- Email: [email protected]
Dopaminergic modulation of pallidal preproenkephalin mRNA
Molecular Brain Research 69 Ž1999. 149–153
Short communication
Dopaminergic modulation of pallidal preproenkephalin mRNA Joseph J. Schuller, Lauren M. Billings, John F. Marshall
)
Department of Psychobiology UniÕersity of California IrÕine, CA, 92697-4550 USA Accepted 9 March 1999
Abstract The present study examines dopaminergic regulation of neuropeptide gene expression within a relatively poorly characterized population of cells, the preproenkephalin ŽPPE. mRNA containing neurons of the globus pallidus ŽGP.. Rats that received 6-hydroxydopamine Ž6-OHDA. lesions or repeated D1 or D2 antagonist administration were compared to control animals. One month after 6-OHDA lesions, PPE mRNA was elevated in the GP ipsilateral to the lesion, with a smaller elevation also being observed in the contralateral GP. Repeated administration of eticlopride, but not SCH 23390, also resulted in elevated PPE mRNA expression in the GP. These data reveal a novel effect of decreased dopamine transmission on the GP, and draw attention to this subpopulation of pallidal neurons. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Globus pallidus; Enkephalin; 6-Hydroxydopamine; Dopamine antagonist
The striatum is a major component of the subcortical neural system known as the basal ganglia. More than 90% of striatal cells are projection neurons, which can be classified according to their efferent targets and neuropeptide gene expression w7x. One such class of projection neurons in rats targets the substantia nigra pars reticulata ŽSNr. and entopeduncular nucleus ŽEP. and expresses the substance P-encoding mRNA, preprotachykinin ŽPPT.. By contrast, neurons projecting to the globus pallidus ŽGP. express the opiate neuropeptide, preproenkephalin ŽPPE.. The expression of both PPE and PPT mRNAs in the striatum is modulated by the neurotransmitter dopamine ŽDA.. Administration of DA receptor agonists attenuate striatal PPE mRNA and elevate PPT mRNA, while decreased DA receptor function as the result of nigrostriatal lesions or DA receptor antagonist treatment causes an elevation of striatal PPE mRNA and attenuation of PPT mRNA Žsee Ref. w1x for review.. Because PPE and PPT mRNAs are expressed for the most part by separate populations of striatal projection neurons, it has been proposed that DA exerts opposite influences over striatopallidal and striatonigral projection neurons w7x. While the GP is well-recognized as the major target of enkephalinergic striatal output neurons, it is less widely
) Corresponding [email protected]
author.
Fax:
q 1-949-824-2447;
E-mail:
known that a subpopulation of neurons within the GP expresses PPE mRNA w8,10,13x. As in the striatum, pallidal gene expression has been shown to be modulated by dopamine. Notably, glutamate decarboxylase ŽGAD. mRNA levels in the GP are elevated by chronic DA antagonist administration w4x or 6-OHDA lesions w21x. Furthermore, administration of DA receptor agonists and antagonists induces the immediate early gene protein product Fos in pallidal subpopulations having different characteristics w19x. In light of this evidence, the goal of the present study was to examine whether pallidal PPE gene expression is modulated by DA. Quantitative in situ hybridization was used to analyze PPE mRNA levels in the GP following either 6-OHDA lesions of the nigrostriatal pathway or repeated administration of the subtype-selective DA receptor antagonists eticlopride ŽD2 antagonist. or SCH 23390 ŽD1 antagonist.. Male Sprague–Dawley rats were individually housed in a temperature- and humidity-controlled environment. All animal procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Chemical lesions of the nigrostriatal dopamine pathway were performed in 12 rats Ž150–175 g. according to previously published methods w14x. Four weeks post-surgery, 6-OHDA lesioned rats and age-matched unlesioned controls Ž n s 5. were decapitated and the brains rapidly removed and frozen to y208C. Sections Ž20 mm. through the striatum and GP were cut on a cryostat, slide-mounted, postfixed in 4%
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 3 8 3 - 9
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J.J. Schuller et al.r Molecular Brain Research 69 (1999) 149–153
paraformaldehyde, and rinsed in 0.1 M phosphate buffer ŽPB. followed by RNase-free water. Coronal sections through the substantia nigra pars compacta ŽSNc. were also collected from 6-OHDA lesioned rats for verification of lesions by in situ hybridization of tyrosine hydroxylase ŽTH. mRNA. Slides were air dried and stored at y208C until used. For the chronic DA antagonist study, 19 rats Ž200–230 g. were injected with either eticlopride Ž1 mgrkg, s.c.., SCH 23390 Ž1 mgrkg, s.c.., or vehicle Ž0.9% saline or water, respectively. twice daily Ž0900 h, 1800 h. for 14 days. Twenty-four hours after the last injection, rats were decapitated and brain sections through the level of the GP were prepared for in situ hybridization according to the procedures described above. Pallidal PPE mRNA was localized using a labeled riboprobe Ž970 bp. transcribed from a cDNA template kindly provided by Dr. S. Sabol, NIH. The cRNA was labeled by incorporation of either 35 S-UTP or digoxigenin ŽDG.-UTP during SP6-mediated transcription. Hybridization procedures involved sequential tissue rinses in 0.75% glycine in 0.1 M PB, 0.1 M PB, 0.25% acetic anhydride in 0.1 M triethanolamine, 2X sodium citrate buffer ŽSSC., a series of ascending alcohols, and chloroform treatment. Riboprobe was diluted to a concentration of 15,000 cpmrml in hybridization buffer Ž24 mM Tris, 1.2 mM EDTA, 360 mM NaCl, 100 mgrml dextran sulfate, 1.2X Denhardt’s solution, 60% formamide, 2.5 mgrml salmon sperm DNA, 6.3 mgrml yeast total RNA. and applied to the tissue in a volume of 200 ml per slide. The slides were incubated in a humid chamber at 558C for 18–20 h. Sections were then rinsed twice in a solution of 50% formamide with 1.6 mgrml sodium thiosulfate in 2X SSC Ž30 min each at 558C., followed by a 15 min rinse in 0.5 M NaCl buffer with 1.6 mgrml sodium thiosulfate at 458C. The tissue was incubated in an RNase A solution Ž20 mgrml in NaCl buffer. at 458C for 30 min, and rinsed at room temperature for 15 min each in 2X SSC Žtwo washes. and 0.1 M PB. Following a brief water rinse, slides were air dried and opposed to Amersham b-Max hyperfilm for 3–10 days. To facilitate cellular analysis of PPE mRNA expression, tissue sections were hybridized with a combination of 35 S-PPE and DG-PPE riboprobes by procedures similar to those described above. After the last 0.1 M PB rinse, double-labeled slides were processed for the chromogenic visualization of the DG-PPE probe. Briefly, the tissue was incubated for 24 h at 48C with an alkaline phosphatase ŽAP.-conjugated sheep anti-DG polyclonal antibody Ž1:500; Boehringer-Mannheim. diluted in 0.1 M phosphatebuffered saline ŽPBS. with 0.1% Tween-20. Following rinses in 0.1 M PBS, the slides were placed in a solution containing the AP substrate BCIPrNBT ŽVector.. When the intensity of the blue reaction product was sufficient to distinguish pallidal neurons under a light microscope Žusually after 2–6 h. the slides were placed in a stop buffer
Ž100 mM NaCl, 100 mM Tris, 10 mM EDTA. for 1 h. After a brief water rinse the slides were air dried and opposed to film for 3–10 days, after which they were dipped in Amersham LM-1 emulsion and stored at 48C for 2–3 weeks. Emulsion-dipped slides were developed with Kodak D-19, fixed, rinsed in water, air-dried, and coverslipped. SNc sections from 6-OHDA rats were hybridized with a DG-labeled TH riboprobe Ž1.2 kb; generous gift of Dr. D. Chikaraishi, Tufts U.. using the same methods as described above for visualization of DG-PPE. Only GP sections from 6-OHDA rats found to have complete loss of TH-positive SNc cells in the injected hemisphere were used in this study. Digitized images of film autoradiograms from 35 S-PPE labeled slides were captured using a CCD camera and analyzed using an MCID image analysis system ŽImaging Research.. An oval sample tool adjusted to include the entire cross-sectional area of the GP was used to measure the proportional area of the GP having grey levels exceeding film background. The results of this analysis are expressed as mean proportional area " S.E.M. for each treatment group. In addition to analysis of the film autoradiograms, cellular-level analysis was performed on emulsion dipped sections from 6-OHDA-treated rats Ž n s 7. as well as rats that received repeated injections of either eticlopride Ž n s 5. or saline Ž n s 5.. Using a Nikon Optiphot microscope, fields through the GP were digitized at 800 = Žon screen magnification. and analyzed with the aid of the image analysis system. Two images of each field were captured, one emphasizing DG-labeled cells, the other 35 S-reduced silver grains in the emulsion layer. By linking the two channels it was possible to select visually identifiable Žblue. PPE-labeled cells in the ‘DG’ image and quantify the grain density overlying those cells in the ‘grain’ image. An oval sample tool Žapproximately 400 mm2 ., adjusted to
Fig. 1. Film autoradiograms of coronal brain sections at the level of the GP, hybridized with 35 S-labeled antisense Ža. or sense Žb. PPE riboprobe. Spotted labeling in the GP contrasts with intense striatal signal Ža.. There was a lack of specific signal in sections hybridized with sense riboprobe Žb..
J.J. Schuller et al.r Molecular Brain Research 69 (1999) 149–153
be slightly larger than each DG-labeled cell profile, was used to quantify the number of pixels per cell which exceeded user-defined relative optical density ŽROD. cutoff levels. For each animal, 25–50 PPE q GP neurons were analyzed. For 6-OHDA rats, 25–50 GP neurons per side were analyzed. Data from cellular level analysis are expressed as mean total grain area Žin pixels. per DG-PPE labeled cell " S.E.M. Statistical effects were assessed using Analysis of Variance ŽANOVA. followed by paired or independent samples t-tests, where appropriate. Confirming previous reports w8,10,13x, hybridization of pallidal tissue sections with 35 S- or DG-labeled antisense PPE riboprobes resulted in a subpopulation of pallidal neurons with cellular labeling, although the intensity of labeling was noticeably less than that in the striatum. The labeling was totally absent when sections were incubated with sense riboprobe, arguing for the specificity of our hybridization ŽFig. 1.. Four weeks after unilateral 6-OHDA lesions of the nigrostriatal pathway, PPE mRNA was elevated in the ipsilateral GP. This effect was apparent on film autoradiograms as an increase in mean proportional area of pallidal PPE signal of 143% compared to the corresponding GP of unlesioned rats ŽTable 1.. Unexpectedly, 6-OHDA lesions also caused a significant elevation of PPE mRNA Ž55%. in the contralateral GP as compared to unlesioned controls. However, the ipsilateral PPE signal was still 47% greater than contralateral levels ŽTable 1.. Analysis of individual labeled GP neurons revealed that the 6-OHDA lesion produced an average increase of 56% in mean pixels per cell ŽFigs. 2 and 3a. as compared to the contralateral GP. For comparison to the GP, striatal PPE levels were also quantified in film autoradiograms from lesioned and unlesioned rats. In agreement with previous reports w5,24x, we
Table 1 Effects of left hemisphere 6-OHDA lesions or repeated DA antagonist administration on mean proportional area Ž"S.E.M.. of pallidal PPE signal on film autoradiograms Nigrostriatal lesions left Žipsilateral. 6-OHDA Ž ns9. Unlesioned Ž ns 5.
0.1597"0.0122 0.0657"0.0155
right Žcontralateral. a,b
0.1101"0.0082 b 0.0711"0.0128
Repeated antagonistsd Eticlopride Ž ns8. SCH 23390 Ž ns 5. Vehicle Ž ns 7. a
0.0930"0.0126 c 0.0233"0.0021 0.0247"0.0017
p- 0.01 compared to contralateral Žright. value, paired samples t-test. p- 0.05 compared to corresponding hemisphere of unlesioned controls, independent samples t-test. c p- 0.05 compared to vehicle control, independent samples t-test. d Repeated antagonist values were collected from both left and right hemispheres. 6-OHDA data cannot be compared to repeated antagonist data since they reflect the results of separate experiments. b
151
Fig. 2. Darkfield photomicrographs from the same rat showing emulsion grain clusters Žarrowheads. corresponding to PPE-expressing pallidal neurons contralateral Ža. and ipsilateral Žb. to a 6-OHDA lesion. Grain clusters are more intense ipsilateral to the lesion. Calibration bar s100 mm.
found a significant 6-OHDA-induced elevation of striatal PPE mRNA ipsilateral to the lesion, but no effect on PPE expression in the contralateral striatum compared to unlesioned controls Ždata not shown.. A 14 day regimen of twice daily injections of the D2 antagonist eticlopride resulted in a 276% increase in the mean proportional area of pallidal PPE mRNA film signal when compared to vehicle-treated controls ŽTable 1.. By contrast, repeated SCH 23390 treatment did not alter the proportional area of pallidal PPE film signal ŽTable 1.. As in the 6-OHDA study, eticlopride-induced elevation of pallidal PPE mRNA was also evident as an increase of 49% in mean pixels per cell in emulsion coated tissue sections as compared to vehicle-treated controls ŽFig. 3b.. The present study demonstrates that pallidal PPE mRNA expression is modulated by DA, and suggests a specific role for the D2 class of DA receptors. Recent work indicates that these PPE mRNA expressing cells comprise a subpopulation of pallidal neurons which are distinguished by their preferential projections to the striatum compared to the subthalamic nucleus ŽSTN., and a lack of parvalbumin immunoreactivity w10x.
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Fig. 3. Cellular analysis of pallidal PPE expression following 6-OHDA lesion Ža. or repeated eticlopride administration Žb.. Mean grain area Žin pixels. per cell was significantly elevated ipsilateral to the 6-OHDA lesion Ž ns 7; paired samples tŽ6. s 3.9, p- 0.01. compared to the contralateral GP. Repeated eticlopride Žtwice daily X 14 days; ns 5. resulted in a significant increase in mean pixels per cell when compared to the vehicle control Ž ns 5; independent tŽ8. sy22.6, p- 0.001..
The upregulation of pallidal PPE mRNA following 6-OHDA lesions or repeated eticlopride treatment may reflect the indirect consequences of interrupting striatal DA neurotransmission. Both nigrostriatal lesions and DA receptor antagonist administration cause a profound disruption of striatal neural function. Several lesion- or antagonist-induced changes, such as elevated PPE and immediate-early gene expression w7,11,18x and D2 receptor binding w14x occur preferentially in striatopallidal projection neurons. There is a corresponding decrease in pallidal firing rates w16x and GABA and m opiate receptor binding w3,15x, suggesting increased release of enkephalin and GABA from striatopallidal terminals resulting in abnormal inhibition of pallidal neurons. Another possible indirect source of pallidal PPE modulation could arise from STN glutamatergic afferents. Nigrostriatal lesions cause hyperactivity in STN neurons w9x and decrease NMDA receptor binding in the GP w17x. Delfs and colleagues have demonstrated that 6-OHDA induced increases in pallidal GAD mRNA could be prevented by lesioning the ipsilateral STN w5x. The notion that pallidal PPE could be modulated by glutamate is further supported by the fact that striatal PPE mRNA is modulated by glutamate receptor activity w22x. The fact that striatal and STN afferents are predicted to have opposing influences over GP activity suggests that
the effects of decreased DA neurotransmission on pallidal gene expression may be complex. Indeed, DA antagonist administration induces the immediate-early gene product Fos in a subset of GP neurons having characteristics different from those expressing Fos in response to DA agonists w19x. Alternatively, elevated pallidal PPE expression following 6-OHDA lesion or repeated eticlopride administration may reflect the loss of DA receptor tone in the GP. A sparse DAergic innervation of the GP by nigral neurons has been reported w12x, and D2 receptor mRNA has been detected in GP neurons w23x. Also, iontophoretic application of DA receptor agonists onto pallidal neurons has been shown to alter their electrophysiological properties w2x, supporting a functional role for the pallidal DA innervation. Repeated administration of the D2 antagonist eticlopride resulted in elevated pallidal PPE mRNA levels, an effect also reported for the striatum following similar treatments w1x. However, the D1 antagonist SCH 23390 had no effect on pallidal PPE mRNA expression in this study, whereas in the striatum repeated SCH 23390 causes a decrease in PPE expression w1x. This suggests that DAergic modulation of pallidal PPE mRNA occurs specifically through the D2-class of DA receptors, possibly related to the fact that D2 mRNA, but not D1 mRNA, is expressed by a subset of pallidal neurons w23x. We observed an unexpected increase in PPE mRNA expression in the contralateral GP following unilateral 6-OHDA lesions. While the mechanisms are poorly understood, effects of unilateral 6-OHDA lesions on the contralateral striatum and GP have been reported for other measures w17,20x. Although there is a minor component of the nigrostriatal pathway which is crossed w6x, this small projection is unlikely to account for the magnitude of the contralateral effect observed in this study. In summary, the data presented here add to the emerging complexity of pallidal organization by demonstrating that long term disruption of DA neurotransmission elevates PPE gene expression within a subpopulation of GP neurons. Recent evidence from our laboratory indicates that the PPE mRNA containing neurons of the GP have characteristics that distinguish them from the majority of GP neurons w10x. Understanding the role of the GP in basal ganglia function and dysfunction will require addressing this heterogeneity.
Acknowledgements The authors would like to thank Mr. Eddie Ibrahim for expert technical assistance with in situ hybridization. This research was supported by NINDS grants NS22698 and NS33670 to J.F.M.
J.J. Schuller et al.r Molecular Brain Research 69 (1999) 149–153
References w14x w1x J.A. Angulo, B.S. McEwen, Molecular aspects of neuropeptide regulation and function in the corpus striatum and nucleus accumbens, Brain Res. Rev. 19 Ž1994. 1–28. w2x D.A. Bergstrom, J.R. Walters, Dopamine attenuates the effects of GABA on single unit activity in the globus pallidus, Brain Res. 310 Ž1984. 23–33. w3x J.M. Delfs, L. Yu, G.D. Ellison, T. Reisine, M.F. Chesselet, Regulation of mu-opioid receptor mRNA in rat globus pallidus: effects of enkephalin increases induced by short- and long-term haloperidol administration, J. Neurochem. 63 Ž1994. 777–780. w4x J.M. Delfs, N.J. Anegawa, M.F. Chesselet, Glutamic acid decarboxylase mRNA in rat pallidum: comparison of the effects of haloperidol, clozapine and combined haloperidol-scopolamine treatments, Neuroscience 66 Ž1995. 67–80. w5x J.M. Delfs, V.M. Ciaramitaro, T.J. Parry, M.F. Chesselet, Subthalamic nucleus lesions: widespread effects on changes in gene expression induced by nigrostriatal dopamine depletion in rats, J. Neurosci. 15 Ž1995. 6562–6575. w6x B. Fass, L.L. Butcher, Evidence for a crossed nigrostriatal pathway in rats, Neurosci. Lett. 22 Ž1981. 109–113. w7x C.R. Gerfen, T.M. Engber, L.C. Mahan, Z. Susel, T.N. Chase, F.J. Monsma Jr., D.R. Sibley, D1 and D2 dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons, Science 250 Ž1990. 1429–1432. w8x R.E. Harlan, B.D. Shivers, G.J. Romano, R.D. Howells, D.W. Pfaff, Localization of preproenkephalin mRNA in the rat brain and spinal cord by in situ hybridization, J. Comp. Neurol. 258 Ž1987. 159–184. w9x O.K. Hassani, M. Mouroux, J. Feger, Increased subthalamic neuronal activity after nigral dopaminergic lesion independent of disinhibition via the globus pallidus, Neuroscience 72 Ž1996. 105–115. w10x B.R. Hoover, J.F. Marshall, Population characteristics of enkephalin mRNA-containing neurons in the globus pallidus of the rat, Neurosci. Lett. Ž1999. in press. w11x M. Jian, W.A. Staines, M.J. Iadarola, G.S. Robertson, Destruction of the nigrostriatal pathway increases Fos-like immunoreactivity predominantly in striatopallidal neurons, Mol. Brain. Res. 19 Ž1993. 156–160. w12x O. Lindvall, A. Bjorklund, Dopaminergic innervation of the globus pallidus by collaterals from the nigrostriatal pathway, Brain Res. 172 Ž1979. 169–173. w13x S. Loughlin, F. Leslie, J. Fallon, Endogenous opioid systems. in: G.
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Paxinos ŽEd.., The Rat Nervous System, Academic Press, San Diego, 1995, pp. 975–998. K.A. Neve, C.A. Altar, C.A. Wong, J.F. Marshall, Quantitative analysis of w3 Hxspiroperidol binding to rat forebrain sections: plasticity of neostriatal dopamine receptors after nigrostriatal injury, Brain Res. 302 Ž1984. 9–18. H.S. Pan, J.B. Penney, A.B. Young, Gamma-aminobutyric acid and benzodiazepine receptor changes induced by unilateral 6-hydroxydopamine lesions of the medial forebrain bundle, J. Neurochem. 45 Ž1985. 1396–1404. H.S. Pan, J.R. Walters, Unilateral lesion of the nigrostriatal pathway decreases the firing rate and alters the firing pattern of globus pallidus neurons in the rat, Synapse 2 Ž1988. 650–656. R.H. Porter, J.G. Greene, D.S. Higgins Jr., J.T. Greenamyre, Polysynaptic regulation of glutamate receptors and mitochondrial enzyme activities in the basal ganglia of rats with unilateral dopamine depletion, J. Neurosci. 14 Ž1994. 7192–7199. G.S. Robertson, S.R. Vincent, H.C. Fibiger, D1 and D2 dopamine receptors differentially regulate c-fos expression in striatonigral and striatopallidal neurons, Neuroscience 49 Ž1992. 285–296. D.N. Ruskin, J.F. Marshall, Differing influences of dopamine agonists and antagonists on Fos expression in identified populations of globus pallidus neurons, Neuroscience 81 Ž1997. 79–92. J. Segovia, U. Tossman, M. Herrera-Marschitz, M. Garcia-Munoz, U. Ungerstedt, gamma-Aminobutyric acid release in the globus pallidus in vivo after a 6-hydroxydopamine lesion in the substantia nigra of the rat, Neurosci. Lett. 70 Ž1986. 364–368. J.J. Soghomonian, M.F. Chesselet, Effects of nigrostriatal lesions on the levels of messenger RNAs encoding two isoforms of glutamate decarboxylase in the globus pallidus and entopeduncular nucleus of the rat, Synapse 11 Ž1992. 124–133. D.L. Somers, R.M. Beckstead, Striatal preprotachykinin and preproenkephalin mRNA levels and the levels of nigral substance P and pallidal Met5-enkephalin depend on corticostriatal axons that use the excitatory amino acid neurotransmitters aspartate and glutamate: quantitative radioimmunocytochemical and in situ hybridization evidence, Mol. Brain Res. 8 Ž1990. 143–158. D.M. Weiner, A.I. Levey, R.K. Sunahara, H.B. Niznik, B.F. O’Dowd, P. Seeman, M.R. Brann, D1 and D2 dopamine receptor mRNA in rat brain, Proc. Natl. Acad. Sci. USA 88 Ž1991. 1859– 1863. W. Young III, T.I. Bonner, M.R. Brann, Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain, Proc. Natl. Acad. Sci. USA 83 Ž1986. 9827–9831.
Short communication
Dopaminergic modulation of pallidal preproenkephalin mRNA Joseph J. Schuller, Lauren M. Billings, John F. Marshall
)
Department of Psychobiology UniÕersity of California IrÕine, CA, 92697-4550 USA Accepted 9 March 1999
Abstract The present study examines dopaminergic regulation of neuropeptide gene expression within a relatively poorly characterized population of cells, the preproenkephalin ŽPPE. mRNA containing neurons of the globus pallidus ŽGP.. Rats that received 6-hydroxydopamine Ž6-OHDA. lesions or repeated D1 or D2 antagonist administration were compared to control animals. One month after 6-OHDA lesions, PPE mRNA was elevated in the GP ipsilateral to the lesion, with a smaller elevation also being observed in the contralateral GP. Repeated administration of eticlopride, but not SCH 23390, also resulted in elevated PPE mRNA expression in the GP. These data reveal a novel effect of decreased dopamine transmission on the GP, and draw attention to this subpopulation of pallidal neurons. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Globus pallidus; Enkephalin; 6-Hydroxydopamine; Dopamine antagonist
The striatum is a major component of the subcortical neural system known as the basal ganglia. More than 90% of striatal cells are projection neurons, which can be classified according to their efferent targets and neuropeptide gene expression w7x. One such class of projection neurons in rats targets the substantia nigra pars reticulata ŽSNr. and entopeduncular nucleus ŽEP. and expresses the substance P-encoding mRNA, preprotachykinin ŽPPT.. By contrast, neurons projecting to the globus pallidus ŽGP. express the opiate neuropeptide, preproenkephalin ŽPPE.. The expression of both PPE and PPT mRNAs in the striatum is modulated by the neurotransmitter dopamine ŽDA.. Administration of DA receptor agonists attenuate striatal PPE mRNA and elevate PPT mRNA, while decreased DA receptor function as the result of nigrostriatal lesions or DA receptor antagonist treatment causes an elevation of striatal PPE mRNA and attenuation of PPT mRNA Žsee Ref. w1x for review.. Because PPE and PPT mRNAs are expressed for the most part by separate populations of striatal projection neurons, it has been proposed that DA exerts opposite influences over striatopallidal and striatonigral projection neurons w7x. While the GP is well-recognized as the major target of enkephalinergic striatal output neurons, it is less widely
) Corresponding [email protected]
author.
Fax:
q 1-949-824-2447;
E-mail:
known that a subpopulation of neurons within the GP expresses PPE mRNA w8,10,13x. As in the striatum, pallidal gene expression has been shown to be modulated by dopamine. Notably, glutamate decarboxylase ŽGAD. mRNA levels in the GP are elevated by chronic DA antagonist administration w4x or 6-OHDA lesions w21x. Furthermore, administration of DA receptor agonists and antagonists induces the immediate early gene protein product Fos in pallidal subpopulations having different characteristics w19x. In light of this evidence, the goal of the present study was to examine whether pallidal PPE gene expression is modulated by DA. Quantitative in situ hybridization was used to analyze PPE mRNA levels in the GP following either 6-OHDA lesions of the nigrostriatal pathway or repeated administration of the subtype-selective DA receptor antagonists eticlopride ŽD2 antagonist. or SCH 23390 ŽD1 antagonist.. Male Sprague–Dawley rats were individually housed in a temperature- and humidity-controlled environment. All animal procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Chemical lesions of the nigrostriatal dopamine pathway were performed in 12 rats Ž150–175 g. according to previously published methods w14x. Four weeks post-surgery, 6-OHDA lesioned rats and age-matched unlesioned controls Ž n s 5. were decapitated and the brains rapidly removed and frozen to y208C. Sections Ž20 mm. through the striatum and GP were cut on a cryostat, slide-mounted, postfixed in 4%
0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 3 8 3 - 9
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J.J. Schuller et al.r Molecular Brain Research 69 (1999) 149–153
paraformaldehyde, and rinsed in 0.1 M phosphate buffer ŽPB. followed by RNase-free water. Coronal sections through the substantia nigra pars compacta ŽSNc. were also collected from 6-OHDA lesioned rats for verification of lesions by in situ hybridization of tyrosine hydroxylase ŽTH. mRNA. Slides were air dried and stored at y208C until used. For the chronic DA antagonist study, 19 rats Ž200–230 g. were injected with either eticlopride Ž1 mgrkg, s.c.., SCH 23390 Ž1 mgrkg, s.c.., or vehicle Ž0.9% saline or water, respectively. twice daily Ž0900 h, 1800 h. for 14 days. Twenty-four hours after the last injection, rats were decapitated and brain sections through the level of the GP were prepared for in situ hybridization according to the procedures described above. Pallidal PPE mRNA was localized using a labeled riboprobe Ž970 bp. transcribed from a cDNA template kindly provided by Dr. S. Sabol, NIH. The cRNA was labeled by incorporation of either 35 S-UTP or digoxigenin ŽDG.-UTP during SP6-mediated transcription. Hybridization procedures involved sequential tissue rinses in 0.75% glycine in 0.1 M PB, 0.1 M PB, 0.25% acetic anhydride in 0.1 M triethanolamine, 2X sodium citrate buffer ŽSSC., a series of ascending alcohols, and chloroform treatment. Riboprobe was diluted to a concentration of 15,000 cpmrml in hybridization buffer Ž24 mM Tris, 1.2 mM EDTA, 360 mM NaCl, 100 mgrml dextran sulfate, 1.2X Denhardt’s solution, 60% formamide, 2.5 mgrml salmon sperm DNA, 6.3 mgrml yeast total RNA. and applied to the tissue in a volume of 200 ml per slide. The slides were incubated in a humid chamber at 558C for 18–20 h. Sections were then rinsed twice in a solution of 50% formamide with 1.6 mgrml sodium thiosulfate in 2X SSC Ž30 min each at 558C., followed by a 15 min rinse in 0.5 M NaCl buffer with 1.6 mgrml sodium thiosulfate at 458C. The tissue was incubated in an RNase A solution Ž20 mgrml in NaCl buffer. at 458C for 30 min, and rinsed at room temperature for 15 min each in 2X SSC Žtwo washes. and 0.1 M PB. Following a brief water rinse, slides were air dried and opposed to Amersham b-Max hyperfilm for 3–10 days. To facilitate cellular analysis of PPE mRNA expression, tissue sections were hybridized with a combination of 35 S-PPE and DG-PPE riboprobes by procedures similar to those described above. After the last 0.1 M PB rinse, double-labeled slides were processed for the chromogenic visualization of the DG-PPE probe. Briefly, the tissue was incubated for 24 h at 48C with an alkaline phosphatase ŽAP.-conjugated sheep anti-DG polyclonal antibody Ž1:500; Boehringer-Mannheim. diluted in 0.1 M phosphatebuffered saline ŽPBS. with 0.1% Tween-20. Following rinses in 0.1 M PBS, the slides were placed in a solution containing the AP substrate BCIPrNBT ŽVector.. When the intensity of the blue reaction product was sufficient to distinguish pallidal neurons under a light microscope Žusually after 2–6 h. the slides were placed in a stop buffer
Ž100 mM NaCl, 100 mM Tris, 10 mM EDTA. for 1 h. After a brief water rinse the slides were air dried and opposed to film for 3–10 days, after which they were dipped in Amersham LM-1 emulsion and stored at 48C for 2–3 weeks. Emulsion-dipped slides were developed with Kodak D-19, fixed, rinsed in water, air-dried, and coverslipped. SNc sections from 6-OHDA rats were hybridized with a DG-labeled TH riboprobe Ž1.2 kb; generous gift of Dr. D. Chikaraishi, Tufts U.. using the same methods as described above for visualization of DG-PPE. Only GP sections from 6-OHDA rats found to have complete loss of TH-positive SNc cells in the injected hemisphere were used in this study. Digitized images of film autoradiograms from 35 S-PPE labeled slides were captured using a CCD camera and analyzed using an MCID image analysis system ŽImaging Research.. An oval sample tool adjusted to include the entire cross-sectional area of the GP was used to measure the proportional area of the GP having grey levels exceeding film background. The results of this analysis are expressed as mean proportional area " S.E.M. for each treatment group. In addition to analysis of the film autoradiograms, cellular-level analysis was performed on emulsion dipped sections from 6-OHDA-treated rats Ž n s 7. as well as rats that received repeated injections of either eticlopride Ž n s 5. or saline Ž n s 5.. Using a Nikon Optiphot microscope, fields through the GP were digitized at 800 = Žon screen magnification. and analyzed with the aid of the image analysis system. Two images of each field were captured, one emphasizing DG-labeled cells, the other 35 S-reduced silver grains in the emulsion layer. By linking the two channels it was possible to select visually identifiable Žblue. PPE-labeled cells in the ‘DG’ image and quantify the grain density overlying those cells in the ‘grain’ image. An oval sample tool Žapproximately 400 mm2 ., adjusted to
Fig. 1. Film autoradiograms of coronal brain sections at the level of the GP, hybridized with 35 S-labeled antisense Ža. or sense Žb. PPE riboprobe. Spotted labeling in the GP contrasts with intense striatal signal Ža.. There was a lack of specific signal in sections hybridized with sense riboprobe Žb..
J.J. Schuller et al.r Molecular Brain Research 69 (1999) 149–153
be slightly larger than each DG-labeled cell profile, was used to quantify the number of pixels per cell which exceeded user-defined relative optical density ŽROD. cutoff levels. For each animal, 25–50 PPE q GP neurons were analyzed. For 6-OHDA rats, 25–50 GP neurons per side were analyzed. Data from cellular level analysis are expressed as mean total grain area Žin pixels. per DG-PPE labeled cell " S.E.M. Statistical effects were assessed using Analysis of Variance ŽANOVA. followed by paired or independent samples t-tests, where appropriate. Confirming previous reports w8,10,13x, hybridization of pallidal tissue sections with 35 S- or DG-labeled antisense PPE riboprobes resulted in a subpopulation of pallidal neurons with cellular labeling, although the intensity of labeling was noticeably less than that in the striatum. The labeling was totally absent when sections were incubated with sense riboprobe, arguing for the specificity of our hybridization ŽFig. 1.. Four weeks after unilateral 6-OHDA lesions of the nigrostriatal pathway, PPE mRNA was elevated in the ipsilateral GP. This effect was apparent on film autoradiograms as an increase in mean proportional area of pallidal PPE signal of 143% compared to the corresponding GP of unlesioned rats ŽTable 1.. Unexpectedly, 6-OHDA lesions also caused a significant elevation of PPE mRNA Ž55%. in the contralateral GP as compared to unlesioned controls. However, the ipsilateral PPE signal was still 47% greater than contralateral levels ŽTable 1.. Analysis of individual labeled GP neurons revealed that the 6-OHDA lesion produced an average increase of 56% in mean pixels per cell ŽFigs. 2 and 3a. as compared to the contralateral GP. For comparison to the GP, striatal PPE levels were also quantified in film autoradiograms from lesioned and unlesioned rats. In agreement with previous reports w5,24x, we
Table 1 Effects of left hemisphere 6-OHDA lesions or repeated DA antagonist administration on mean proportional area Ž"S.E.M.. of pallidal PPE signal on film autoradiograms Nigrostriatal lesions left Žipsilateral. 6-OHDA Ž ns9. Unlesioned Ž ns 5.
0.1597"0.0122 0.0657"0.0155
right Žcontralateral. a,b
0.1101"0.0082 b 0.0711"0.0128
Repeated antagonistsd Eticlopride Ž ns8. SCH 23390 Ž ns 5. Vehicle Ž ns 7. a
0.0930"0.0126 c 0.0233"0.0021 0.0247"0.0017
p- 0.01 compared to contralateral Žright. value, paired samples t-test. p- 0.05 compared to corresponding hemisphere of unlesioned controls, independent samples t-test. c p- 0.05 compared to vehicle control, independent samples t-test. d Repeated antagonist values were collected from both left and right hemispheres. 6-OHDA data cannot be compared to repeated antagonist data since they reflect the results of separate experiments. b
151
Fig. 2. Darkfield photomicrographs from the same rat showing emulsion grain clusters Žarrowheads. corresponding to PPE-expressing pallidal neurons contralateral Ža. and ipsilateral Žb. to a 6-OHDA lesion. Grain clusters are more intense ipsilateral to the lesion. Calibration bar s100 mm.
found a significant 6-OHDA-induced elevation of striatal PPE mRNA ipsilateral to the lesion, but no effect on PPE expression in the contralateral striatum compared to unlesioned controls Ždata not shown.. A 14 day regimen of twice daily injections of the D2 antagonist eticlopride resulted in a 276% increase in the mean proportional area of pallidal PPE mRNA film signal when compared to vehicle-treated controls ŽTable 1.. By contrast, repeated SCH 23390 treatment did not alter the proportional area of pallidal PPE film signal ŽTable 1.. As in the 6-OHDA study, eticlopride-induced elevation of pallidal PPE mRNA was also evident as an increase of 49% in mean pixels per cell in emulsion coated tissue sections as compared to vehicle-treated controls ŽFig. 3b.. The present study demonstrates that pallidal PPE mRNA expression is modulated by DA, and suggests a specific role for the D2 class of DA receptors. Recent work indicates that these PPE mRNA expressing cells comprise a subpopulation of pallidal neurons which are distinguished by their preferential projections to the striatum compared to the subthalamic nucleus ŽSTN., and a lack of parvalbumin immunoreactivity w10x.
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Fig. 3. Cellular analysis of pallidal PPE expression following 6-OHDA lesion Ža. or repeated eticlopride administration Žb.. Mean grain area Žin pixels. per cell was significantly elevated ipsilateral to the 6-OHDA lesion Ž ns 7; paired samples tŽ6. s 3.9, p- 0.01. compared to the contralateral GP. Repeated eticlopride Žtwice daily X 14 days; ns 5. resulted in a significant increase in mean pixels per cell when compared to the vehicle control Ž ns 5; independent tŽ8. sy22.6, p- 0.001..
The upregulation of pallidal PPE mRNA following 6-OHDA lesions or repeated eticlopride treatment may reflect the indirect consequences of interrupting striatal DA neurotransmission. Both nigrostriatal lesions and DA receptor antagonist administration cause a profound disruption of striatal neural function. Several lesion- or antagonist-induced changes, such as elevated PPE and immediate-early gene expression w7,11,18x and D2 receptor binding w14x occur preferentially in striatopallidal projection neurons. There is a corresponding decrease in pallidal firing rates w16x and GABA and m opiate receptor binding w3,15x, suggesting increased release of enkephalin and GABA from striatopallidal terminals resulting in abnormal inhibition of pallidal neurons. Another possible indirect source of pallidal PPE modulation could arise from STN glutamatergic afferents. Nigrostriatal lesions cause hyperactivity in STN neurons w9x and decrease NMDA receptor binding in the GP w17x. Delfs and colleagues have demonstrated that 6-OHDA induced increases in pallidal GAD mRNA could be prevented by lesioning the ipsilateral STN w5x. The notion that pallidal PPE could be modulated by glutamate is further supported by the fact that striatal PPE mRNA is modulated by glutamate receptor activity w22x. The fact that striatal and STN afferents are predicted to have opposing influences over GP activity suggests that
the effects of decreased DA neurotransmission on pallidal gene expression may be complex. Indeed, DA antagonist administration induces the immediate-early gene product Fos in a subset of GP neurons having characteristics different from those expressing Fos in response to DA agonists w19x. Alternatively, elevated pallidal PPE expression following 6-OHDA lesion or repeated eticlopride administration may reflect the loss of DA receptor tone in the GP. A sparse DAergic innervation of the GP by nigral neurons has been reported w12x, and D2 receptor mRNA has been detected in GP neurons w23x. Also, iontophoretic application of DA receptor agonists onto pallidal neurons has been shown to alter their electrophysiological properties w2x, supporting a functional role for the pallidal DA innervation. Repeated administration of the D2 antagonist eticlopride resulted in elevated pallidal PPE mRNA levels, an effect also reported for the striatum following similar treatments w1x. However, the D1 antagonist SCH 23390 had no effect on pallidal PPE mRNA expression in this study, whereas in the striatum repeated SCH 23390 causes a decrease in PPE expression w1x. This suggests that DAergic modulation of pallidal PPE mRNA occurs specifically through the D2-class of DA receptors, possibly related to the fact that D2 mRNA, but not D1 mRNA, is expressed by a subset of pallidal neurons w23x. We observed an unexpected increase in PPE mRNA expression in the contralateral GP following unilateral 6-OHDA lesions. While the mechanisms are poorly understood, effects of unilateral 6-OHDA lesions on the contralateral striatum and GP have been reported for other measures w17,20x. Although there is a minor component of the nigrostriatal pathway which is crossed w6x, this small projection is unlikely to account for the magnitude of the contralateral effect observed in this study. In summary, the data presented here add to the emerging complexity of pallidal organization by demonstrating that long term disruption of DA neurotransmission elevates PPE gene expression within a subpopulation of GP neurons. Recent evidence from our laboratory indicates that the PPE mRNA containing neurons of the GP have characteristics that distinguish them from the majority of GP neurons w10x. Understanding the role of the GP in basal ganglia function and dysfunction will require addressing this heterogeneity.
Acknowledgements The authors would like to thank Mr. Eddie Ibrahim for expert technical assistance with in situ hybridization. This research was supported by NINDS grants NS22698 and NS33670 to J.F.M.
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