Area Striatum Olfactory Pituitary Inferior colliculus Superior colliculus Cerebellum Cortex Hippocampus Hypothalamus Olfactory bulb Spinal cord Thalamus
3 Month
24 Month
63 28 16 ND ND ND ND ND ND ND ND ND
27 S 50 ND ND ND ND ND ND ND ND ND
Brains from 3- and 24-month-old rats were dissected, the RNA extracted and the D~mRNA analyzed by Northern analysisusing the D2mRNA bridgeprobe. The autoradiogramswerequantified using a DUMAS image analyzer. ND = not detectable.
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and intermediate lobes o f pituitary glands irom 24m o n t h - o l d rats than in 3-month-old animals (I igurcs 7 and 8). N o significant differences were seen in the posterior lobe o f the pituitary which had a very weak signal in b o t h 3-month-old and in 24-month-okl rats. A cellular analysis o f these in situ hybridization studies revealed that the higher signal in the anterior and intermediate lobes o f the pituitary in 24-month-old rats was attributed not only to the larger size o f the gland but also to a greater signal per cell. This latter result may be due, in part, to the cellular hypertrophy which was evident in the anterior and intermediate lobes o f the pituitary o f 24-month-old rats. Work from a n u m b e r o f laboratories indicates that there is a reduction o f b o t h D~ and D2 receptors in aged rat brain. Studies by N o r m a n et al. (1987) have shown that in aged rats both the level as well as the turnover o f the D 2 d o p a m i n e receptors are reduced in striatum. Other studies showed not only that there was a reduction in spiroperidol binding in the striatum and olfactory tubercle, but that there was an increase in the pituitary gland (Govoni et al., 1980; Arita et al. 1984). These results correlate very well with our studies o f the relative age-related changes in the D2 d o p a m i n e receptor m R N A in the various brain regions. Earlier work from our laboratory showed that the ability o f beta adrenergic receptors to adapt to neuronal, h o r m o n a l or environmental stimuli is impaired in brain o f aged rats (Greenberg & Weiss, 1978; Weiss et al., 1979; Greenberg & Weiss, 1979). M o r e recent studies showed the ability ofD~ d o p a m i n e receptors to respond to light is also reduced in the
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24 ~ T H Fig. 7. Effect of age on D2 dopamine receptor mRNA in rat pituitary using in situ hybridization histochemistry. Pituitary glans from 3- and 24-month-old rats were removed, 10 p M sections prepared on a cryostat, and fixed sections analyzed for the D2 dopamine receptor mRNA by in situ hybridization using the [3sS]-D2dopamine receptor mRNA common probe. AL = anterior lobe; IL = intermediate lobe; PL = posterior lobe.
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Fig. 8. Comparison of the D 2 receptor mRNA in different lobes of the pituitary gland of 3- and 24-month-old rats. Pituitary glands were prepared as described in the legend to Figure 7. The signals on the autoradiograms were quantitated using the DUMAS image analyzer. * P < 0.05 compared with the corresponding area of 3-month-old rat pituitary. ** P < 0.01 compared with the corresponding area of 3-month-old rat pituitary.
Dopamine 90 retina of senescent rats (Porceddu et al., 1990). Still other studies showed the rate of synthesis o f receptor for a number of neurotransmitters, including the beta adrenergic receptors (Greenberg et al., 1985), the alpha 1 and alpha 2 adrenergic receptors (Zhou et al., 1986), and the muscarinic acetylcholine receptors (Pintor et al., 1990) are also reduced in aging. These results suggest that a reduced synthesis of receptors for neurotransmitters might represent a general defect associated with the aging process. Whether alterations in specific dopaminergic behaviors are explainable by changes in the D2 dopamine receptor m R N A is still an open question. Repeated administration of dopaminergic agents have thus far yelded inconsistent results. Thus, continuous administration of the D2 agonist quinpirole reduced the D2 dopamine receptor m R N A in mouse striatum (Weiss et al., 1990), and repeated treatment of rats with the dopamine antagonist haloperidol increased the D2 receptor m R N A in the intermediate lobe of the pituitary (Auteliano et al., 1989). By contrast, others (van Tol et al., 1990) have found no significant change in the level of D2 receptor m R N A in rat striatum following chronic treatment with a number of antipsychotic drugs. More detailed studies, particularly on the time course of drug treatment, may help resolve these issues. Finally, there appears to be a correlation between the induction of dopaminergically mediated supersensitive behavior and the decrease in dopamine cell bodies in the substantia nigra as measured by the reduction in D2 m R N A in this area. However, it is still unclear what specific alterations of receptors or postreceptors events occur in the striatum that explain these changes in dopaminergic behavior. In this regard, recent studies from our laboratory show that following repeated treatment with the D2 dopamine agonist quinpirole there is not only a reduction in D2 dopamine receptors in striatum but also a reduction in the activity of the calmodulin-sensitive protein kinase (Zhou, Zhang and Weiss, unpublished). Surely there are many factors involved in the modulation of dopaminergic responses. Acknowledgement--Supported by Grant MH 42148 awarded by the National Institutes of Mental Health. REFERENCES
Arita, J., Reymond, M.J. and Porter, J.C. (1984) Evidence for alteration in the processing of dopamine in the anterior pitutitary gland of aged rats: Receptors and intracellular compartmentalization of dopamine. Endocrinology 114, 974-979.
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Auteliano, D.J., Snyder, L., Sealfon, S.C. and Roberts, J.L. (1989) Dopamine D2 receptor messenger RNA is differentially regulated by dopaminer~ agents in rat anterior and neurointermediate pituitary. Mol. Cell Endocrinol. 67, 101-105. Broaddus, W.C. and Bennet, P.J. (1990) Postnatal development of striatal dopamine function. II. Effects of neonatal 6-hydroxydopamine treatment on Dt and D2 receptors, adenylate cyclase activity and presynaptic dopamine function. Develop. Brain Res. 52, 273-277. Bunzow, J.R., Van Tol, H.H.M., Grandy, D.K., Albert, P., Salon, J., Christie, M., Machida, C.A., Neve, K.A. and Civelli, O. (1988) Cloning and expression of a rat D2 dopamine receptor cDNA. Nature 336, 783-787. Chio, C.L., Hess, G.F., Graham, R.S. and Huff, R.M. (1989) A second molecular form of D2 dopamine receptor in rat and bovine caudate nucleus. Nature 343, 266-269. Chromczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phenolchloroform extraction. Annal. Biochem. 162, 156-159. Dal Toso R., Sommer B., Ewert M., Herb A., Pritchett D.B., Bach A., Shivers B.D. and Secburg P.H. (1989) The dopamine D2 receptor: Two molecular forms generated by alternative splicing. EMBO J. 8, 4025--4043. Davis L.G., Dibner M.D. and Battery J.F. (1986) Basic Methods of Molecular Biology, pp. 73-135. Elsevier, New York. Duncan G.E., Criswell H.E., Mc Cown T.J., Paul I.A., Mueller R.A. and Breese G.R. (1987) Behavioral and neurocbemical responses to haloperidol and SCH-23390 in rats treated neonatally or as adults with 6hydroxydopamine. J.Pharmacol. Exptl. Therap. 243, 1027-1043. Feigenbraum J.J. and Yanai J. (1984) Normal and abnormal determinants of dopamine receptor ontogeny in the central nervous system. Prog. Neurobiol. 23, 191-225. Gelbard H.A., Teicher M.H., Faedda G. and Baldessarini R.J. (1989) Postnatal development of dopamine D~ and D2 receptor sites in rat striatum. Brain Res. 49, 123-130. Giros, B., Sokoloff, P., Martres, M.P., Riou, J.F., Emorine, L.J. and Schwartz, J.C. (1989) Alternative splicing directs the expression of two D2 dopamine receptor isoform. Nature 342, 923-926. Govoni, S., Memo, M., Saiani, L., Spano, P.F. and Trabucchi, M. (1980) Impairment of brain neurotransmitter receptors in aged rats. Mech. Age. Develop. 12, 39-46. Greenberg, L.H. and Weiss, B. (1978) Beta adrenergic receptors in aged brain: reduced number and capacity of pineal gland to develop supersensitivity. Science 201, 6163. Greenberg, L.H. and Weiss, B. (1979) Ability of aged rats to alter beta adrenergic receptors of brain in response to repeated administration of reserpine and desmethylimipramine. J. Pliarmacol. Exptl. Therap. 211, 309-316. Greenberg, L.H., Brunswick, D.J. and Weiss, B. (1985) Effect of age on the rate of recovery of beta-adrenergic receptors in rat brain following desmethylimipramine-induced subsensitivity. Brain Res. 328, 81-88. Kostrzewa, R.M. and Saleh, M.I. (1989) Impaired ontogeny of striatal dopamine D~ and D 2 binding sites after postnatal treatment of rats with SCH-23390 and spiroperidol. Develop. Brain Res. 45, 95-101.
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Monsma, F.J. Jr, MeVittie, L.D., Gerfen, C.R., Mahan, L.C. and Sibley, D.R. (1989) Multiple D2 dopamine receptors produced by alternative splicing. Nature 342,926-929. Morelli, M., Mennini, T., Cagnotto+ A., Toffano, G. and DiChiara, G. (1990) Quantitative autoradiographical analysis of the age-related modulation of central dopamine D, and D 2 receptors. Neuroscience 36, 403-410. Norman, S.A.B., Battaglia, G. and Creese, I. (1987) Differential recovery rates of rat D~ dopamine receptors as a function of aging and chronic reserpine treatment following irreversible modification: A key to receptor regulatory mechanisms. J. Neurosci. 7, 1484-1491+ Pintor, A., Fortuna, S., De Angelis, S. and Michalek, H. (1990) Impaired recovery of brain muscarinic receptor sites following an adaptive down-regulation induced by repeated administration of diisopropylfluorophosphate in aged rats. Life Sci. 46, 1027-1036. Porceddu, M.L., De Montis, G., Pepitoni, S., Toffano, G. and Biggio, G (1990) Failure of dark adaptation to upregulate D, dopamine receptors in retina of senescent rats. Neurobiol. Aging 11, 105-109. Sales, N., Martres, M.P., Bouthenet, M.L. and Schwartz, J.C. (1989) Ontogeny of dopaminergic D z receptors in rat nervous system: Characterization and detailed autoradiographic mapping with ['2~l]iodosulpiride. Neuroscience 28, 673-700. Severson, J.A. and Randall, P.K. (1985) D z dopamine receptors in aging mouse striatum: Determination of high- and low-affinity agonist binding sites. J. Pharmacol. Exptl. Therap. 233,361-368. Van Tol, H.H.M., Riva, M., Civelli, O. and Creese, I. (1990) Lack of effect of chronic dopamine receptor blockade on
D 2 dopamine receptor mRNA level. Neuroscl Left. l l~ 303-308. Weiss, B., Greenberg, L. and Cantor, E. (1979) Age-rclated alterations in the development of adrenergic denervation suoersensitivity. Fed. Proc. 38, 19t5 1921. Weiss, B., Clark, M.B. and Greenberg, L.H. (1984) Modulation of cathecolaminergic receptors during development and aging. In: Handbook of Neurochemistry (Lajtha A., ed.), pp. 595-627. Plenum Publishing Corporation. Weiss, B., Cimino, M., Winkter, J.D. and Chen, J.F. (1989) Behavioral, biochemical and molecular biological correlates of modulating dopaminergic responses. In: Neurochemical Pharmacology- A Tribute to B.B. Brodie (Costa E., ed), pp. 149-164. Raven Press, New York. Weiss, B., Zhou, L.W., Chen, J.F., Szele, F. and Bai, G+ (1990) Distribution and modulation of the D, dopamine receptor mRNA in mouse brain: Molecular and behavioral correlates. Adv. Biosci. 77, 4- 26. Winkler, J.D. and Weiss, B. (1989) Effect ot+ continuous exposure to selective D~ and D 2dopaminergic agonists on rotational behavior in supersensitive mice. J.Pharmacol+ Exptl. Therap. 249, 507-516. Winkler, J.D., Callison, K., Cass, S.A. and Weiss, B. (1988) Selective down-regulation of D, dopamine mediated rotational behavior in supersensitive mice. Neuropharmacology 27,439-442. Zhou, L.W., Freilich, J.S., Weiss, B. and Greenberg, L.tt. (1984) Impaired recovery of alpha 1 and alpha 2 adrenergic receptors in brain of aged rats. J. Gerontol. 39,538 546.
0197-0186/92 $5.00+0.00 Copyright© 1992PergamonPressplc
DEVELOPMENTAL A N D AGE-RELATED CHANGES IN THE D2 D O P A M I N E RECEPTOR m R N A SUBTYPES IN RAT BRAIN BENJAMIN WEISS, JANG FAN CHEN, SU1POZHANG and LONG-Wu ZHOU Division of Neuropsychopharmacology,Department of Pharmacology, Medical College of Pennsylvania at Eastern Pennsylvania Psychiatric Institute, 3200 Henry Avenue, Philadelphia, PA, U.S.A.
Abstract--The influence of ontogeny and aging on the D2 dopamine receptor mRNA in rat brain were examined using/n situ hybridization histochemistry and Northern analysis utilizing oligonueleotide probes complementary to the different D2 mRNA subtypes. At birth, there was a high level of D2 dopamine receptor mRNA in corpus striatum relative to that found in the cerebral cortex and other brain areas. The hybridization signal of striatum (using a probe that hybridizes to both the D2Aand D2s mRNA) increased during the first two postnatal weeks, reached a peak at day 16, then declined slightly. The D2A mRNA showed a similar distribution and developmental pattern. Intracisternal injection of 6-hydroxydopamine into neonates did not significantly alter the increase of the D 2 dopamine receptor mRNAs, suggesting that neuronal input does not influence the ontogenetic development of this mRNA. In striatum, olfactory tubercule and inferior colliculus, the D2AmRNA declined between 3 and 24 months of age. By contrast, there was an age-related increase in the D2^ mRNA in the anterior and intermediate lobes of the pituitary. The mRNA for the D2a dopamine receptor showed very low but nevertheless detectable levels in striatum, olfactory tubercule and pituitary. Like with the D~ mRNA, in 24-month-old rats the D2BmRNA declined in striatum and olfactory tubercule and increased in pituitary. These results show that there are differential tissue-related changes in the mRNAs for the D 2 dopamine receptor during both development and aging.
An important challenge in neurobiology is to explain at a biochemical and molecular level the mechanisms by which dopaminergically mediated behaviors are modulated. Earlier studies showed that the behavioral supersensitivity induced in mice by unilaterally lesioning the corpus striatum with 6- hydroxydoparnine could be overcome by continuously infusing the mice with dopaminergic agonists selective for the D~ or D2 dopamine receptor (Winkler et al., 1988; Winkler and Weiss, 1989; Weiss et al., 1990). The behavioral downregulation induced by the D2 dopamine agonist quinpirole was accompanied by a reduction in the level of D2 dopamine receptor and a decrease in the m R N A for the D2 dopamine receptor (Weiss et al., 1990). Since very young and very old animals have a reduction in certain dopaminergic responses (Weiss et al., 1984; Morelli et aL, 1990; Feigenbraum & Yanai, 1984), we have begun a series of studies designed to examine the effect of age (from birth to senility) on the level of the D2 dopamine receptor m R N A in the brain of rodents. The original publication of the e D N A sequence for the D2 dopamine receptor reported by Bunzow et al. (1988) was soon followed by evidence that there was more than one subtype of this receptor, two of which are derived by alternative splicing (Chio et al., 1989; Dal Toso et al., 1989; Giros et al., 1989; Monsma et al.,
1989). Indeed, the more recently described receptor subtype, which has a 29 amino acid insert in the third intracellular loop, the region of the receptor thought to be involved in the binding of G-proteins, appears to be the major form of the D2 dopamine receptor. To study the different subtypes of the D2 dopamine receptor m R N A , we have developed oligonucleotide probes selective for these dopamine receptor mRNAs. Using these probes and utilizing the techniques of in situ hybridization histochemistry and Northern analysis, we have examined the ontogenetie development of these D2 dopamine receptor mRNAs in rat brain, the changes that take place during advanced age, and some of the factors that influence the development of these mRNAs. EXPERIMENTALPROCEDURES
Developments of probes We have developed three oligodeoxynucleotide probes: one is a 39 mer sequence complementary to the region on the mRNA that codes for the second extracelluiarloop (common probe); another is a 39 mcr sequence complementary to the region coding for part of the 29 amino acid insert sequence on the purported G-protein binding site of the receptor (insert probe, specific for one D~ mRNA); and the third is a 43 mer sequence complementary to the region coding for that area that spans the site where the insert sequence wouldbe (bridge probe, specific for the D2B mRNA).
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In situ hybridization histochemistry Animals were quickly decapitated and the brains were removed and immediately frozen on dry ice. Ten micron sections were cut on a cryostat and thaw-mounted onto gelain-coated slides. The slides were kept at 70 °C until hybridization. For hybridization, the sections were warmed to room temperature, fixed for 5 min in buffered 4% paraformaldehyde, and briefly rinsed in 0.1 M phosphate buffered saline and 2 X SSC(1 x SSC = 0.15 M sodium chloride/ 0.025 M sodium citrate). Some sections were pretreated with unlabeled probes or RNase A (Sigma Chemical Corp.) to study the probe specificity. The sections were dehydrated in ethanol and then prehybridized at 32°C for 3 hours in the hybridization buffer, consisting of 4 X SSC, 1X Denhardt's solution, 0.5 mg/ml salmon sperm DNA, 0.25 mg/ml yeast tRNA, 50% formamide, 10% dextran sulfate and 10 mM dithiothreitol. This was followed by an overnight hybridization at 32"C with approximately 0.4 nM[~SS]-labelled probe (about 1.5x106 cpm/300 ,ul/slide) in hybridization buffer. After hybridization, the slides were washed in I X SSC at room temperature for 2 hr and then in decreasing salt concentrations to a final stringency of0.5X SSC at 48°C for 1 hour, each wash containing 14 ,uM 2-mercaptoethanol and I% sodium thiosulphate. The slides were then dehydrated and delipidated, first in ethanol, then xylene, then ethanol again. Washed tissue sections were dried and placed in an X-ray cassette with Kodak X-ray film (XAR-5) placed directly over the mounted sections. The X-ray film was developed after approximately 5 days. Subsequent densitometric analyses of the X-ray film were performed on a Drexel University Image Analysis System (DUMAS). Following exposure to X-ray films, the slides were dipped into photographic emulsion (NTB2, Kodak, diluted 1:1 with distilled water), allowed to dry, and stored in light-tight slides boxes at 4"C. After about 40 days exposure, the photographic emulsion was developed. The slides were then counter-stained with eosin-hematoxylin and examined microscopically for the cellular localization of autoradiographic grains.
Northern blot analysis The total RNA was extracted from rat and mouse striata using either a guanidium thiocyanate/CsC12 gradient as described by Davis et al (1986) or by lithium chloride: chloroform extraction as described by Chromczynski and Sacchi (1987). Poly (A)~RNA was isolated with oligo d(T) cellulose columns as described by Davis et al. (1986). RNA content was quantified by measuring the absorbance at 260 nm or from the fluorescence ofethidium bromide (0.1 ,ug/ml) at an excitation wavelength of 250 nm and an emission wavelength 600 nm. Samples of total RNA (6ug) were subjected to electrophoresis on a 1% agarose gel containing 0.66 M formaldehyde. The RNA was then transferred onto nitricellulose membranes by capillary transfer overnight in 10x SCC. The blots were immobilized and prehybridized for 2 hr at room temperature in 4X SCC, 10x Denhardt's solution and 100/tg/ ml salmon sperm DNA, and then hybridized at 65°C overnight with [32P]-dATP-labeled oligonucleotide probe (I,5 XI06 cpm/ng probe; 0.13 nm) in the same buffer. The blots were washed three times at 55"C and once at 65"C (30 min. each) with a solution of 2X SCC and exposed to X-ray film with an intensifying screen at 70°C.
RESULTS AND DIS(T[ SSION
Spec(ficity of probes Figure 1 shows a N o r t h e r n blot o f the D2 m R N A using the three oligonucleotide probes. As can be seen, in each case there was a single b a n d at 2.9 kb c o r r e s p o n d i n g to the molecular weight o f the D, d o p a m i n e receptor m R N A (no measurable differences were seen a m o n g the 3 probes for the two different m R N A s ; this is not surprising since the extra 87 base sequence accounts for too small a portion of the total m R N A sequence to be differentiated). The figure also shows t h a t the radiolabeled insert p r o b e was displaced by a d d i n g 100 fold excess o f the unlabeled insert probe but not by excess bridge probe. Similarly, the radiolabeled bridge p r o b e was displaced by adding 100 fold excess o f the unlabeled bridge probe but not by the insert probe.
Distribution olD: dopamine receptor mRNAs In situ hybridization studies of the distribution of the d o p a m i n e receptor m R N A in m o u s e brain using the c o m m o n p r o b e (Weiss et aL, 1990) yielded results similar to those found by others in studies of the rat. The highest levels of the D2 d o p a m i n e receptor m R N A in mouse brain were in regions c o n t a i n i n g the cells bodies a n d their projection fields of the nigrostriatal, mesolimbic a n d mesocortical dopaminergic systems, with particularly high levels in the corpus striatum a n d olfactory tubercule. Studies o f other brain sections showed high levels in substantia nigra pars compacta, ventral tegmental area, a n d anterior and intermediate lobes of the pituitary gland, and lower levels in the cerebral cortex, dentate gyrus a n d pyramidal cell layer of the h i p p o c a m p u s , certain thalamic nuclei, inferior colliculus, a n d medulla. Little or no signal was seen in the s u b s t a n t i a nigra pars reticulata, cerebellum, or posterior lobe of pituitary. The small but nevertheless detectable levels seen in cerebral cortex a n d h i p p o c a m p u s using in situ hybridization histochemistry was confirmed by N o r t h e r n analysis o f p o l y (A) + m R N A isolated from brain areas by oligo d (T) columns. Large quantities of D2 m R N A were found in striatum, and small but significant quantities in h i p p o c a m p u s and cerebral cortex. Surprisingly, relatively high c o n c e n t r a t i o n s were also found in spinal cord. N o detectable levels, however, were seen in cerebellum or liver (Chen, Qin, Szele; Bai and Weiss, u n p u b l i s h e d observations). Cellular analysis olD: doparnine receptor mRNA A cellular analysis o f the D2 d o p a m i n e receptor m R N A in m o u s e corpus striatum or s u b s t a n t i a nigra
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Fig. I. Northern analysis ofdopamine D2receptor mRNA in mouse brain. Total cellular RNA was extracted from mouse striatum using the method of Chromczynski and Sacchi (1987). Poly (A) + RNA was isolated with oligo d(T) cellulose columns as described by Davis et al. (1986). The yield of RNA was about 2.7% of total RNA. For Northern blots, 3/zg of poly (A)* RNA was fractioned on 1% agarose gel containing 2.2 M formaldehyde and blotted onto nitrocellulose membranes. The mRNA was hybridized with labeled probe in the absence or presence of 100 fold excess unlabeiled oligonucleotide probe (displacer) as described in the section on Experimental Procedures. The autoradiography was carried out after exposing the X-ray film to the hybrid at-70°C for 17 days. The figure demonstrates specificdisplacement of the hybridization signal of D2 dopamine receptor mRNA by the corresponding insert or bridge probe. C = common probe, I = insert probe, B = bridge probe. pars compacta showed that the signal was associated with cell bodies; there were relatively few grains evident in the cellular matrix (Weiss et al. 1990). The cellular location of the m R N A was examined further using selective lesions induced with 6hydroxydopamine. Rats were unilaterally lesioned in the corpus striatum or substantia nigra with 6hydroxydopamine, and at various times after these lesions in situ hybridization studies were performed using the 02 m R N A common probe. The results of the studies showed that lesioning the substantia nigra greatly reduced the signal in the nigra but failed to substantially alter the signal in the striatum (Figure 2). Similarly, lesioning the striatum had little effect on the hybridization signal in striatum but greatly reduced the signal in the nigra and ventral tegmental area on the lesioned side (not shown). Taken together, these results suggest: (1) that the D 2 dopamine receptor m R N A in substantia nigra pars compacta is localized largely in dopaminergic cell bodies whose terminal projections lie in the striatum, and that this m R N A might code for D2 autoreceptors; (2) that the D2 dopamine receptor m R N A of striatum is in non-dopaminergic cell bodies intrinsic to the striatum and probably codes for post-synaptic D2 dopamine receptors.
Relationship between alterations of D: mRNA and behavior To determine the possible relationship between the loss of dopamine cell bodies in substantia nigra, with dopaminergic behavioral supersensitivity, we
administered 6-hydroxydopamine unilaterally into the striatum of mice. We then determined their rotational response to apomorphine and the levels of D2 dopamine receptor m R N A in substantia nigra. As can be seen (Figure 3), there was a significant correlation between these two events. Animals that evidenced the greatest rotational activity showed the greatest reduction in the levels of D2 receptor mRNA. These results show that the reduction of cell bodies containing D 2 receptor m R N A in the substantia nigra correlates well with the development of dopaminergically mediated behavioral supersensitivity.
Ontogenesis olD: dopamine receptor mRNA In studing the ontogenetic development of the D2 dopamine receptor m R N A in brain using in situ hybridization histocbemistry, it is important to note that the levels of D E m R N A are relatively low, particularly in newborn rats, and that there was significant non-specific binding of the probe. Therefore, all the studies were performed in the absence and presence of excess unlabeled oligonucleotide probe to displace the radiolabeled probe from specific binding sites on the D2 mRNA. For example, adding 100 fold excess D2 probe to brain sections of 2-day-old rats abolished the signal in olfactory bulb and striatum but had little influence on the signal in cortex or cerebellum. Similar results were found when adult brain was studied. Using greater stringency conditions, such as higher posthybridization wash temperatures, also reduced the non-specific hybridization.
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Fig. 2. Effect of 6-hydroxydopamine lesions on D~ dopamine receptor mRNA in corpus striatum and substantia nigra.Mice were unilaterally lesioned in the substantia nigra with microinjections of 6hydroxydopamine. The animals were killed 6 days after lesioning,the brains were removed and immediately frozen on dry ice. Ten micron sections were subjected to in situ hybridization histochemistry as described in Experimental Procedures• The results showed that lesioning the substantia nigra almost completely abolished the hybridization signal in substantia nigra pars compacta ipsilateral to the lesion, but had no significant effect on the hybridization signal in the striatum.
Figure 4 shows the ontogenetic development of the D 2 m R N A in striatum of rats 1 to 32 days of age. In these experiments, in situ hybridization studies were performed with the D2 m R N A c o m m o n probe and the results quantified with an image analyzer• As can be see, the D 2 m R N A increased soon after birth, reached a maximum at 16 days, then declined slightly. Studies
of the D2Am R N A using the insert probe showed an identical distribution and developmental pattern (not shown). The time course for the expression of the D2 dopamine receptor m R N A correlates well with the ontogenetic development of the D2 receptor (Feigenbraum & Yanai, 1984; Sales e t al., 1989; Gelbard et al., 1989).
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D, dopamine receptor mRNA in substantia nigra with rotational response to apomorphine. Mice were unilaterally lesioned in the substantia nigra with microinjections of 6hydroxydopamine. After one week the mice were challenged with apomorphine and rotational behavior was determined. The animals were then killed and the D2 mRNA was measured by/n situ hybridization histochemistry. The results show a positive correlation between the decrease of D2 mRNA in substantia nigra with the increased rotational response to apomorphine. 0.06
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striatum, mice were administered 6-hydroxydopamine intracerebroventriculady at 2 and 6 days of age. Animals were then killed at various times and the D2 m R N A determined by in situ hybridization histochemistry and by Northern analysis. The results showed that the administration of 6-hydroxydopamine failed to alter the development of the D2 mRNA, suggesting that this m R N A and, therefore, presumably the D2 dopamine receptor, can develop in the absence of dopaminergic input. Similar results were noted earlier for the beta adrenergic receptor where sympathetically denervating the pineal gland from birth still allowed the development of the beta adrenergic receptor (Weiss et al., 1984). In contrast with the results with the m R N A for the D: dopamine receptor, 6-hydroxydopamine treatment to newborn rats caused an increase in the enkephalin m R N A in striatum (not shown). These results on the development of the D: m R N A are consistent with those of Duncan et al. (1987) who showed that treating neonates with 6-hydroxydopamine produced little changes in D2 receptors of adult striatum. By contrast, Broaddus and Bennett (1990) reported about a 50% reduction in D 2receptors in caudate of adult rats following 6-hydroxydopamine treatment of neonates. Other investigators (Kostrzewa and Seleh 1989) reported that treatment of newborn rats with spiroperidol impaired the development of D 2 receptors.
Cellular analysis of ontogenetic development of D: dopamine receptor mRNA
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Fig. 4. Ontogenetic development of 02 dopamine receptor mRNA in rat striatum. Brains from rats at various postnatal ages were removed and parasagital sections at the level of the corpus striatum were prepared for /n situ hybrLdiTation histochemistry using the [35S]-labelledD~dopamine common probe. After high stringency post-hybridization washings, the hybridization signals were detected by film autoradiography and analyzed with the DUMAS image analysis system. To examine the effect of dopaminergic neuronal input on the development of the D2 m R N A in
Figure 5 shows the cellular distribution of the D2 dopamine receptor m R N A in rat striatum during postnatal development. As may be seen, at postnatal day 2 (P2), the hybridization signal for the D2 m R N A was scattered throughout the field, with only relatively few cellular elements labeled; those that were labeled were of relatively low density. By P32 only a certain subpopulations of cells were labeled, and these cells were considerabily more heavily labeled. These results showing that the increase in D2 m R N A during ontogeny is caused by a selectiveincrease in the density of specific types of cells in the striatum suggests that the development expression of the D2 dopamine receptor m R N A in certain striatal neurons may be associated with the maturation and differentiation of these neurons.
Effects of advancedage on D2 dopamine receptor mRNA During aging, there is a reduced level of D:
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Fig. 6. Northern blot analysis of D 2 dopamine receptor mRNA in the brains of young and old rats (insert probe). Brains of 3- and 24-month-old Fisher-344 rats were dissected, and total RNA was extracted. The RNA was subjected to Northern blot analysis using the D2 dopamine receptor insert probe. IC --~ inferior colliculus; OT = olfactory tubercle; ST = corpus striatum; PIT = pituitary gland.
dopamine receptors in various brain regions of the rat (Weiss et al., 1984; Severson and Randall, 1985; Morelli et al., 1990). To determine whether or not this decrease in receptors might be caused by a reduction in their synthesis, we have studied the effects of advanced age on the D2 dopamine receptor mRNA in several brain regions. In these studies, we have compared the levels of the D2 m R N A in 3- and 24 month-old rats by Northern blot analysis using both the insert and bridge probes. As can be seen (Figure 6), using the insert probe, 24-month-old rats had a significantly lower level of D2 dopamine receptor mRNA in corpus striatum, olfactory tubercle and inferior colliculus. By contrast, in pituitary the levels of D2 dopamine receptor mRNA was higher in 24-month-old rats than in 3-month-old rats. These results of Northern blot analysis in several brain areas of 3- and 24-month-old rats using the 'insert D2 mRNA probe are quantified and summarized in Table 1. The data show that both in 3- and 24-month-old rats, striatum has more than two times the level of D2 dopamine receptor mRNA than olfactory tubercle or pituitary and about 10 to 20 times more than the inferior and superior colliculi. Several other tissues showed non-detectable levels using these analyses. Tissues from aged rats had lower levels of the D2 mRNA in all brain areas examined, with the notable exception of pituitary gland. In striatum and olfactory tubercule, there was about 50% reduction in 24-month-old rats, in inferior colliculus there was about a 34% reduction, and in the superior colliculus the m R N A was the insert probe, the use of the bridge probe indicated that 24-
Table 1. Distributio~of D2mRNA(Insert)in 3-month-oldand 24month-oldrat brain D2mRNA(fmol/Gmtotal RNA) Area Striatum Olfactory Pituitary Inferiorcolliculus Superiorcolliculus Cerebellum Cortex Hippocampus Hypothalamus Olfactorybulb Spinalcord Thalamus
3 Month
24 Month
430 150 140 32 21 ND ND ND ND ND ND ND
220 73 310 21 ND ND ND ND ND ND ND ND
Brains from 3- and 24-month-oldrats were dissected, the RNA extractedand the I)2mRNAanalyzedbyNorthernanalysisusing the D2mRNAinsertprobe.The autoradiogramswerequantified usinga DUMASimaseanalyzer. ND = not detectable.
month-old rats had significantly less of the d2 bridge m R N A in striatum and olfactory tubercle, and more of this subtype of the D, mRNA in pituitary. A calculation of the ratio of the two subtypes of D2 mRNA shows thatthere was from 6 to 9 times more signal using the Dz m R N A insert probe than the D2 mRNA bridge probe in all tissues regardless of the age of the animal. Further examination of the effect of age on the D2 mRNA in pituitary using in situ hybridization histochemistry confirmed that there was a significantly higher density of the D2 mRNA signal in the anterior
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Table 2. Distribution of D2mRNA (Bridge) in 3-month-oldand 24month-old rat brain D:, mRNA (fmol;Gm total RNA)
Area Striatum Olfactory Pituitary Inferior colliculus Superior colliculus Cerebellum Cortex Hippocampus Hypothalamus Olfactory bulb Spinal cord Thalamus
3 Month
24 Month
63 28 16 ND ND ND ND ND ND ND ND ND
27 S 50 ND ND ND ND ND ND ND ND ND
Brains from 3- and 24-month-old rats were dissected, the RNA extracted and the D~mRNA analyzed by Northern analysisusing the D2mRNA bridgeprobe. The autoradiogramswerequantified using a DUMAS image analyzer. ND = not detectable.
IL
3 MONTH
and intermediate lobes o f pituitary glands irom 24m o n t h - o l d rats than in 3-month-old animals (I igurcs 7 and 8). N o significant differences were seen in the posterior lobe o f the pituitary which had a very weak signal in b o t h 3-month-old and in 24-month-okl rats. A cellular analysis o f these in situ hybridization studies revealed that the higher signal in the anterior and intermediate lobes o f the pituitary in 24-month-old rats was attributed not only to the larger size o f the gland but also to a greater signal per cell. This latter result may be due, in part, to the cellular hypertrophy which was evident in the anterior and intermediate lobes o f the pituitary o f 24-month-old rats. Work from a n u m b e r o f laboratories indicates that there is a reduction o f b o t h D~ and D2 receptors in aged rat brain. Studies by N o r m a n et al. (1987) have shown that in aged rats both the level as well as the turnover o f the D 2 d o p a m i n e receptors are reduced in striatum. Other studies showed not only that there was a reduction in spiroperidol binding in the striatum and olfactory tubercle, but that there was an increase in the pituitary gland (Govoni et al., 1980; Arita et al. 1984). These results correlate very well with our studies o f the relative age-related changes in the D2 d o p a m i n e receptor m R N A in the various brain regions. Earlier work from our laboratory showed that the ability o f beta adrenergic receptors to adapt to neuronal, h o r m o n a l or environmental stimuli is impaired in brain o f aged rats (Greenberg & Weiss, 1978; Weiss et al., 1979; Greenberg & Weiss, 1979). M o r e recent studies showed the ability ofD~ d o p a m i n e receptors to respond to light is also reduced in the
IL
0.3
I--7 A n t e r i o r Lobe
T
I n t e r m e d i a t e Lobe F=~ Posterior Lobe
// ~_~ 0.2
t/
C
0
24 ~ T H Fig. 7. Effect of age on D2 dopamine receptor mRNA in rat pituitary using in situ hybridization histochemistry. Pituitary glans from 3- and 24-month-old rats were removed, 10 p M sections prepared on a cryostat, and fixed sections analyzed for the D2 dopamine receptor mRNA by in situ hybridization using the [3sS]-D2dopamine receptor mRNA common probe. AL = anterior lobe; IL = intermediate lobe; PL = posterior lobe.
g
0.1
0.0 1
month
24-month
Fig. 8. Comparison of the D 2 receptor mRNA in different lobes of the pituitary gland of 3- and 24-month-old rats. Pituitary glands were prepared as described in the legend to Figure 7. The signals on the autoradiograms were quantitated using the DUMAS image analyzer. * P < 0.05 compared with the corresponding area of 3-month-old rat pituitary. ** P < 0.01 compared with the corresponding area of 3-month-old rat pituitary.
Dopamine 90 retina of senescent rats (Porceddu et al., 1990). Still other studies showed the rate of synthesis o f receptor for a number of neurotransmitters, including the beta adrenergic receptors (Greenberg et al., 1985), the alpha 1 and alpha 2 adrenergic receptors (Zhou et al., 1986), and the muscarinic acetylcholine receptors (Pintor et al., 1990) are also reduced in aging. These results suggest that a reduced synthesis of receptors for neurotransmitters might represent a general defect associated with the aging process. Whether alterations in specific dopaminergic behaviors are explainable by changes in the D2 dopamine receptor m R N A is still an open question. Repeated administration of dopaminergic agents have thus far yelded inconsistent results. Thus, continuous administration of the D2 agonist quinpirole reduced the D2 dopamine receptor m R N A in mouse striatum (Weiss et al., 1990), and repeated treatment of rats with the dopamine antagonist haloperidol increased the D2 receptor m R N A in the intermediate lobe of the pituitary (Auteliano et al., 1989). By contrast, others (van Tol et al., 1990) have found no significant change in the level of D2 receptor m R N A in rat striatum following chronic treatment with a number of antipsychotic drugs. More detailed studies, particularly on the time course of drug treatment, may help resolve these issues. Finally, there appears to be a correlation between the induction of dopaminergically mediated supersensitive behavior and the decrease in dopamine cell bodies in the substantia nigra as measured by the reduction in D2 m R N A in this area. However, it is still unclear what specific alterations of receptors or postreceptors events occur in the striatum that explain these changes in dopaminergic behavior. In this regard, recent studies from our laboratory show that following repeated treatment with the D2 dopamine agonist quinpirole there is not only a reduction in D2 dopamine receptors in striatum but also a reduction in the activity of the calmodulin-sensitive protein kinase (Zhou, Zhang and Weiss, unpublished). Surely there are many factors involved in the modulation of dopaminergic responses. Acknowledgement--Supported by Grant MH 42148 awarded by the National Institutes of Mental Health. REFERENCES
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