Localization of an endoplasmic reticulum calcium ATPase mRNA in rat brain by in situ hybridization

Neurosuence Vol 43, No 1, pp 1-9, 1991

Printed m Great Britain

0306-4522/91 $3 00 + 0 00

Pergamon Press plc ,c, 1991 IBRO

LOCALIZATION OF A N ENDOPLASMIC RETICULUM CALCIUM ATPase m R N A IN RAT BRAIN BY IN S I T U HYBRIDIZATION K K MILLER, A. VERMA, S H SNYDER* a n d C A Ross Departments of Neurosoence, Pharmacology and Molecular Soences, Psychmtry and Behavmral Sciences, Johns Hopkins Umverslty School of Medicine, 725 North Wolfe Street, Baltimore, MO 21205, U S A Abstraet--SERCA-2 is an endoplasmlc ret~culum Ca 2+ ATPase present m brain [Gunteskl-Hambhn el al (1988) J b m l C h e m 263, 15032 15040] We sought to map the dlstnbutmn of this pump m the rat brain and investigate its relationship to C a 2+ uptake by brain endoplasmlc retlculum Using m sttu hybridization and Northern blots with antlsense ohgonucleotlde probes, we found that SERCA-2 is concentrated most densely m the cerebellum, especmlly m Purkmje cells, and m the hlppocampus, with heavy labehng also in cortex, thalamus, pontme nuclei and the mltral cell layer of the olfactory bulb ~SCa~+ uptake d~splayed a s~mllar pattern w~th heaviest accumulation m cerebellum, hippocampus, cortex, thalamus and olfactory bulb In corpus striatum and substantm mgra, relative 45Ca~* accumulatmn was greater than SERCA-2 mRNA Thus, SERCA-2 appears to be involved m Ca z+ uptake into endoplasm~c ret~culum m brain for release by mos~tol 1,4,5-trlsphosphate and other agents A-M

Levels of free mtracellular Ca 2+ m e d m t e diverse processes in neurons, including regulation of neurot r a n s m i t t e r release a n d responses to receptor actlv a t l o n . 8'22"23 C a :+ &spositlon varies strikingly in different n e u r o n a l p o p u l a t i o n s t h r o u g h o u t the b r a i n Receptors for mosltol 1 , 4 , 5 - t n s p h o s p h a t e (IP3), which release mtracellular stores of Ca 2+ into the cytoplasm, exist m m u c h higher densities m Purkmje cells o f the cerebellum t h a n in any o t h e r brain 100 2~2s P u r k m j e cells are also greatly enriched in various C a 2+ binding proteins 24 a n d display a unique Ca 2+ action p o t e n t m l in their dendrites ~8 Cytoplasmic levels of free Ca 2+ are regulated by activity of the Ca 2+ A T P a s e o f the endoplasmlc reticulum (ER), the organelle which c o n t a i n s the largest stores of Ca 2+ within cells 8 Intracellular A T P - d e p e n d e n t sequestration o f 45Ca 2÷ by brain ttssue has been d e m o n s t r a t e d in a n u m b e r of recent

W e sought to locahze m R N A for Ca 2+ p u m p s of i m p o r t a n c e for n e u r o n a l Ca 2~ d l s p o s m o n m the brain Molecular cloning has revealed at least three separate genes for E R Ca 2+ ATPases: one expressed m fast twitch skeletal muscle (sarcoplasmlc a n d endoplasmlc retlculum calcium ATPase, S E R C A - I ) but n o t m b r a i n y ° one which is present m the brain as well as cardmc, slow-twitch skeletal muscle, a n d s m o o t h muscle (SERCA-2), m52~a n d one which occurs in m a n y tissues but apparently with relatively low levels in the brain ( S E R C A - 3 ) 7 Two lsoforms exist of the S E R C A - 2 protein which are coded for by several m R N A species In o u r present studies we endeavored to localize, by m s~tu hybr~dlzatmn, m R N A for the S E R C A - 2 C a 2~ A T P a s e

studies 21214 However, there is relatively limited mform a t l o n on the regional distribution m brain o f 4SCa 2+ uptake Recently, we localized C a 2+ u p t a k e assoclated with E R by a u t o r a d l o g r a p h y with 4SCa~+ in

Rats (175-250 g, male Sprague Dawleyl were killed by decap~tatmn For some experiments the rats were deeply anesthetized w~th pentobarbltal and 20 mg of ~botemc acid in 1/~1 0 1 N NaOH was rejected stereotaxlcally into the left hlppocampus one week prmr to use Brains were rapidly removed, embedded m Tlssue-Tek (Miles, Napervflle, IL) or brain paste and frozen on dry ice Sections (12#m thick) were cut m a cryostat onto slides coated wath gelatin/chrome alum, or poly-L-lyslne, and stored at - 7 0 ' C In s~tu hybridization was done by methods previously described with minor modifications H 2529 Solutions were made with demnlzed dmtflled water treated with 0 1% dlethylpyrocarbamate and autoclaved Sections were fixed m 4% freshly depolymenzed paraformaldehyde m phosphate-buffered sahne (PBS) for 5 mm and then rinsed twice in PBS At this point, some control sections were incubated with RNAase A (0 2 mg/ml m PBS) for 60 mm at 37 'C, then washed twice with PBS All sections were acetylated with 0 25% acetic anhydride/0 1%

rat b r a i n sections ~.7 Llmlted l m m u n o h l s t o c h e m l c a l studies indicate high densities o f a Ca ~+ A T P a s e in cerebellar P u r k m j e cells,~7 but lsoforms o f this enzyme have not been localized *To whom all correspondence should be addressed DTT, dithiothreltol, EDTA, ethylenedlammetetra-acetate, ER, endoplasmie retlculum, HEPES, N-2-hydroxyethylpxperazme-N'-2-ethanesulfomc acid, IP3, mosltol 1,4,5-tnsphosphate, PBS, phosphatebuffered saline, PEG, polyethylene glycol, PLC, phosphohpase C, SERCA, sarcoplasmlc and endoplasmlc retlculum calcmm ATPase, SSC, sodmm/sallne otrate

Abbret,tanons

EXPERIMENTAL PROCEDURES

2

k. K MII.LER el ai

trlethanolamlnem09% NaCl(pHS.0)for t0mln Sections were then dehpldated m chloroform for 5- 10 mm (preceded and followed by a graduated series of ethanols) and dried Ohgonucleotlde probes were synthestzed m an Apphed Blosystems Model 380B or a MulUgene/Blosystems Model 7500 DNA synthesizer and purified by high performance hquld chromatography All probes were 45 bases long Three SERCA-2 sequences were chosen emptoymg sequences common to SERCA-2A and B Probe I corresponded to nucleotJdes 1606-1650 of rat brain cDNA RB 2 5 : Probe 2 corresponded to nucleotldes 2056 2 1 0 0 Probe 3 corresponded to nucleotldes 2506 2550 A fourth probe, corresponding to nucleotldes 4627--4671 of RB 4 17) 5 is present m class 2, 3 and 4 mRNA and codes selectively for the SERCA-2B xsoform For each sequence mRNA, both complementary (antisense) and identical (sense) onentatlon probes were synthesized Probes were Y-end-labeled with terminal transferase (50 70 units) (Bethesda Research Laboratories) and [~sS] dATP (1200C1/mmol, NEN/Dupont, Boston, MA) to a specific actwny of approximately 9600 Cl/mmol and separated from unincorporated nucleotides by column chromatography (NENSORB 20 column, NEN/Dupont) For labehng reactions, probes were labeled separately or equal amounts of the three anUsense or three sense probes were mixed together (10pmol of probes with 100pmol labeled dATP) Probes were suspended m hybndlzauon buffer [4 x sodmm/sahne citrate (SSC),'I x Denhardt's solution,' l mM EDTA/2OmM phosphate buffer, pH 7 4/0 1% denatured salmon sperm DNA/0 1% yeast tRNA/10% dextran sulfate, 50% delomzed formamlde (Bethesda Research Laboratories)] (SSC = 0 15M NaCI/0.015 M sodmm c~trate, 1 x Denhardt's soluuon = 0 02% bovine serum atbumm/O 02% Ficol1400/0 02% potywnylpyrrolidone) and 106 c p m of probe m 100 ,ul was apphed per section The hybrtdlzation buffer over the secuons was covered w~th paraffin film and sections were incubated in a humid atmosphere overmght at 37-C In some control experiments, th~s incubation was carried out m the presence of 100-fold excess unlabeled probe The next day, sections were washed m three changes of 1 x SSC at 5YC for a total of I h In melt curve experiments, sechons were washed m l x SSC at 35 'C, 45°C or 55°C or m 1 x SSC at 55-'C plus various amounts of fonnamlde 15% (approximating a wash temperature of 65~C), 30% (approximating 75~C), 45% (approximating 85 'C) or 60% (approxlmatmg 95~C) Sections were washed for a further 30 mm m t x SSC at room temperature, dipped once m cold water to nnse offsalt and dned under a stream of cool air Sections were apposed to beta-sensmve film (Amersham Betamax) for five to 15 days and developed m Kodak DI9 Some sections were &pped m Kodak NTB2 emulsion diluted 1 1 in water, exposed for three to stx weeks and developed m Dl9 Autora&ograms were quantzfied using an image analysis system (RAAS 1000, Amersham, Arhngton Heights, IL) For Northern blots, probes were labeled with [~32p]dATP (5000 C1/mmol) using tenmnal transferase, to a specific actlvay of about 40,000 Cl/mmol RNA was purified by acid guamdmmm/phenol/chloroform extraction,9 fractlonated on a 1% agarose gel w~th 0.66 M formaldehyde as denaturant ~t and transferred to nylon membranes (Nytran, 045/am, Schleicher and Schuell, Keene, NH) Membranes were incubated with the labeled probe at 37~C overmght in hybndizat~on buffer (4 x SSC/5 x Denhardt's solution/ 0 1% sodium dodecyl sulfate/100 mg per ml salmon sperm DNA/50% deiomzed formam~de) Wash condmons were ~dentlcal to those used for m sttu expenments. Membranes were allowed to dry and apposed to beta-sensmve film for three to seven days Autora&ograms were quantified with an ~mage analys~s system using eth~dmm bromide fluorescence of ribosomal RNA to correct for amount of RNA loaded per lane (RAAS 1000 Amersham)

For the 4~Ca2~ uptake assay. Irozen rat brain ~et.hoxl, were allowed to thaw and then premcubated m permeablhzatlon buffer containing 0 I M KCt, I0 mM HEPES Tr~, pH 7 5, 3% polyethylene glycol 1PEG, average mol ,~t 8000), l mM dlthlothreltol (DTT) and 10 #M dlgltonm for 10 mm at 25:C, to selectively permeabdlze plasma membrane, but leave the ER membrane intact Secuons were then transferred into uptake buffer containing 0 15 M KCI, 10mM HEPES-Tns, p H 7 5 . 3% PEG, 2ram MgCI~, 2mM K~ATP, I0mM phosphocreatme. 20#g,:m/creatme phosphokmase, 100,uM total CaC1z, 0 l#Cl,'ml 4'CaCI., (NEN-DuPont), 2 mM DTT and mdtcated addttlons Free calcmm concentrations were adjusted to desired ~alues with [3H]-EGTA using a Ca -'~ sensltwe electrode (Onon, Charlestown, MA) Incubations were performed m plastic slide marling vessels (Evergreen, Los Angeles, CA) or m glass staining chambers Buffer volumes were adjusted to gwe approxtmately 50 ug/ml protein m the assa~ At m&cated Umes slides were removed from the uptake medmm and transferred into wash buffer containing 0 l M KCI, 10 mM NaCI, 10 mM HEPES-KOH. pH 7 0, 3% PEG and 1 mM EDTA at 4' C After washing m this buffer tbr 5 mm, sections were dried under a cool air stream and either apposed to beta sensmve film (Beta Max, Amersham) or d~pped m Kodak NTB2 emulsion dduted 1 1 an water and developed m Kodak de,mloper DI9 followmg 12 24h exposure RESULTS

Northern blot analysis o / S E R C A - 2 and pertpheral ttssues

rnRNA m brain

Burk et al 7 m e a s u r e d m R N A for the two forms o f S E R C A - 2 by N o r t h e r n blot S E R C A - 2 A m R N A levels were extremely high in heart and skeletal

muscle with very low levels in brain and other Ussues, whereas S E R C A - 2 B levels were quite htgh m brain and numerous other tissues. 7 Our Northern blots employed a probe that reacts wath both SERCA-2A and S E R C A - 2 B (Fig. 1) We observed discrete bands at 4 4, 6.2 and 7.5 kb similar to results o f Burk et al. 7 The 4.4 b a n d was m o s t intense in heart but also occurred in skeletal muscle, stomach and kidney with neghglble levels m hver. The 6 2 kb band was prominent m the brain with htghest levels m the cerebellum, next highest levels in the h l p p o c a m p u s and pons, and

progresswely lower levels m other brain regtons and httle or no expression m peripheral tissues. The 7.5 kb b a n d was famter than the other two b a n d s and had a widespread &stribution with some enrxchment m bram. The three different probes were m~xed together for the blot m Fig. l W h e n blotted separately on mixed regions o f bram, each o f the probes revealed all three bands (4.4, 6 2 and 7.5) In N o r t h e r n blots to c o m p a r e S E R C A - 2 and S E R C A - 3 h y b n & z a t i o n to R N A from mixed b r a m regtons, S E R C A - 2 hybrldizatton was substantmlly more intense (data not shown). In s~tu hybrtdizatton locahzation o f S E R C A - 2 m R N A

tn peripheral ttssues o f fetal rat and in adult rat bram To assess the &stributton o f S E R C A - 2 m R N A

throughout the b o d y m developing ammals, we conducted in situ hybridization on whole body sections o f the 21-day fetal rat (Fig. 2). G r a m density was

Endoplasmlc retlculum calcmm ATPase mRNA

3

=9.5 "7.5 -4.4

-2.4 -18S -1.4 Fig 1 Tissue &stnbutlon of the mRNAs encodmg SERCA-2 mRNA from the mdtcated tissues and regions of rat brain was analysed by Northern blot Northern blot hybridization was performed as described in Experimental Procedures The posttlons and sizes (m kb) of the RNA markers and the 18 S and 28 S ribosomal RNA subumts are shown on the right highest by far in the heart with grains more concentrated m the ventricle than the adjacent aorta, By contrast, neghgible gram levels were apparent m the hver lnterme&ate gram densities were present m skeletal muscle of the tongue and smooth muscle of the intestines. The nasal eplthehum &splayed high gram densities. Moderate levels of grams were observed throughout the brain with no special concentrat~on m the ~mmature cerebellum, m which Purklnje cells have not yet developed. This gram distribution approximates to the relative levels of S E R C A - 2 m R N A by Northern blot analysis in the adult, with highest levels m the heart, negligible levels in the kidney and intermediate values in skeletal muscle 7 The specificity of the zn s l t u hybn&zatlon techtuque was shown by elimination of label in samples treated with 100-fold excess of unlabeled probe or pretreated with RNAase, and m the lack of label in sections incubated with ra&olabeled sense probe of the same specific actlwty (data not shown) In melt

curve analysis, probes dissociated at 65-70 C with a sharp transition point consistent w~th specific labehng. In adult rat brain striking differences were apparent in various regions (Figs 3A, 4 and 5) The highest labehng in the brain was m cerebellum, with grain densities most highly concentrated over the Purkmje cell layer, substantially lower levels m the granule cell layer and even lower levels in the molecular layer Photomicrographs disclosed extremely dense label selectively over Purklnje cell somata (Fig 5A, B) Most of the bramstem, especially the reticular formatlon, displayed very low grain densities with the striking exception of very high densities in the pontine nuclei (Figs 3A and 4 A - F ) The superficml layer of the superior colliculus also had moderate labehng (Figs 3A and 4D, E) G r a m densities were low m most of the hypothalamus with the exception of the supraoptlc and paraventricular nuclei By contrast, the thalamus possessed substantial labeling (Fig 4H, I) The

4

K K MILLER et al

Fig 2 Locahzatlon of SERCA-2 mRNA by m s'ttu hybn&zatlon in 21-day-old rat fetus The print wd,, made directly from the X-ray film so the hybrldizaUon signal is white The region of the heart wa,s underexposed to prevent fogging of nearby regaons In sttu hybri&zatlon was performed as described 1:) Experimental Procedures Ad, adrenal, Ao, aorta, Cb, cerebellum, Cx, cortex, D, dorsal forebram, H, hmdbram, He, heart, LI, liver, Lu, lung, LI, large intestine, LV, lateral ventricle, M, mldbraln NE, nasal eplthehum, P, pituitary, SI. small intestine, Th, thymus, To, tongue

i!

J Fig. 3. Localization of SERCA-2 mRNA and 45Ca2+ uptake in saglttal section of rat brain In sztu hybndlzatton and 4SCa2+ uptake were performed as described m Experimental Procedures Representatwe sections are shown. (A) In sttu hybrtdlzatlon, posmve signal is white. Non-specific binding On presence of RNAase or excess unlabeled probe, or use of sense probe) m all cases was not above film background. (B) Accumulation of 4SCa2+ into mtracellular stores of brain sections When performed m presence of A23187, result was not above film background Cbl, cerebellum, Ctx, cortex, H, hlppocampus; M, medulla; OB, olfactory bulb, PN, pontme nucleus, Str, stnatum; SN, substantm mgra, Th, thalamus

Endoplasmlc retlculum calcmm ATPase mRNA

•

5

DTN

E

pN

t

-')C

'RN F

/

DG

~,~'

Pyr

G

S

k

CC "

"/~at

Fig 4 Locahzatlon of SERCA-2 mRNA in coronal sections of rat brain In sttu hybridization was performed as described m Experimental Procedures. SecUons run caudal (A) to rostral (L). AC, anterior commissure, BLA, basolateral amygdaloid nucleus, CC, corpus callosum; CP, chorold plexus; Cb, cerebellum, DG, dentate gyrus, DTN, dorsal tegmental nucleus, FC, frontal cortex; FN, facial nucleus, IC, inferior colhculus, LS, lateral septum, NLOT, nucleus of the lateral olfactory tract, PN, pontme nucleus, PVN, periventncular nucleus, Plr, preform cortex, Pyr, pyramidal cell layer of hlppocampus, RN, red nucleus, SC, superior colhculus; SON, supraopt~c nucleus, STT, spinal tngemmal tract, Str, strmtum, Thai, thalamus, VMN, ventromedxal nucleus of the hypothalamus

6

K tq MILLERet

al

j.

Fng 5 Cellular locahzatnon of SERCA-2 mRNA m cerebellum, olfactory bulb and hlppocampus In ~'~tu hybn&zatnon and autoradlograpby were performed as described A and B are cerebellum C and D arc olfactory bulb E and F are bippocampus A, C and E are brightfield xmages B, D and F are darkfield images. CP, chorold plexus, DCN, deep cerebellar nuclei; DG, dentate gyrus, GCL, granule cell layer, EPL, external plexnform layer, Go, glomeruli; MCL, mltral cell layer, ML, molecular layer, PCL, Purkmje cell layer

caudate--putamen also had heavy labeling, with lower levels over the globus pallidus (Figs 3 and 4J-L) The hlppocampus displayed very high gram densities over the pyramidal cell layers Granule cells

of the dentate gyrus were also heavily labeled but somewhat less than pyramidal cells (Figs 3, 4 F - H , 5E, F). The lateral septal nucleus, which contams cells of origin for tracts that innervate the hippocampus,

Endoplasmlc reUculum calcium ATPase mRNA was also heavily labeled (Figs 3A and 4K) The olfactory tubercle and basal forebraln displayed moderate levels of label (Figs 3A and 4K, L) Grain densities were moderate throughout the cerebral cortex, with higher levels in the pyriform cortex. Densities were approximately equal in the different cortical layers (Figs 3A and 4E L) The olfactory bulb had moderate levels of label, with high accumulations of grains selectively over the mltral cells (Figs 3A and 5L, D) Label over white matter areas throughout the brain was low (Figs 3 and 4). Label over chorold plexus of the lateral and fourth ventricle was moderate, while labeling over ependymal cells was low (Figs 4B, 5 and data not shown) We quantified a representative hybridized saglttal section (Table l) Relative levels resembled the Northern blot analysis with cerebellum showing the highest density of S E R C A - 2 and hlppocampus the second highest, Table l Quantltation of SERCA-2 mRNA in various regions of rat brain from in ~ttu hybridized saglttal sections Optical density Brain region units Cerebellum cortex 32 deep cerebellar nucleus 8 Hlppocampus pyramidal cell layer 25 dentate gyrus 14 total 17 Frontal cortex 20 Thalamus ventrobasal complex 24 medial dorsal 19 Amygdala basolateral 19 Lateral septum 19 Medial septum 7 Caudate putamen 17 Hypothalamus paraventrlcular nucleus 17 ventromedlal nucleus 13 anterior hypothalamlc area 13 supraoptlc nucleus 13 Medial geniculate body 15 Dorsal tegmental nucleus 15 Olfactory bulb 14 Bed nucleus 14 Facial nucleus 14 Cochlear nucleus 13 Nucleus of the diagonal band 13 Pontlne nucleus 12 reticular formauon 6 total 9 Vestibular nucleus 12 Zona lnserta 12 Spinal trlgemlnal nucleus 10 Chorold plexus 10 Inferior colhculus 9 Superior colliculus 8 Mldbram central gray 7 reticular formation 6 Nucleus of stria termmahs 6 Medulla reucular formauon 6 Inferior ohvary nucleus 5 Corpus callosum 5 SERCA-2 mRNA was quantltated as described in Experimental Procedures A representative in ~itu hybridized saglttal section of rat brain was used

7

In sttu hybridization of rat brain saglttal sections using probe 4 which corresponds to m R N A codmg for the longer C-terminus found only in S E R C A - 2 B lsoform was also performed (data not shown) The distribution was very similar to that shown above for the other probes with cerebellum and hlppocampus most heavily labeled Comparison o f Ca -,+ uptake and S E R C A - 2

mRNA

locahzattons

Recently we have developed a technique that permlts the visualization of 45Ca:+ accumulated by the E R Ca 2+ ATPase in brain slices. 27 We have compared the locahzatIons of S E R C A - 2 m R N A and accumulated 45Ca2+ (Fig. 3A, B) Overall the locahzatlons were quite similar with 45Ca2+ uptake and S E R C A - 2 m R N A both most concentrated in the cerebellum aSCa2+ uptake extended into the molecular layer where S E R C A - 2 m R N A was lower, presumably reflecting the restriction of m R N A to cell bodies, whereas the Ca :+ pump was also expressed in Purklnje cell dendrites in the molecular layer 45Ca2~ was relatively high in the substantla nlgra, basal forebraln and strlatum, while S E R C A - 2 m R N A levels m these regions were relatively low The bralnstem displayed low levels of both aSCap' ~ accumulation and SERCA-2 m R N A (Fig 3) High levels of both 45Ca 2+ uptake and S E R C A - 2 m R N A were observed in the hlppocampus, cerebral cortex and caudate-putamen The hypothalamus had markedly lower levels of both 45Ca2+ and SERCA-2 labeling than the thalamus Within the thalamus, highest levels of both label were in the ventrobasal complex ~SCa-'+ levels, like those of S E R C A - 2 m R N A , were moderate in the basal forebrain and olfactory bulb mltral cell layer Throughout the brain, 45Ca2 ~ was lOW In white matter, as for SERCA-2 mRNA DISCUSSION The m sttu hybridization technique employed here appears to selectively label S E R C A - 2 m R N A Lack of label with excess unlabeled antlsense probe, after pretreatment with R N A a s e or with sense probe of the same specific activity, support the specificity of labeling Moreover, the distribution of labeling in peripheral tissues and in regions of the brain parallels m R N A levels determined by Northern blot analysis Three genes for E R or sarcoplasmlc retlculum Ca-" + ATPases have been distinguished by molecular cloning 4 15,1920,21 S E R C A - I predominates in skeletal muscle and IS not expressed in the brain 4 SERCA-3 occurs in numerous peripheral tissues with relatively low levels In the brain 7 S E R C A - 2 appears to be the ~

predominant Ca :+ ATPase of brain and, accordingly, was the focus of our m 5ttu hybridization study The S E R C A - 2 probes used for most of our experiments bind equally well to m R N A encoding the S E R C A - 2 A and S E R C A - 2 B lsoforms The 6 2 kb band we

8

K K MILLERet al

observed m Northern blots, present only m brain, reflects S E R C A - 2 B class 4 m R N A , while the 4 4 kb band, reflecting both S E R C A - 2 A class 1 m R N A S E R C A - 2 B class 2 m R N A is highly concentrated in peripheral tissues. 7 The 7.5 kb band which is faint but present m all tissues reflects S E R C A - 2 B class 3 m R N A S E R C A - 2 A class 1 m R N A corresponds to Gunteskl-Hambhn el al ~s RS 8-17. S E R C A - 2 B class 2 m R N A corresponds to R K 7-12. S E R C A - 2 B class 3 m R N A corresponds to RB 4-1 7. S E R C A - 2 B class 4 m R N A corresponds to RB 2-5 It is hkely that the autoradlographlc grains observed in our tn sltu hybridization studies predominantly demonstrate S E R C A - 2 B m R N A , the principal E R Ca 2+ pump of brain. This fits with our finding that probe 4, selective for SERCA-2B, displays the same locahzatlons obtained with probes that recogmze both S E R C A - 2 A and 2B Because of the speoal ~mportance of calcmm ions in the regulation of excitability in neuronal cells, 8'18 the brain Is enriched m lntracellular calcmm uptake and release mechamsms. Recent evidence suggests there are at least two E R Ca 2+ p o o l s - - a n IP 3 releasable, and an IP 3 lnsensmve pool. ~° ~3.27However, whether there is similar compartmentahzatlon of c a l o u m uptake mechamsms is uncertain. In the present study, the distribution m brain of S E R C A - 2 m R N A is very slmdar, though not ldentlcal, to that of total ATP-dependent c a l o u m uptake into E R of brain sections. That these two patterns are simdar is consistent with the idea that the calcium pump encoded by the S E R C A - 2 m R N A is physiologically important in brain intracellular Ca 2+ handling That there are some differences (e.g m caudate-putamen, basal forebrain and pontine nuclei) suggests functional subspecialization of calcium uptake pumps, m brain, as has been suggested in adrenal medulla 6 Perhaps other Ca: + pumps, such as the S E R C A - 3 gene product are especially important m these regions. In addition, the differences may also reflect varmtlons m the techniques S E R C A - 2 m sltu hybndlzauon locahzes the m R N A that codes for the pump and is therefore limited to cell bodies, while 4SCa 2+ uptake can occur throughout the cells" processes, Our results suggest that the calcmm pump encoded by the S E R C A - 2 m R N A causes calcium entry into both the IP~ sensmve and insensitive pools In most

areas of the brain with high S E R C A - 2 levels, 4~Ca2 entry is at least partly IP 3 sensltwe, e.g the cerebellure, cortex and caudate-putamen. 27 However. m some regions with high SERCA-2 levels, 4SCa2" uptake is almost entirely IP 3 insensitive, e g the lateral septum, the pontme nuclei and portions of the hlppocampus 27 In regions with both high SERCA-2 levels and hlgh IPa-sensltwe 4SCa2+ uptake levels, the S E R C A - 2 gene product may be revolved m calcium handhng related to phospholnositlde turnover. We have previously found high levels of phospholipase C (PLC) lsozyme m R N A m regions with high expression of S E R C A - 2 m R N A , e.g. PLC~ m the cerebellar Purkmj¢ cells and PLCfl in cortex and caudate-putamen. 2~ These regions contain receptors known to actwate phosphomosltlde t u r n o v e r - - m e t a b o t r o p l c glutamate receptors in the cerebellum, 3 and muscanmc chohnerglc receptors m the cortex and caudate-putamen -~ The selectively high levels of S E R C A - 2 m R N A autoradlographic grains in cerebellar Purkmje cells fit with an increasing body of evidence pointing to a uniquely important role of Ca 2+ m Purkmje cells 24 The Ca 2+ ATPase has also been localized to Purkmje cells at very high levels using immunofluorescence ~7 Purkmje cells possess a Ca 2 ) action potential in their dendrites, which transfers large amounts of Ca 2+ into the interior of the cell. 1't6'18 One function of the E R Ca 2. pump would be to sequester this Ca 2~ Additionally, E R Ca 2+ is the source for IP3 releasable stores. IP3 receptors are enriched in Purkmje cells much more than any other central nervous system neuron 26,28 ip 3 can release the majority of E R stores in Purkinje cells 27 A slmdarly high concentration of IP3 receptors along with 4SCa2* accumulation and Ca 2+ pump m R N A occurs m the hlppocampus Like the Purkmje cells of the cerebellum, ventricles of the heart are greatly ennched in Ca 2' pump m R N A and possess a prominent Ca 2. action potentml, which determines cardiac contractility This supports a role of the E R Ca 2 ~ pump in sequestering Ca 2+ entering the cell wa the Ca 2 + action potential A c k n o w l e d g e m e n t s - - A V is supported by USPHS training

grant ES 07141 S H.S ~s supported by USPHS grant MH-18501, Research Scientist Award DA-00074 and a gift of the Bristol-Myers-Squibb Company C A R. is supported by NIHM MH 43040, a grant from NARSAD and he is a Pew Scholar m the Biomedical Soences

REFERENCES

1. Andrews S B, Leapman R. D , Landis D M D and Reese T S (1988) Activity-dependent accumulation ofcalcmm in Purkmje cell dendritic sprees Proc natn Acad Sct USA 85, 1682-1685. 2 Berrldge M J and Irvlne R F (1989) Inosltol phospates and cell s~gnalhng Nature 341, 197-205 3 Blackstone C. D., Supattapone S. and Snyder S H. (1989) Inosltot phospholipid-linked glutamate receptors medmte cerebellar parallel-fiber-Purkmje-cell synaptic transmms~on Proc. natn Acad. ScL USA 86, 4316-4320. 4 Brandl C J , Green N M , Korczak B and MacLennan D H (1986) Two Ca ~+ ATPase genes: homolognes and mechamst~c lmphcat~ons of deduced amino acid sequences. Cell 44, 597-607 5. Buckley N J , Bonner T. I and Brann M. R (1988) Locahzatlon of a family of muscarimc receptor mRNAs in rat brain J Neurosct 8, 4646-4652 6 Burgoyne R D , Cheek T R., Morgan A , O'Sullivan A J , Moreton R. B., Berrldge M J , Mata A. M , Colyer J , Lee A. G and East J M (1989) Dlstnbut~on of two chstmct Ca2+-ATPase-hke proteins and their relationships to the agomst-sens~twe calcium store m adrenal chromaffin cells Nature 342, 72-74

Endoplasmlc reticulum calcium ATPase mRNA

9

7 Burk S E . Lytton J , MacLennan D. H and Shull G E (1989) cDNA cloning, functional expression and mRNA tissue distribution of a third organellar Ca 2+ pump J btol Chem 264, 18561 18568 8 Carafoh E (1987)Intracellular calcium homeostasis A Rev Blochem 56, 395-433 9 Chomczynski P and Sacchi N (1987) Analyt. Blochem 162, 156-159. 10 Daniell L C and Harris R A (1989) Ethanol and mositol 1,4,5-trlphosphate release calcium from separate stores of brain microsomes J Pharmac exp Ther 250, 875-881 11 Davis L G , Dibner M O and Battey J F (1986) Basu" Methods m Molecular Biology Elsevier, New York 12 Gandhi C R and Ross D H (1987) Inosltol 1,4,5-trasphosphate induced mobilization of Ca 2+ from rat brain synaptosomes Neurochem Res 12, 67 72 13 Ghosh T K , Mullaney M M , Tarazi F I and Gill D L (1989) GTP-activated communication between &stlnct lnositol 1,4,5-trlphosphate-sensitive and -insensitive calcium pools Nature 340, 236-238 14 Gill D L , Chueh S H and Whittow C L (1984) Functional importance of the synaptlc plasma membrane calcium pump and sodium-calcium exchanger. J btol Chem 259, 10807-10813 15 Gunteskl-Hambhn A - M , Greeb J and Shull G E (1988) A novel Ca -,+ pump expressed in brain, kidney and stomach is encoded by an alternative transcript of the slow-twitch muscle sarcoplasmlc reticulum Ca-ATPase gene J htol Chem 263, 15032 15040 16 Hockberger P E , Tseng H -Y and Connor J A (1989) Fura-2 measurements of cultured rat Purklnje neurons show dendritic localization of Ca 2+ influx. J Neurosct 9, 2272 2284 17 Kaprlehan Z , Campbell A M and Fambrough D M (1989) Identification of a Ca2+-ATPase in cerebellar Purklnje cells Molec Brain Res 6, 55-60 18 Lhnas R and Suglmori M (1980) Electrophysiological properties of tn vitro Purkinje cell dendrites in mammalian cerebellar slices J Phystol, Lond 305, 197~13 19 Lytton J , ZaraIn-Herzberg A , Penasamy M and MacLennan D H (1989) Molecular cloning of the mammalian smooth muscle sarco(endo)plasmic reticulum Ca2+-ATPase J btol Chem 264, 7059 7065 20 Lytton J and MacLennan D H (1988) Molecular cloning ofcDNAs from human kidney coding for two alternatively splJced products of the cardiac Ca2+-ATPase gene J btol Chem 263, 15024-15031 21 MacLennan D H , Brandl C J , Korczak B and Green N M (1985) Amino-acid sequence of a Ca 2+ + M g 2+dependent ATPase from rabbit muscle sarcoplasmlc retlculum, deduced from its complementary DNA sequence Nature 316, 696-700 22 Meldolesl J , Volpe P and Pozzan T (1988) The lntracellular distribution of calcium Trends Neuro~cl 11, 449 452 23 Miller R J (1988) Calcium signalling in neurons Trends Neuroscl 11, 415 419 24 Ross C , Bredt D and Snyder S (1990) Messenger molecules in the cerebellum Trends Neuroscl 13, 216-222 25 Ross C A , MacCumber M W , Glatt C E and Snyder S H (1989) Brain phosphohpase C isoenzymes differential mRNA locahzations by in sttu hybridization Pro~ natn Acad S~I USA 86, 2923-2927 26 Ross C A , Meldolesi J , Mllner T A , Satoh T , Supattapone S and Snyder S H (1989) Inosltol 1.4,5-trlsphosphate receptor localized to ER in cerebellar PurkInje neurons Nature 339, 468-470 27 Verma A , Ross C A , Verma D , Supattapone S and Snyder S H (1990) Rat brain endoplasmlc retlculum calcium pools are anatomically and functionally segregated Cell Regulation 1, 781 790 28 Worley P F , Baraban J M and Snyder S H (1989) Inosltol 1,4.5-trlsphosphate receptor binding autoradlograph~c locahzatlon in rat brain J Neuroscz 9, 339-346 29 Young W S I I I . Mezey E and Siegel R E (1986) Neuroses Lett 70, 198-203 (Accepted 6 January 1991)