Molecular Brain Research, 8 (1990) 63-68 Elsevier
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Detection of CCK m R N A in the motor nucleus of the rat trigeminal nerve with in situ hybridization histochemistry E l l e n L. Sutin 1'2'* a n d D a v i d M. J a c o b o w i t z 1'2 1Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD 20892 ( U.S.A.) and 2University of California, San Diego, La Jolla, CA 92093 (U.S.A.) (Accepted 16 January 1990) Key words: Cholecystokinin; Nucleus of the trigeminai nerve; Dorsal raphe nucleus; Acetylcholinesterase; In situ hybridization histochemistry; Rat brain
The regional distribution of dorsal pontine tegmental neurons expressing cholecystokinin (CCK) messenger RNA (mRNA) was examined using in situ hybridization histochemical techniques. A distribution of neurons containing CCK mRNA was observed in the motor nucleus of the trigerninal nerve, a region which has not been shown to contain CCK by immunocytochemical means. In addition, CCK mRNA was seen in the caudal portion of the dorsal raphe nucleus which correlates with prior immunocytochemical observations. The presence of CCK mRNA in the cholinergic motor trigeminal nucleus suggests that these cells express the CCK gene and have gone undetected in previous immunocytochemical studies. It is also suggested that CCK may have an influence on muscular contraction at sites of trigeminal nerve innervation (e.g. muscles of mastication).
INTRODUCTION Cholecystokinin ( C C K ) o c t a p e p t i d e is present in high concentrations in the m a m m a l i a n nervous system associated with sensory and m o t o r pathways 34. A n involvem e n t of the C C K system has also been p r o p o s e d in neurological and psychiatric diseases such as Parkinson's disease, H u n t i n g t o n ' s disease and schizophrenia 1'3. Furt h e r m o r e , C C K has b e e n implicated in the central control of eating b e h a v i o r 8'22. N e u r o n a l p e r i k a r y a containing C C K - i m m u n o r e a c t i v i t y are located in the cerebral cortex, h i p p o c a m p u s , ventral t e g m e n t a l a r e a and periaqueductal grey m a t t e r 11'14"2°'21'33'34. The aim of this study was to characterize the distribution of the messenger R N A ( m R N A ) coding for C C K in the dorsal pontine t e g m e n t u m and to c o m p a r e this p a t t e r n with that of the neuropeptide. In the present study we have utilized a 48-base synthetic oligonucleotide D N A p r o b e c o m p l e m e n t a r y to C C K m R N A in o r d e r to detect specific C C K gene transcripts by in situ hybridization histochemistry. This p r o b e was r a d i o l a b e l e d by using terminal deoxynucleotidyl transferase to introduce multiple [35S]dATP residues to the 3" terminus of the p r o b e . We r e p o r t here the anatomical localization of cells expressing the C C K gene
in the m o t o r trigeminal nucleus and the caudal portion of the dorsal r a p h e nucleus. MATERIALS AND METHODS Synthesis and labeling of CCK oligonucleotide probe The probe for CCK was constructed by solid phase synthesis on an applied Biosystems 380A DNA synthesizor (Foster City, CA) and purified on an 8% polyacrylamide/8 M urea preparative sequencing gel. The CCK probe was complementary to bases 315-362 of the rat cholecystokinin mRNA 9. This probe has previously been demonstrated to be specific by Burgunder and Young4 by using several controls including the use of a control probe of messenger sense coding for the last 16 amino acids of the rat vasopressin glycopeptide15. Burgunder and Young4 also performed a Northern analysis on total RNA isolated from dissected rat thalamus and found a single band of about 800 bases, in agreement with previous studies. This oligonucleotide probe was radiolabeled with 35S using the 3" end tailing method. The probe (5 nmol; final concentration 0.1 ~M) was incubated with [35S]dATP (spec. act. 1200 Ci/mmol; New England Nuclear (NEN), Boston, MA) at a final concentration of 1.0 aM (50 nmol), 150 units of the enzyme terminal deoxynucleotidyl transferase (Bethesda Research Labs [BRL], Gaithersburg, MD) in a solution containing 5x DNA tailing buffer (BRL) in a final volume of 50 #1. The reaction mixture was incubated for 10 min at 37 °C. The labeled probe was separated from the enzyme and unincorporated radiolabeled dATP residues by phenol/chloroform extraction and ethanol precipitation. In situ hybridization histochemistry In situ hybridization histochemistry was performed by using a
* Present address: Stanford University School of Medicine, Department of Psychiatry, Sleep Research Center, Stanford University, Palo Alto, CA 94305, U.S.A. Correspondence: D. Jacobowitz, Laboratory of Clinical Science, Building 10, Room 3D-48, NIMH, Bethesda, MD 20892, U.S.A. 0169-328X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
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Fig. 1. A: distribution of the selective labeling after in situ hybridization histochemistry with a 35S-labeled CCK probe at the level of the locus coeruleus. The caudal dorsal rapbe nucleus (small arrow) and the motor nucleus of the trigeminal nerve (large arrows) are labeled in this film autoradiogram which was developed after 20 days. B: photomicrograph of individually-labeled cells of the caudal dorsal raphe nucleus after an 8 day exposure to film emulsion (x310) and C: motor nucleus of the trigeminal nerve (nV) after 21 days of exposure (× 170).
65 slight modification of protocols which have been utilized successfully with other oligonucleotide probes3s. Adult male Sprague-Dawley rats were killed by decapitation and brains were removed, frozen quickly on dry ice and warmed to -18 °C in a cryostat. 12-/~m sections were cut through the dorsal pontine tegmentum and were then thaw-mounted onto twice-coated gelatin chrome alum slides. The slides were then either stored desaccated at -80 °C until use or processed immediately. Before hybridization, sections were warmed to room temperature and allowed to dry. Sections were then fixed in 4% formaldehyde in phosphate-buffered saline (PBS, 4 °C) for 10 min. Slides were rinsed twice (1 min each) in PBS and incubated in 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCI (pH 8) for 10 min at room temperature and then transferred successively through 70% (1 rain), 80% (1 min), 95% (2 min), 100% ethanol (1 min), 100% chloroform (5 min), 100% (1 min), and 95% ethanol (2 min) and air-dried. 500,000 to 1 million CPM oligonucleotide probe in 50 gl hybridization buffer containing 0.10% D T r (dithiothreitol, Sigma, St. Louis, MO), 50% formamide, 10% dextran sulfate, 4x SSC, 1x Denhardt's solution (0.02% ficoll/0.02% polyvinylpyrrolidine/ 0.02% bovine serum albumin), 500 gg/ml denatured salmon sperm DNA, and 250/~g/ml yeast tRNA were applied to each section. Sections were then covered with a parafilm coverslip and incubated overnight at 37 °C in a humid chamber. The coverslips were removed and the sections rinsed twice in l x SSC at room temperature. Slides were then washed in four 15-min changes of 2 x SSC containing 50% formamide at 40 °C, then for two 30-min washes at room temperature in 1 x SSC, rinsed in water, dehydrated in 70% and then 95% ethanol and air-dried. A separate set of tissue preparations were treated with an RNase A solution (Sigma, St. Louis, MO; 25 #g/ml at 42 °C for 1 h) before hybridization. Competition experiments were also performed in which a 5-fold excess (25 nmol or 0.1 gM) of non-radioactive probe was prehybridized onto the sections prior to hybridization with the 35S-labeled CCK probe. In addition, to demonstrate the unique and differential hybridization distributions produced with the CCK probe, hybridizations were also performed on similar sets of tissue sections using 5 other synthetic oligonucleotide probes. These other antisense probes were complementary to substance P, (bases 124-171, Krause et al)9), corticotropin-releasing factor (CRF, bases 496-543, Jingami et a1.17); galanin (bases 115-153, Rrkaeus and Brownstein25), tyrosine hydroxylase (bases 1441-1488, Grima et al)°), and CGRP (bases 259-315, Amara et al.2). Each of these probes have been demonstrated to be specific for their respective target mRNA 12'26'36'37. Autoradiographic localization of bound probe was performed by exposure of the sections to X-ray film (XAR-5, Kodak, Rochester, NY) for 1-6 weeks. For higher resolution, the sections were dipped into NTB3 nuclear tract emulsion (Kodak) diluted 1:1 with water. Exposures ranged from 1 to 6 weeks after which the slides and/or films were developed by using Kodak D-19 developer followed by Kodak fixer and counterstained with 0.1% thionin. RESULTS B o t h brightfield and darkfield optics were used to e x a m i n e the a u t o r a d i o g r a m s and to d e t e r m i n e the distribution of l a b e l e d cells. T h e C C K oligonucleotide p r o b e revealed transcripts coding for the C C K peptide in the caudal dorsal r a p h e nucleus and m o t o r nucleus of the trigeminal nerve. Fig. 1A shows a film a u t o r a d i o g r a m in which both the midline dorsal raphe nucleus (small arrow) and the the bilateral m o t o r nucleus (large arrows) can be seen. A t the individual cell level the labeled cells in the caudal dorsal raphe nucleus a p p e a r e d to be
m o d e r a t e l y l a b e l e d and were localized p r i m a r i l y towards the midline and ventral aspect of the central gray (Fig. 1B). In m o r e rostral sections, at the level of the main serotonergic r a p h e , no cells were o b s e r v e d to contain C C K m R N A transcripts. A l m o s t all of the large m o t o r neurons in the nucleus s e e m e d to be labeled, although some cells a p p e a r e d m o r e densely l a b e l e d with grains than others (Fig. 1C). A longer e x p o s u r e of the film a u t o r a d i o g r a m s also r e v e a l e d l a b e l e d cells in the pontine reticular formation and nucleus of the t r a p e z o i d body. H o w e v e r these regions were not characterized at the individual cell level for this report. Both p r e t r e a t m e n t of the tissue sections with R N A s e and hybridization with excess cold p r o b e before hybridization with the l a b e l e d C C K p r o b e gave no autoradiographic signal, indicating that the positive signal o b t a i n e d was derived from o l i g o n u c l e o t i d e - m R N A hybrids ( d a t a not shown). Labeling with the 5 o t h e r antisense oligonucleotide probes r e v e a l e d 5 different and unique patterns of hybridizations in the dorsal p o n t i n e tegmental region of the rat brain (data not shown). Tyrosine hydroxylase m R N A was localized to the locus coeruleus and subcoeruleus only, while galanin m R N A was found in the locus coeruleus only. T h e substance P and C R F probes both hybridized specifically to the laterodorsal tegmental nucleus, while the substance P p r o b e also labeled the p e d u n c u l a r pontine t e g m e n t a l nucleus in m o r e rostral sections and the C R F p r o b e l a b e l e d cells in the dorsal p a r a b r a c h i a l nucleus. C G R P m R N A was d e m o n s t r a t e d only in the ventrolateral subdivision of the dorsal p a r a b r a c h i a l nucleus. N o n e of these 5 additional oligonucleotides labeled cells in the m o t o r nucleus of the trigeminal nerve or the caudal dorsal r a p h e nucleus, yet were found in the a p p r o p r i a t e region w h e r e its respective p e p t i d e is k n o w n to be contained. DISCUSSION We have used a synthetic oligonucleotide p r o b e comp l e m e n t a r y to rat C C K m R N A to d e m o n s t r a t e C C K gene expression in cells of the caudal dorsal raphe nucleus and m o t o r nucleus of the trigeminal nerve of the rat brain. Since the c D N A encoding rat preprocholecystokinin was cloned and sequenced by D e s c h e n n e s et al. 9, only 5 publications have r e p o r t e d on the localization of the C C K gene in rat brain with in situ hybridization histochemical techniques. Siegel and Young 3° first detected C C K m R N A c o m p l e m e n t a r y R N A p r o b e s and e x a m i n e d transcripts in the h y p o t h a l a m u s , amygdala, and striatum and these findings were replicated by O ' B r i e n et al. 23 using a c o m p l e m e n t a r y D N A p r o b e to p r e p r o C C K . Two m o r e recent reports by B u r g u n d e r and Young 4 and Savasta et al. 27 who used synthetic olig0nucleotide
66 probes to different coding regions of the CCK gene described distributions which further correlated with the earlier reports. The distribution of cells containing the mRNA for CCK in the caudal dorsal raphe is in good agreement with previous immunocytochemical studies 2°' 33,34
The findings of CCK mRNA in the motor nucleus of the trigeminal nerve is an important one in that the CCK peptide has not been mapped there previously with immunocytochemical techniques. Furthermore, since the cranial motor neurons are cholinergic, it can be inferred that CCK coexists with acetylcholine (ACh) in the trigeminal motor cells. In addition, calcitonin generelated peptide (CGRP) has been shown to be present in a sub-population of the trigeminal motor cells 16"32. Therefore a triple coexistence of ACh, CCK and CGRP potentally exists in cells of the trigeminal motor nucleus, although to substantiate this a detailed co-localization analysis would be required. Similarly, it is likely that CCK coexists with serotonin in the caudal dorsal raphe nucleus. CCK has been demonstrated to coexist with one major neurotransmitter, dopamine, in cell bodies of the substantia nigra and ventral tegmental area ~ and, more recently, with tyrosine hydroxylase (TH) and vasoactive intestinal polypeptide (VIP)/peptide histidine-isoleucine (PHI) in the rat ventral tegmental area and supramammillary region 18. A subpopulation of dopaminergic neurons in the rat ventral mesencephalon has also been found to contain both CCK and neurotensin e9. In addition, CCK has been found to coexist with substance P in the central grey31 and dorsal root ganglion 7 and with VIP in the rat hippocampus TM and visual cortex24. The absence of reported CCK-containing cell bodies in the motor nucleus of the trigeminal nerve in immunocytochemical studies is difficult to explain although not unprecedented. Recent papers utilizing in situ hybridization histochemistry have reported several CCK mRNAcontaining cells in the thalamus which immunocytochemical reports failed to mention 4'3°'35. There are several possible explanations for the disparity between the results obtained from these two techniques. The possibility of differential splicing of the CCK primary transcript, followed by the synthesis of a totally different peptide, is ruled out because a single species corresponding to the same size found in the rest of the brain is seen in the Northern analysis of rat thalamic tissue 4. A second possibility is differential processing of the precursor peptide which could yield a peptide species not recognized by the CCK antiserum used. Neurons in these regions could conceivably synthesize mRNA for CCK but this mRNA may not be efficiently translated into peptide, or the peptide product may be synthesized and then
rapidly released or degraded. According to Burgunder and Young, the most likely possibility is simply that in situ hybridization histochemistry is more sensitive in recognizing CCK cells compared to immunocytochemistry. The results and conclusions of this investigation rely on the specificity of the probes used for in situ hybridization histochemistry. Several factors argue for the specificity of the probes used in this study. First of all, discrete localization of the CCK probe occurred in several specific brain areas; second, the distribution of CCK mRNAs observed with in situ hybridization histochemistry is in general agreement with the localization of the peptide as reported in previous immunocytochemical studies. Third, the CCK probe was previously used for Northern blot analysis of total mRNA preparations under conditions of comparable stringency as used for in situ hybridization histochemistry, and a single band of appropriate length was observed 4. Fourth, pretreatment of tissue sections with enzymes which specifically degrade RNA eliminated true hybridization signals. And fifth, the pattern of hybridization seen with the CCK probe was unique compared to the cellular labeling obtained with 5 other oligonucleotide probes of similar length in the dorsal pontine tegmentum. The functional significance of coexistence of a classical neurotransmitter and a peptide is at present unclear, but previous observations point to at least one function of neuropeptides, namely to modulate the effects of coexisting classical transmitters. For example, behavioral analyses of the role of CCK in the mesolimbic pathway have employed microinjections of CCK into the nucleus accumbens alone, and in combination with central or systemic administration of dopaminergic agonists, have been recently reviewed 5"6. Administered alone at a wide range of doses, CCK had no effect on locomotor activity. However, when given in combination with dopamine, CCK potentiated dopamine-induced hyperlocomotion and apomorphine-induced stereotypy6. CCK, therefore, appears to act as a modulator of dopamine when given in low doses into the nucleus accumbens, having no effect alone, but potentiating the behavioral effects of dopamine. Since the trigeminal nerve is known to innervate the muscles of mastication, the tensor tympani and the tensor veli palatini, it would be of interest to study a possible modulatory influence of CCK on the contraction of these muscles. After submission of this paper, another report appeared that also described the presence of CCK mRNAcontaining cells in several brain regions including the motor nucleus of the trigeminal nerve 13. These results considerably substantiate the present observations.
67 Acknowledgements. The probes for CCK, substance P, CRF, galanin and TH were generously supplied by Drs. W. Scott Young III and Michael J. Brownstein, Laboratory of Cell Biology, NIMH, Bethesda, MD 20892. The CGRP probe was provided by Dr.
Michael J. Iadorola, Neurobiology and Anesthesiology Branch, NIDR, NIH, Bethesda, MD 20892. E.L.S. was supported by NIMH National Research Service Traineeship in Psychiatry, University of California, San Diego, Neurosciences 5 T32 MH18398-02.
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68 dorsal tegmental nucleus and adjacent area, J. Comp. Neurol., 270 (1988) 243-270. 34 Vanderhaeghen, J.J., Neuronal cholecystokinin. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy Vol. 4, GABA and Neuropeptides in the CNS, Part I, Elsevier, Amsterdam, 1985, pp. 406-435. 35 Voigt, M.M. and Uhl, G.R., Preprocholecystokinin mRNA in rat brain: regional expression includes thalamus, Mol. Brain Res., 4 (1988) 247-253. 36 Warden, M.K. and Young III, W.S., Distribution of cells containing mRNAs encoding substance P and neurokinin B in
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Detection of CCK m R N A in the motor nucleus of the rat trigeminal nerve with in situ hybridization histochemistry E l l e n L. Sutin 1'2'* a n d D a v i d M. J a c o b o w i t z 1'2 1Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD 20892 ( U.S.A.) and 2University of California, San Diego, La Jolla, CA 92093 (U.S.A.) (Accepted 16 January 1990) Key words: Cholecystokinin; Nucleus of the trigeminai nerve; Dorsal raphe nucleus; Acetylcholinesterase; In situ hybridization histochemistry; Rat brain
The regional distribution of dorsal pontine tegmental neurons expressing cholecystokinin (CCK) messenger RNA (mRNA) was examined using in situ hybridization histochemical techniques. A distribution of neurons containing CCK mRNA was observed in the motor nucleus of the trigerninal nerve, a region which has not been shown to contain CCK by immunocytochemical means. In addition, CCK mRNA was seen in the caudal portion of the dorsal raphe nucleus which correlates with prior immunocytochemical observations. The presence of CCK mRNA in the cholinergic motor trigeminal nucleus suggests that these cells express the CCK gene and have gone undetected in previous immunocytochemical studies. It is also suggested that CCK may have an influence on muscular contraction at sites of trigeminal nerve innervation (e.g. muscles of mastication).
INTRODUCTION Cholecystokinin ( C C K ) o c t a p e p t i d e is present in high concentrations in the m a m m a l i a n nervous system associated with sensory and m o t o r pathways 34. A n involvem e n t of the C C K system has also been p r o p o s e d in neurological and psychiatric diseases such as Parkinson's disease, H u n t i n g t o n ' s disease and schizophrenia 1'3. Furt h e r m o r e , C C K has b e e n implicated in the central control of eating b e h a v i o r 8'22. N e u r o n a l p e r i k a r y a containing C C K - i m m u n o r e a c t i v i t y are located in the cerebral cortex, h i p p o c a m p u s , ventral t e g m e n t a l a r e a and periaqueductal grey m a t t e r 11'14"2°'21'33'34. The aim of this study was to characterize the distribution of the messenger R N A ( m R N A ) coding for C C K in the dorsal pontine t e g m e n t u m and to c o m p a r e this p a t t e r n with that of the neuropeptide. In the present study we have utilized a 48-base synthetic oligonucleotide D N A p r o b e c o m p l e m e n t a r y to C C K m R N A in o r d e r to detect specific C C K gene transcripts by in situ hybridization histochemistry. This p r o b e was r a d i o l a b e l e d by using terminal deoxynucleotidyl transferase to introduce multiple [35S]dATP residues to the 3" terminus of the p r o b e . We r e p o r t here the anatomical localization of cells expressing the C C K gene
in the m o t o r trigeminal nucleus and the caudal portion of the dorsal r a p h e nucleus. MATERIALS AND METHODS Synthesis and labeling of CCK oligonucleotide probe The probe for CCK was constructed by solid phase synthesis on an applied Biosystems 380A DNA synthesizor (Foster City, CA) and purified on an 8% polyacrylamide/8 M urea preparative sequencing gel. The CCK probe was complementary to bases 315-362 of the rat cholecystokinin mRNA 9. This probe has previously been demonstrated to be specific by Burgunder and Young4 by using several controls including the use of a control probe of messenger sense coding for the last 16 amino acids of the rat vasopressin glycopeptide15. Burgunder and Young4 also performed a Northern analysis on total RNA isolated from dissected rat thalamus and found a single band of about 800 bases, in agreement with previous studies. This oligonucleotide probe was radiolabeled with 35S using the 3" end tailing method. The probe (5 nmol; final concentration 0.1 ~M) was incubated with [35S]dATP (spec. act. 1200 Ci/mmol; New England Nuclear (NEN), Boston, MA) at a final concentration of 1.0 aM (50 nmol), 150 units of the enzyme terminal deoxynucleotidyl transferase (Bethesda Research Labs [BRL], Gaithersburg, MD) in a solution containing 5x DNA tailing buffer (BRL) in a final volume of 50 #1. The reaction mixture was incubated for 10 min at 37 °C. The labeled probe was separated from the enzyme and unincorporated radiolabeled dATP residues by phenol/chloroform extraction and ethanol precipitation. In situ hybridization histochemistry In situ hybridization histochemistry was performed by using a
* Present address: Stanford University School of Medicine, Department of Psychiatry, Sleep Research Center, Stanford University, Palo Alto, CA 94305, U.S.A. Correspondence: D. Jacobowitz, Laboratory of Clinical Science, Building 10, Room 3D-48, NIMH, Bethesda, MD 20892, U.S.A. 0169-328X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
64
Fig. 1. A: distribution of the selective labeling after in situ hybridization histochemistry with a 35S-labeled CCK probe at the level of the locus coeruleus. The caudal dorsal rapbe nucleus (small arrow) and the motor nucleus of the trigeminal nerve (large arrows) are labeled in this film autoradiogram which was developed after 20 days. B: photomicrograph of individually-labeled cells of the caudal dorsal raphe nucleus after an 8 day exposure to film emulsion (x310) and C: motor nucleus of the trigeminal nerve (nV) after 21 days of exposure (× 170).
65 slight modification of protocols which have been utilized successfully with other oligonucleotide probes3s. Adult male Sprague-Dawley rats were killed by decapitation and brains were removed, frozen quickly on dry ice and warmed to -18 °C in a cryostat. 12-/~m sections were cut through the dorsal pontine tegmentum and were then thaw-mounted onto twice-coated gelatin chrome alum slides. The slides were then either stored desaccated at -80 °C until use or processed immediately. Before hybridization, sections were warmed to room temperature and allowed to dry. Sections were then fixed in 4% formaldehyde in phosphate-buffered saline (PBS, 4 °C) for 10 min. Slides were rinsed twice (1 min each) in PBS and incubated in 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCI (pH 8) for 10 min at room temperature and then transferred successively through 70% (1 rain), 80% (1 min), 95% (2 min), 100% ethanol (1 min), 100% chloroform (5 min), 100% (1 min), and 95% ethanol (2 min) and air-dried. 500,000 to 1 million CPM oligonucleotide probe in 50 gl hybridization buffer containing 0.10% D T r (dithiothreitol, Sigma, St. Louis, MO), 50% formamide, 10% dextran sulfate, 4x SSC, 1x Denhardt's solution (0.02% ficoll/0.02% polyvinylpyrrolidine/ 0.02% bovine serum albumin), 500 gg/ml denatured salmon sperm DNA, and 250/~g/ml yeast tRNA were applied to each section. Sections were then covered with a parafilm coverslip and incubated overnight at 37 °C in a humid chamber. The coverslips were removed and the sections rinsed twice in l x SSC at room temperature. Slides were then washed in four 15-min changes of 2 x SSC containing 50% formamide at 40 °C, then for two 30-min washes at room temperature in 1 x SSC, rinsed in water, dehydrated in 70% and then 95% ethanol and air-dried. A separate set of tissue preparations were treated with an RNase A solution (Sigma, St. Louis, MO; 25 #g/ml at 42 °C for 1 h) before hybridization. Competition experiments were also performed in which a 5-fold excess (25 nmol or 0.1 gM) of non-radioactive probe was prehybridized onto the sections prior to hybridization with the 35S-labeled CCK probe. In addition, to demonstrate the unique and differential hybridization distributions produced with the CCK probe, hybridizations were also performed on similar sets of tissue sections using 5 other synthetic oligonucleotide probes. These other antisense probes were complementary to substance P, (bases 124-171, Krause et al)9), corticotropin-releasing factor (CRF, bases 496-543, Jingami et a1.17); galanin (bases 115-153, Rrkaeus and Brownstein25), tyrosine hydroxylase (bases 1441-1488, Grima et al)°), and CGRP (bases 259-315, Amara et al.2). Each of these probes have been demonstrated to be specific for their respective target mRNA 12'26'36'37. Autoradiographic localization of bound probe was performed by exposure of the sections to X-ray film (XAR-5, Kodak, Rochester, NY) for 1-6 weeks. For higher resolution, the sections were dipped into NTB3 nuclear tract emulsion (Kodak) diluted 1:1 with water. Exposures ranged from 1 to 6 weeks after which the slides and/or films were developed by using Kodak D-19 developer followed by Kodak fixer and counterstained with 0.1% thionin. RESULTS B o t h brightfield and darkfield optics were used to e x a m i n e the a u t o r a d i o g r a m s and to d e t e r m i n e the distribution of l a b e l e d cells. T h e C C K oligonucleotide p r o b e revealed transcripts coding for the C C K peptide in the caudal dorsal r a p h e nucleus and m o t o r nucleus of the trigeminal nerve. Fig. 1A shows a film a u t o r a d i o g r a m in which both the midline dorsal raphe nucleus (small arrow) and the the bilateral m o t o r nucleus (large arrows) can be seen. A t the individual cell level the labeled cells in the caudal dorsal raphe nucleus a p p e a r e d to be
m o d e r a t e l y l a b e l e d and were localized p r i m a r i l y towards the midline and ventral aspect of the central gray (Fig. 1B). In m o r e rostral sections, at the level of the main serotonergic r a p h e , no cells were o b s e r v e d to contain C C K m R N A transcripts. A l m o s t all of the large m o t o r neurons in the nucleus s e e m e d to be labeled, although some cells a p p e a r e d m o r e densely l a b e l e d with grains than others (Fig. 1C). A longer e x p o s u r e of the film a u t o r a d i o g r a m s also r e v e a l e d l a b e l e d cells in the pontine reticular formation and nucleus of the t r a p e z o i d body. H o w e v e r these regions were not characterized at the individual cell level for this report. Both p r e t r e a t m e n t of the tissue sections with R N A s e and hybridization with excess cold p r o b e before hybridization with the l a b e l e d C C K p r o b e gave no autoradiographic signal, indicating that the positive signal o b t a i n e d was derived from o l i g o n u c l e o t i d e - m R N A hybrids ( d a t a not shown). Labeling with the 5 o t h e r antisense oligonucleotide probes r e v e a l e d 5 different and unique patterns of hybridizations in the dorsal p o n t i n e tegmental region of the rat brain (data not shown). Tyrosine hydroxylase m R N A was localized to the locus coeruleus and subcoeruleus only, while galanin m R N A was found in the locus coeruleus only. T h e substance P and C R F probes both hybridized specifically to the laterodorsal tegmental nucleus, while the substance P p r o b e also labeled the p e d u n c u l a r pontine t e g m e n t a l nucleus in m o r e rostral sections and the C R F p r o b e l a b e l e d cells in the dorsal p a r a b r a c h i a l nucleus. C G R P m R N A was d e m o n s t r a t e d only in the ventrolateral subdivision of the dorsal p a r a b r a c h i a l nucleus. N o n e of these 5 additional oligonucleotides labeled cells in the m o t o r nucleus of the trigeminal nerve or the caudal dorsal r a p h e nucleus, yet were found in the a p p r o p r i a t e region w h e r e its respective p e p t i d e is k n o w n to be contained. DISCUSSION We have used a synthetic oligonucleotide p r o b e comp l e m e n t a r y to rat C C K m R N A to d e m o n s t r a t e C C K gene expression in cells of the caudal dorsal raphe nucleus and m o t o r nucleus of the trigeminal nerve of the rat brain. Since the c D N A encoding rat preprocholecystokinin was cloned and sequenced by D e s c h e n n e s et al. 9, only 5 publications have r e p o r t e d on the localization of the C C K gene in rat brain with in situ hybridization histochemical techniques. Siegel and Young 3° first detected C C K m R N A c o m p l e m e n t a r y R N A p r o b e s and e x a m i n e d transcripts in the h y p o t h a l a m u s , amygdala, and striatum and these findings were replicated by O ' B r i e n et al. 23 using a c o m p l e m e n t a r y D N A p r o b e to p r e p r o C C K . Two m o r e recent reports by B u r g u n d e r and Young 4 and Savasta et al. 27 who used synthetic olig0nucleotide
66 probes to different coding regions of the CCK gene described distributions which further correlated with the earlier reports. The distribution of cells containing the mRNA for CCK in the caudal dorsal raphe is in good agreement with previous immunocytochemical studies 2°' 33,34
The findings of CCK mRNA in the motor nucleus of the trigeminal nerve is an important one in that the CCK peptide has not been mapped there previously with immunocytochemical techniques. Furthermore, since the cranial motor neurons are cholinergic, it can be inferred that CCK coexists with acetylcholine (ACh) in the trigeminal motor cells. In addition, calcitonin generelated peptide (CGRP) has been shown to be present in a sub-population of the trigeminal motor cells 16"32. Therefore a triple coexistence of ACh, CCK and CGRP potentally exists in cells of the trigeminal motor nucleus, although to substantiate this a detailed co-localization analysis would be required. Similarly, it is likely that CCK coexists with serotonin in the caudal dorsal raphe nucleus. CCK has been demonstrated to coexist with one major neurotransmitter, dopamine, in cell bodies of the substantia nigra and ventral tegmental area ~ and, more recently, with tyrosine hydroxylase (TH) and vasoactive intestinal polypeptide (VIP)/peptide histidine-isoleucine (PHI) in the rat ventral tegmental area and supramammillary region 18. A subpopulation of dopaminergic neurons in the rat ventral mesencephalon has also been found to contain both CCK and neurotensin e9. In addition, CCK has been found to coexist with substance P in the central grey31 and dorsal root ganglion 7 and with VIP in the rat hippocampus TM and visual cortex24. The absence of reported CCK-containing cell bodies in the motor nucleus of the trigeminal nerve in immunocytochemical studies is difficult to explain although not unprecedented. Recent papers utilizing in situ hybridization histochemistry have reported several CCK mRNAcontaining cells in the thalamus which immunocytochemical reports failed to mention 4'3°'35. There are several possible explanations for the disparity between the results obtained from these two techniques. The possibility of differential splicing of the CCK primary transcript, followed by the synthesis of a totally different peptide, is ruled out because a single species corresponding to the same size found in the rest of the brain is seen in the Northern analysis of rat thalamic tissue 4. A second possibility is differential processing of the precursor peptide which could yield a peptide species not recognized by the CCK antiserum used. Neurons in these regions could conceivably synthesize mRNA for CCK but this mRNA may not be efficiently translated into peptide, or the peptide product may be synthesized and then
rapidly released or degraded. According to Burgunder and Young, the most likely possibility is simply that in situ hybridization histochemistry is more sensitive in recognizing CCK cells compared to immunocytochemistry. The results and conclusions of this investigation rely on the specificity of the probes used for in situ hybridization histochemistry. Several factors argue for the specificity of the probes used in this study. First of all, discrete localization of the CCK probe occurred in several specific brain areas; second, the distribution of CCK mRNAs observed with in situ hybridization histochemistry is in general agreement with the localization of the peptide as reported in previous immunocytochemical studies. Third, the CCK probe was previously used for Northern blot analysis of total mRNA preparations under conditions of comparable stringency as used for in situ hybridization histochemistry, and a single band of appropriate length was observed 4. Fourth, pretreatment of tissue sections with enzymes which specifically degrade RNA eliminated true hybridization signals. And fifth, the pattern of hybridization seen with the CCK probe was unique compared to the cellular labeling obtained with 5 other oligonucleotide probes of similar length in the dorsal pontine tegmentum. The functional significance of coexistence of a classical neurotransmitter and a peptide is at present unclear, but previous observations point to at least one function of neuropeptides, namely to modulate the effects of coexisting classical transmitters. For example, behavioral analyses of the role of CCK in the mesolimbic pathway have employed microinjections of CCK into the nucleus accumbens alone, and in combination with central or systemic administration of dopaminergic agonists, have been recently reviewed 5"6. Administered alone at a wide range of doses, CCK had no effect on locomotor activity. However, when given in combination with dopamine, CCK potentiated dopamine-induced hyperlocomotion and apomorphine-induced stereotypy6. CCK, therefore, appears to act as a modulator of dopamine when given in low doses into the nucleus accumbens, having no effect alone, but potentiating the behavioral effects of dopamine. Since the trigeminal nerve is known to innervate the muscles of mastication, the tensor tympani and the tensor veli palatini, it would be of interest to study a possible modulatory influence of CCK on the contraction of these muscles. After submission of this paper, another report appeared that also described the presence of CCK mRNAcontaining cells in several brain regions including the motor nucleus of the trigeminal nerve 13. These results considerably substantiate the present observations.
67 Acknowledgements. The probes for CCK, substance P, CRF, galanin and TH were generously supplied by Drs. W. Scott Young III and Michael J. Brownstein, Laboratory of Cell Biology, NIMH, Bethesda, MD 20892. The CGRP probe was provided by Dr.
Michael J. Iadorola, Neurobiology and Anesthesiology Branch, NIDR, NIH, Bethesda, MD 20892. E.L.S. was supported by NIMH National Research Service Traineeship in Psychiatry, University of California, San Diego, Neurosciences 5 T32 MH18398-02.
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