The Journal of Neuroscience, December 1992, 12(12): 47934799

Regulation of Brain-derived Neurotrophic Factor and Nerve mRNA in Primary Cultures of Hippocampal and Astrocytes

Francisco Zafra, Dan Lindholm, Eero Cast&n, Jukka Hartikka, and Hans Thoenen Department of Neurochemistry, Max Planck Institute for Psychiatry, 8033 Planegg-Martinsried, Germany

Brain-derived neurotrophic factor (BDNF) and NGF are both distribution, cellular localization, and developmental expres- expressed by neurons in the hippocampus. In previous stud- sion (seeBarde, 1991; Thoenen, 1991). Besidescompleting the ies, it has been demonstrated that both BDNF and NGF mRNA spectrum of biological actions of and identifying levels are regulated by neuronal activity. Upregulation is and characterizing their receptors, the elucidation of the regu- predominantly regulated by the glutamate (NMDA and non- lation of their synthesis is essentialfor a comprehensive un- NMDA receptors); downregulation, predominantly by the derstanding of the physiological functions of the neurotrophins. GABA system (Zafra et al., 1990, 1991). In neuronal cultures In the CNS, neurotrophins [NGF, brain-derived neurotrophic of the rat hippocampus, potassium depolarization and kainic factor (BDNF), and -3 (NT-3)] are predominantly acid-mediated increases in BDNF and NGF mRNA were elim- expressedin neurons.However, there are marked differencesin inated in a dose-dependent manner by the calcium channel their pattern of distribution and time course of their develop- blocker nifedipine. Conversely, calcium ionophores (Bay- mental expression(Ernfors et al., 1990a,b; Hofer et al., 1990; K8844 and ionomycin) augmented BDNF and NGF mRNA Maisonpierre et al., 1990; Phillips et al., 1990; Wetmore et al., levels by a calmodulin-mediated mechanism. In view of the 1990). In previous studies,we have shown that both NGF and fact that many potential modulators (conventional transmit- BDNF mRNA are regulatedby neuronalactivity predominantly ters and ) of neuronal and astrocytic BDNF by the glutamate and GABA systems(Zafra et al., 1990, 199 1). and NGF mRNA synthesis may act via the adenylate cyclase Preliminary tissue culture experiments suggestedthat other system, we studied the effect of forskolin, an activator of transmitter systemsand neuropeptidesare also involved in the adenylate cyclase. Indeed, forskolin enhanced the effects regulation of the neuronal synthesisof BDNF and NGF (Zafra of calcium ionophores and kainic acid on BDNF and NGF et al., 1990). mRNA levels. , such as -1 and trans- Although the main sitesof NGF expressionin the CNS are forming growth factor-fll, which have previously been shown specific populations of neurons (Ayer-LeLievre et al., 1988; to increase NGF mRNA markedly in astrocytes, were without Whittemore et al., 1988; Gall and Isackson, 1989; Bandtlow et effect on neuronal BDNF and NGF mRNA levels. In contrast al., 1990; Ernfors et al., 1990b), the first reports on the regulation to neuronal cultures, where the regulation of BDNF and NGF of NGF synthesisin the CNS referred to astrocytes in culture mRNA was generally very similar, the regulation in astro- (Lindsay, 1979; Furukawa et al., 1986). The role played by these cytes was distinctly different. All the cytokines that produce cells under physiological conditions is unclear, since in situ hy- a marked increase in NGF mRNA were without effect on bridization experiments have been unable to provide evidence astrocyte BDNF mRNA levels, which under basic conditions for the substantialexpression of neurotrophins in non-neuronal were below the detection limit. However, pro- cells of the CNS under physiological conditions (Ayer-LeLievre duced a marked elevation of BDNF mRNA in astrocytes, an et al., 1988; Whittemore et al., 1988; Gall and Isackson, 1989; effect that was further enhanced by glutamate receptor ag- Bandtlow et al., 1990; Emfors et al., 1990b). However, it is onists. In contrast, the response of NGF mRNA under the possiblethat astrocyteshave a more important role under patho- same culture conditions (low-serum medium) did not re- physiological conditions. As a consequenceof tissue damage, spond either to norepinephrine or to forskolin. However, the regulatory mechanisms,which are similar to those shown to responsiveness of NGF mRNA in astrocyte cultures for CAMP regulate NGF synthesisin cultured astrocytes (Furukawa et al., is very much dependent on culture conditions. 1986; Sprangeret al., 1990; Lu et al., 199 1; Yoshida and Gage, 1991), may come into play. The detection of new membersof the NGF genefamily (referred As a first step toward understanding the regulation of the to as neurotrophins) has resulted in the rapid accumulation of synthesisof neurotrophins in integrated intact systems,we com- information on the spectrumof their biological actions, regional pared the regulation of NGF and BDNF mRNA in primary cultures of hippocampal neurons and cerebral astrocytes. Since the basallevels of BDNF mRNA in hippocampal cultures are Received May 1, 1992; revised June 29, 1992; accepted July 1, 1992. about 20-30 times higher than those of NGF mRNA (Hofer et F.Z. was a recipient of EMBO long-term fellowship. E.C. is a fellow of the Alexander van Humboldt Foundation. We thank Dr. Tony Hughes and Ms. al., 1990; Zafra et al., 1990, 1991) and as it seemsthat the Lorraine Bale for linguistic revision. mechanismsof regulation of NGF and BDNF mRNAs in neu- Correspondence should be addressed to Hans Thoenen, Max Planck Institute for Psychiatry, Department of Neurochemistry, Am Klopferspitz 18A, 8033 Pla- ronsare generallyvery similar, the main emphasisof the present negg-Martinsried, Germany. study wasto determinethe regulationof neuronalBDNF mRNA. Copyright 0 1992 Society for Neuroscience 0270-6474/92/124793-07$05.00/O To quantify the distinctly lower basallevels of NGF mRNA, it 4794 Zafra et al. l Regulation of BDNF and NGF mRNA in Neurons and Astrccytes

ElControl a + EC 28s -

BDNF

C 0 0,1 l,o 10,o C KA W7 UA w7 Nifedipin OJ M 1

Figure 1. Effect of nifedipine on the potassium- and kainic acid-in- duced expression of BDNF mRNA in hippocampal neurons. Neurons b were incubated for 3 hr in the presence of 50 mM KC1 or 30 PM kainic acid and with the indicated concentrations of nifedipine. Cellular RNA was extracted and analyzed as described in Materials and Methods. Values are the mean f SEM of three different experiments. was necessary to use a quantitative PCR method that has been previously described in detail (Zafra et al., 1990). Calcium influx and the subsequentactivation of calcium/ calmodulin-activated kinase(s)seem to play an essential role in the regulation of neuronal NGF and BDNF mRNA levels. Cytokines and growth factors that had previously been shownto increaseNGF mRNA in primary cultures of astrocytes and that also enhancethe production of NGF protein (Spranger et al., 1990, Yoshida and Gage, 1992) were without effect on C C TPATPA BDNF mRNA levels, either in astrocytes or in hippocampal Figure 2. a, The effect of calmodulin inhibitor on BDNF mRNA in neurons.Thus, although the mechanismsthat regulatethe levels hippocampal neurons. Hippocampal neurons were incubated without additions (C) or for 4 hr with kainic acid (KA; 30 PM) in the presence of neuronal BDNF and NGF mRNA are very similar, there are or absence of a calmodulin inhibitor W7 (1 PM). Total RNA was ex- marked differences in the regulation of these mRNAs in astro- tracted and analyzed as described in Materials and Methods. The up- cytes. permost band represents 28s ribosomal RNA, the two lower bar& (4 and 1.5 kb) correspond to BDNF mRNA (Hofer et al., 1990). b, Seven- Materials and Methods day-old hippocampal neurons were incubated for 3 hr without (C) or Materials. Interleukin- 1 (IL- 1) was obtained from Biogen Inc.; Dulbec- with protein kinase C activator TPA (100 rig/ml). RNA was extracted co’s Modified Eagle’s Medium (DMEM) and fetal calf serum, from and analyzed by Northern blot analysis. GIBCO, transforming growth factor-@1 (TGF+l), from British Bio- technology; basic (basic FGF), from Boehringer shaking, and the astrocytes were replated onto plastic dishes. More than Mannheim; H7, W7, calmidazolium, nifedipine, and Bay-K8644, from 95% of the cells were astrocytes as judged by staining with glial fibrillary Research Biochemicals Inc.; (S,)- and (RJ-adenosine 3’:5’-cyclic phos- acidic protein (results not shown). Astrocytes were incubated for 4 hr phorothioate [(S,)- and (&,)-CAMPS], from Biolog Life Science Institute after addition of various growth factors and agents, and the NGF and (Bremen, Germany); and all other reagents, from Sigma. BDNF mRNA levels were determined by Northern blot analysis. CAMP Cell cultures. Neurons were prepared from hippocampus of 17-d-old levels were determined by a from Amersham and corrected for rat embryos as previously described (Zafra et al., 1990). Briefly, the protein content. hippocampi were dissected and incubated for 20 min at 37°C in phos- RNA preparation and Northern blot analysis. RNA was extracted from phate-buffered saline (PBS) in a solution containing 10 mM glucose, 1 cells as described by Chomnynski and Sacchi ( 1987) after the addition mg/ml albumin, 6 ccg/ml DNase, and 12 U/ml papain. After washing, of a shortened [700 (bp)] BDNF cRNA recovery standard (10 the cells were dissociated and collected by centrifugation, and resus- pg). Total cellular RNA was electrophoresed through a 1.3% agarose pended in DMEM supplemented with 10% fetal calf serum. The cells gel and transferred to Hybond N filters. After blotting, the filters were were plated onto plastic culture dishes (0.5 x lo6 cells per 35 mm dish) hybridized with a ‘*P-labeled cRNA probe specific for mouse BDNF that were precoated with poly-DL-omithine (0.5 mgml). The neurons (Zafm et al., 1990) as described earlier (Heumann and Thoenen, 1986). were incubated in a serum-free medium lacking glutamate and con- The filters were washed for 20 min at high stringency (0.2x saline- taining the supplements as described by Brewer and Cotman (1989). sodium citrate at 7X) and exposed to x-ray film. Autoradiography was Within a few days of incubation, the cultures exhibited a dense network performed for various periods of time depending on the intensity of the of , and the cells were usually used for the experiments at day signals. A laser scanner (LKB) was used to estimate the amount of 7 after plating. The neurons were incubated for various periods of time specific BDNF transcripts in the samples. in the presence of growth factors and additions as indicated in the figure Quantitative PCR. Total neuronal RNA and a shortened NGF cRNA captions. BDNF mRNA levels were subsequently determined by North- standard (153 bp) were reverse transcribed into cDNA using a specific em blot analysis, whereas a quantitative PCR method (see below) was 3’-primer for NGF (24-mer, S’TGTACGCCGATCAAAAACGCAGT used to estimate NGF mRNA levels in the neurons. GG-3’) and avian myeloblastosis virus-reverse transcriptase. The DNA Astrocytes were prepared from newborn rat brains and purified as was subsequently subjected to 18 cycles of PCR using the above-men- described earlier (Spranger et al., 1990). In brief, microglia and oligo- tioned primer and another S’-primer (24-mer, 5’-CAGCATGGTGGA dendrocytes were removed from confluent primary glial cell cultures by GTFITGGCCTGT-3’) (Zafra et al., 1990). The amplification was linear The Journal of Neuroscience, December1992, 12(12) 4795

a

BDNF-mRNA -I-

STANDARD - .:

C KA H7KA H7 Figure 3. The effect of protein kinase C inhibitor on BDNF mRNA in hippocampal neurons. The neurons were incubated in the presence of 30 PM kainic acid (KA), with 25 PM of protein kinase C inhibitor H7 b or without additions(C). RNA was extracted and analyzed by Northern blot analysis. The two upper bun& are the two transcripts of BDNF, and the lower bund(700 bp) represents 5 pg of the BDNFcRNA recovery standard (see Materials and Methods). b 10.0 - E for up to 20 cycles. The products obtained (203 bp for NGF and 153 P bp for recovery standard) were run on a 3% NuSieve/agarose gel and I.? 7.5 - blotted onto Hybond N+ membranes (Amersham), and the filters were 2 hybridized with a mouse NGF cRNA probe as described previously *E s.o- (Zafm et al., 1990). (r p 2.5 - Results D 0-I Role played by calcium in the increaseof BDNF mRNA levels 0 2s 5.0 7.5 100 in hippocampal neuronsinitiated by high potassiumand BAY-K 8fi44 (PM) kainic acid In previous experiments, we have demonstrated that depolar- Figure 4. The effect of forskolin on the calcium- or kainic acid-induced expression of BDNF mRNA. a, Hippocampal neurons were incubated ization by high KC1 (50 mM) increased the levels of BDNF for 3 hr in the presence of 50 PM kainic acid (KA), with 20 PM forskolin mRNA in cultured hippocampal neurons in a calcium-depen- (FK), or with both drugs (kX+FK). Values represent mean + SEM of dent manner (Zafm et al., 1990). Of all the transmitter sub- three experiments. b, Hippocampal neurons were incubated for 3 hr in stancesor their analogstested, kainic acid, a glutamate receptor the presence of the indicated concentrations of Bay-K8644 in the pres- ence (solid circles) or absence (open circles) of 20 PM forskolin. Total agonist, had by far the greatesteffect on BDNF mRNA in these RNA was extracted and analyzed as described in Materials and Meth- cells. To investigate the role ofcalcium in more detail, nifedipine ods. Values represent mean rt SEM of three experiments. (a calcium channel blocker) was used on the one hand and activators of calcium channelson the other. As shown in Figure 1, nifedipine impaired both the potassium-and the kainic acid- Involvement of adenylate cyclasein the kainic acid-mediated mediated increasein BDNF mRNA in a concentration-depen- increasein BDNF mRNA dent manner. Nifedipine not only interfered with the high KCl- Forskolin, an activator of adenylate cyclase, alone increased and kainic acid-mediated BDNF mRNA increasebut, at con- BDNF mRNA only slightly, but it markedly enhancedthe kainic centrations of 1 and 10KM, also reducedthe basallevel of BDNF acid-mediated BDNF mRNA increasein hippocampalneurons mRNA significantly (P < 0.05; Fig. 1).Conversely, Bay-K8644, (Fig. 4a). Forskolin also increasedthe BDNF mRNA increase a compound that increasesintracellular calcium concentration mediated by Bay-K8644 (Fig. 4b). Likewise, forskolin poten- by opening L-type calcium channels,augmented BDNF mRNA tiated the effect of the calcium ionophore ionomycin in increas- levels in these neurons (see Fig. 4b, lower curve). W7, a cal- ing BDNF mRNA levels in theseneurons (data not shown). 8- modulin antagonist (Hidaka et al., 1991), blocked the kainate- Bromo-CAMP and (Q-CAMPS, which also activate protein ki- induced increasein BDNF mRNA (Fig. 2a). Similar resultswere naseA, produced similar effectsas forskolin. The effect of (S,)- obtained with another calmodulin antagonist, calmidazolium CAMPS could be suppressedby a 20-fold excessof (R&S, (Van Belle, 1981) (results not shown). H7, a relatively specific an inactive stereoisomer(data not shown)@‘Brian et al., 1982). inhibitor of protein kinase C (Hidaka et al., 1991), did not Thus, the effect of kainic acid on BDNF mRNA levels could interfere with the kainic acid-mediated increasein BDNF mRNA involve both an increasein calcium influx and an activation of in hippocampal neurons(Fig. 3). Thesedata indicate that kainic adenylate cyclase.To evaluate whether kainic acid doesindeed acid induces BDNF mRNA in neurons by increasingintracel- affect adenylate cyclaseactivity, we studied the levels of CAMP lular calcium, which in turn acts through calcium/calmodulin- in hippocampal neurons incubated with kainic acid. Table 1 dependent protein kinases.However, protein kinaseC may also showsthat there is no significant increasein CAMP. Forskolin, have a role in the regulation of BDNF mRNA in cultured hip- a direct activator of adenylate cyclase,served as a positive con- pocampal neurons, becauseTPA, a phorbol ester, which acti- trol and resulted in a 5.5-fold increasein CAMP (Table 1). The vates protein kinase C, slightly increasedBDNF mRNA levels absenceof a direct action of CAMP in the kainic acid-initiated in these neurons (Fig. 2b). increasein BDNF mRNA was further establishedby the effect 4796 Zafra et al. * Regulation of BDNF and NGF mRNA in Neurons and Astrccytes

Table 1. CAMP content in primary cultures of hippocampal neurons Table 2. Effect of growth factors and cytokines on BDNF mRNA levels in hippocampal neurons CAMP Additions (nmol/me motein) BDNF mRNA Addition Amount (% of controls) None 12? 6 (n=4) Forskolin (20 PM) 65+ ll(n=4) None 100 -+ 4 Kainic acid (30 PM) 16 t 8 (n = 4) IL-la 30 U/ml 103 + 7 IL-10 30 U/ml 97 * 7 The neurons were incubated for 5 min in the presence of the indicated additions. CAMP was determined as indicated in Materials and Methods. TGF-01 2 @ml 110 + 6 PDGF 5 r&ml 107 + 5 EGF 5 &ml 102 ?z 6 of (R,)-CAMPS. This compound competeswith CAMP in bind- bFGF 5 @ml 94 + 4 ing to the regulatory subunit of the protein kinaseA, but does The neurons were incubated for 4 hr in the absenceor presence of various growth not activate it @‘Brian et al., 1982).@,)-CAMPS neither changed factors in the concentrations indicated. The levels of BDNF mRNA were deter- the basal level of BDNF mRNA nor affected the kainic acid- mined by Northern blot analysis (seeMaterials and Methods). The valuesrepresent the means + SEM of three or more determinations. PDGF, platelet-derived growth mediated increase (data not shown). Thus, CAMP is not re- factor. sponsiblefor the kainic acid-mediated increasein BDNF mRNA levels in hippocampal neurons. Effect of cytokines on NGF and BDNF mRNA levelsin Regulation of NGF mRNA in hippocampal neurons hippocampalneurons Since the levels of NGF mRNA in cultures of hippocampal Previous experiments have demonstratedthat a number of cy- neurons are 20-30 times lower than those of BDNF mRNA, it tokines produce an increasein the levels of NGF mRNA and wasnecessary to usea sensitivePCR method for the quantitative NGF protein in primary cultures of rat astrocytes (Lindholm et determination of NGF mRNA. As demonstrated in previous al., 1990; Spranger et al., 1990; Lu et al., 1991; Yoshida and experiments, NGF mRNA levels in hippocampal neuronscan Gage, 1991, 1992). In view of the fact that thesemolecules are be increasedby depolarization with high KC1 and kainic acid expressedin brain cells (neuronsand/or glial cells)after damage (Zafra et al., 1990, 1991). The responsesof NGF mRNA are of neuronal tissue, it was of interest to see whether the same generally smaller than those of BDNF mRNA. Moreover, ni- molecules,which induce NGF mRNA in astrocytes, also affect fedipine, a calcium channel blocker, and EGTA abolished the neuronal BDNF mRNA. However, none of the moleculesin- kainic acid-mediated increasein NGF mRNA (data not shown) vestigated significantly changed the level of BDNF mRNA in asdemonstrated for BDNF (Fig. 1).Bay-K8644, which increases neurons(Table 2). intracellular calcium levels by activating L-type calcium chan- The effects of the same moleculeson NGF mRNA in hip- nels,elevated NGF mRNA in hippocampalneurons in a manner pocampal cultures were not reliable, as it was not possible to similar to that seenfor BDNF mRNA (Figs. 4b, 5). Forskolin produce pure neuronal cultures without contamination by small alone had a small (2.5fold increase) effect on NGF mRNA numbers of astrocytes; thus, the weak and inconsistent effects levels, which was additive to that observed with Bay-K8644 on NGF mRNA in thesecultures may result from a small num- (Fig. 5). This is in contrast to the actions seenon BDNF mRNA, ber of contaminating astrocytes. where the effects of forskolin alone had little effect (Fig. 4a) but markedly potentiated the effect of Bay-K8644 (Fig. 4b) As seen Regulation of NGF and BDNF mRNAs in cultured astrocytes above for BDNF mRNA (Fig. 2b), TPA also increasedthe levels In confirmation of previous experiments (Lindholm et al., 1990; of NGF mRNA in hippocampal neurons (Fig. 5). Sprangeret al., 1990), IL- 1 and TGF-/I both produce a marked increasein NGF mRNA levels. However, they did not affect the very low levels of BDNF mRNA (Fig. 6). Thus, unlike the situation in cultured hippocampal neurons, BDNF and NGF mRNAs in cultured astrocytes are regulated in distinctly dif- ferent manners.In contrast to observationsin hippocampal neu- rons, the basal levels of BDNF mRNA in control astrocytes were very low and could not be increasedby any other growth factor tested, such as FGF, TGF-P, or (EGF; data not shown). Both BDNF and NGF mRNA levels wereincreased by TPA, ionomycin, and forskolin (Fig. 6). How- ever, forskolin increasedBDNF mRNA levels substantiallymore than those of NGF mRNA. As in hippocampal neurons, for-

4 skolin enhancedthe stimulatory effect of the calcium ionophore C FK BAY ITPA inomycin on BDNF mRNA levels in cultured astrocytes, sug- gesting that BDNF regulation might involve similar mecha- Figure 5. The effect of forskolin and increased calcium on NGF mRNA nisms in both neurons and glia cells. levels in hippocampalneurons. The neuronswere incubated for 3 hr in the presence of 20 PM forskolin (FK) and 1 PM Bay-K8644 (BA y? or Regulation of NGF and BDNF mRNA levelsin astrocytes by with both agents together (FK BAY). In some cultures, the phorbol ester neurotransmitters TPA (100 @ml) was used. RNA was extracted and NGF mRNA levels were determined by the quantitative PCR method as described in Ma- With the knowledgethat neuronal activity regulatesBDNF and terials and Methods. The values represent means f SEM (n = 3). NGF mRNA expressionin neurons and since astrocytes also The Journal of Neuroscience. December 1992. 12(12) 4797

NGF-mRNA w

Figure 6. The effectof variousgrowth factorsand agents on NGF andBDNF mRNA levelsin astrocytes.Confluent astrocyteswere incubatedfor 4 hr in the absence(c) or presenceof various additions:IL-1 ULI: 30 U/ml): TGF- BDNF-mRNA j31(TGF; 4 n&l); ?PA (lOO’hg/ml); forskolin(FE, 20 PM);ionomycin (ION, 1 &ml). Total cellularRNA wasex- tractedand analyzedby Northernblot STANDARD - analysisin the upperpanel. The 1.3 kb bandrepresents NGF mRNA, whereas C IL1 TGF TPA FK ION FK -STANDARD- the lower panel representsthe two ION mRNA transcriptsfor BDNF. expressneurotransmitter receptors, we investigated a seriesof kainic acid administration or induction of limbic seizureby the physiological transmitters or their synthetic analogson cultured stimulation of entorhinal cortex, BDNF mRNA levels in the astrocytes.Norepinephrine significantly increasedBDNF mRNA hippocampusare markedly increasedboth in the granule cells levels in astrocytes (Fig. 7). and to a lesserextent of the dentate gyrus and in the pyramidal neuronsof the CA l- epinephrine also increased BDNF mRNA (data not shown). CA3 regions(Ballarin et al., 1991; Emfors et al., 1991; Isackson Glutamate alone had no effect on BDNF mRNA, but it poten- et al., 1991). In contrast, the changesin NGF mRNA are re- tiated the effect of norepinephrine (Fig. 7). Quisqualic acid, stricted to the granule cells of the dentate gyrus. Neurons ex- which activates both ionotropic and metabotropic glutamate pressingNGF mRNA scatteredin the pyramidal cell layer, the receptors, exhibited effects similar to glutamate (not shown). stratum oriens, and the hilus do not appearto be responsiveto Norepinephrine also increasedthe levels of NGF mRNA in kainic acid (Gall et al., 1991; E. CastrCn, unpublished obser- cultured astrocytes, but this effect appearedto be dependenton vations). In the cerebellum, BDNF mRNA is expressedin the the batch of serumused (see Discussion). In contrast, histamine, granular cell layer (Hofer et al., 1990) whereasthe presenceof 5-HT, and carbachol did not influence the expressionof either NGF mRNA could not be unambiguouslydemonstrated by in BDNF or NGF mRNA in cultured astrocytes under our exper- situ hybridization (Bandtlow et al., 1990). In addition to the imental conditions (data not shown). differencesin the basal levels and the different patterns of ex- pressionafter systemic stimulation, the increasein the BDNF Discussion mRNA after injection of kainic acid in vivo is absolutely and The goal of the present investigation was to compare the reg- relatively larger than that of NGF mRNA both in hippocampus ulation of BDNF and NGF in primary cultures of hippocampal and cortex (Zafra et al., 1990). An additional difference between neurons and brain astrocytes. The investigations were confined the regulation of NGF and BDNF mRNA is their regulation by to the determination of the mRNA of the two neurotrophins, glucocorticoids in vivo and in primary cultures of rat hippocam- as a sufficiently sensitive and specific immunoassayfor BDNF pus. Glucocorticoids and higher concentrations of mineralo- protein is not yet available. corticoids increaseNGF mRNA both in vivo and in vitro (Lind- In agreement with previous experiments (Zafra et al., 1990, 199l), BDNF and NGF mRNA levels in cultured hippocampal neurons were regulated in a similar manner, although some differenceswere observed. In contrast, the regulation of BDNF and NGF mRNAs in cultured astrocytes proved to be distinctly different. \ 1.5. D&erences betweenthe regulation of BDNF and NGF mRNA E in hippocampal neurons Qz 1.0 - Although the regulation of BDNF and NGF mRNAs showmany @z E similarities both in vitro and in vivo (Zafra et al., 1990, 1991; ( OS. present results), there are also some clear differences:the basal 2 levels of NGF mRNA both in primary cultures of brain neurons s - J and in brain tissue in vivo are 20-30 times lower than those of C Glu QA NE BDNF mRNA (Hofer et al., 1990; Zafra et al., 1990, 1991). Moreover, the distribution of NGF mRNA in the rodent brain Figure 7. Theeffect of norepinephrineand glutamate agonists on BDNF is generally more restricted both under normal conditions and mRNA levelsin astrocytes.Cells were incubated for 4 hr in the presence of 0.5 mM glutamate(Cl@, 25 PM quisqualate(QA), 25 PM norepi- after stimulation by kainic acid or limbic seizures (Gall and nephrine(NE), or norepinephrinewith glutamateor quisqualate.RNA Isackson, 1989; Ballarin et al., 1991; Ernfors et al., 1991; Gall wasextracted and analyzedas describedin Materialsand Methods. et al., 1991: Isacksonet al., 1991). For instance, after systemic Valuesrepresent the meanf SEM of threeexperiments. 4799 Zafra et al. - Regulation of BDNF and NGF mRNA in Neurons and Astrocytes holm et al., 1992a). Under the same experimental conditions, displaces CAMP from the regulatory unit of protein kinase A there is no detectable effect on BDNF mRNA levels (Lindholm without activating it (O’Brian et al., 1982). Thus, the regulation et al., 1992a). of BDNF and NGF mRNA levels by kainic acid does not seem to involve CAMP directly. However, the potentiating effect of Effect of various cytokines on the levels of BDNF and NGF forskolin is interesting considering that in integrated neuronal mRNAs in primary cultures of hippocampal neurons systems, neuropeptides and transmitter substances, which reg- After previous experiments had shown that a series of lym- ulate CAMP, may modulate the predominantly glutamate-me- phokines and cytokines produce a strong increase in NGF mRNA diated increase in BDNF and NGF mRNA levels in neurons. in cultured astrocytes (Lindholm et al., 1990; Spranger et al., 1990; Yoshida and Gage 199 1, 1992), we investigated the effect Comparison between the regulation of BDNF and NGF of the same molecules on the levels of BDNF and NGF mRNAs mRNA in primary culture of astrocytes in hippocampal cultures. All factors that markedly increased In contrast to the many similarities in the regulation of BDNF amounts of NGF mRNA in astrocyte cultures produced no and NGF mRNA in hippocampal neurons, the regulation for change in BDNF mRNA levels in hippocampal cultures. The these two neurotrophins in primary cultures of astrocytes is in effects of the factors on NGF mRNA levels in the latter cultures many respects distinctly different. NGF mRNA levels are in- were not absolutely clear in that small and inconsistent increases creased by a variety of cytokines, such as IL- 1, EGF, FGF, and in the levels of NGF mRNA were seen. These small increases the most potent TGF-B (Lindholm et al., 1990; Spranger et al., could be attributed to the response of the contaminating non- 1990, Yoshida and Gage, 199 1, 1992). None of these molecules neuronal cells. This would also explain the results reported by had any detectable effect on BDNF mRNA levels. In this con- Friedman et al. (1990) showing that IL- 1 increases NGF mRNA text, it is of interest to note that there is a distinct difference in mixed cultures from hippocampus. not only between the regulation of NGF mRNA in neurons and astrocytes, but also between astrocytes, Schwann cells, and fi- Regulation of BDNF and NGF mRNA levels in neuronal broblasts (Lindholm et al., 1988; Matsuoka et al., 1991). For cultures of rat hippocampus: involvement of calcium and instance, TGF-j3 is by far the strongest inducer of NGF mRNA CAMP in astrocytes (Lindholm et al., 1990), whereas it has no effect The increase in both BDNF and NGF mRNA levels in hip- on NGF mRNA levels in primary cultures of fibroblasts and pocampal neurons following high-potassium depolarization or Schwann cells (Matsuoka et al., 199 1). Indeed, in Schwann cells addition of kainic acid depends on calcium influx. On the one TGF-/3 abolishes the increases in NGF mRNA mediated by hand, the effects of depolarization and kainic acid can be mim- other means (Matsuoka et al., 199 1). icked by calcium ionophores, and on the other, the increase in Norepinephrine, which under our experimental conditions BDNF mRNA levels initiated by these treatments can be abol- had only a small effect on NGF mRNA in astrocytes, markedly ished in a dose-dependent manner by nifedipine. Nifedipine is increased the extremely low levels of BDNF mRNA in unstim- thought predominantly to block voltage-gated L-type calcium ulated astrocytes. The fact that not only norepinephrine but also channels, and the effects of both kainic acid and high potassium forskolin initiated an increase in BDNF mRNA levels in astro- on calcium influx are mediated by a voltage-gated mechanism. cytes indicates that these effects are mediated by CAMP. However, recent experiments have demonstrated that, depend- In agreement with our previous experiments (Spranger et al., ing on the relative unit composition of the non-NMDA gluta- 1990), neither ,&agonists, protein kinase A activation by non- mate receptors (Hollmann et al., 199 l), these -gated chan- hydrolyzable derivatives of CAMP, nor activation of adenyl nels regulate not only the flux of monovalent, predominantly cyclase by forskolin produced a consistent increase in NGF sodium, ions but also divalent calcium ions. The complete mRNA levels in astrocytes. This is in contrast to the observa- blockade of the kainic acid-mediated BDNF mRNA increase tions of Schwartz and Mishler (1990) who reported an increase and, at higher concentrations, the reduction of the basal levels in NGF mRNA, not only in C6 glioma cells but also in primary of BDNF mRNA by nifedipine raise the question of whether cultures of astrocytes. We did observe a small increase in NGF nifedipine also blocks the ligand-gated calcium influx via the mRNA by norepinephrine with some but not all batches of non-NMDA glutamate receptors. serum that we used, which could explain the discrepancy be- The calcium influx-induced by kainic acid seems to be me- tween our results and those of Schwartz and Mishler (1990). It diated by the calcium/calmodulin-dependent protein kinase. This has been reported that different batches of serum may influence is supported by the fact that W7 and calmidazolium, compounds expression of voltage-gated calcium channels in primary astro- that block calmodulin (Van Belle, 198 1; Hidaka et al., 199 l), cytes (Barres et al., 1989). In agreement with these observations, block the kainic acid-induced increase in BDNF mRNA. More- Yoshida and Gage (1992) have recently reported that under over H7, a protein kinase inhibitor that predominantly blocks defined conditions, CAMP increases NGF mRNA only in the protein kinase C (Hidaka et al, 1991) did not interfere with presence of basic FGF. the kainic acid-mediated increase in BDNF mRNA. In conclusion, the regulation of the levels of BDNF and NGF In view of the fact that a variety of transmitter substances mRNAs in hippocampal neurons in many respects shows a high and neuropeptides regulate CAMP in positive or negative man- degree of similarity both in vitro and in vivo. However, there ners, we investigated the effect of forskolin on the levels of are also distinct differences, in particular their regulation by BDNF and NGF mRNAs. In neuronal cultures, forskolin in- glucocorticoids (Lindholm et al., 1992b). Both BDNF and NGF creased the NGF mRNA levels about 2.5-fold, whereas there mRNA levels are regulated by calcium influx, which seems to was only a slight effect on BDNF mRNA. However, the effects act via calcium/calmodulin-dependent protein kinases, which of the calcium ionophor Bay-K8644 and ionomycin on BDNF are additionally modulated by CAMP via protein kinase A. These mRNA were markedly potentiated by forskolin. Kainic acid effects of CAMP might be of considerable importance for the alone did not change the levels of CAMP; neither was the effect modulatory role by other transmitter systems and neuropeptides of kainic acid affected by the CAMP antagonist (R,)-CAMP, which that activate adenyl cyclase or interfere with its activation. In The Journal of Neuroscience, December 1992, 12(12) 4799 contrast to the high degree of similarity between NGF and BDNF distribution of brain-derived neurotrophic factor mRNA in the adult mRNA regulation in neurons,the regulationof thesetwo mRNA mouse brain. EMBO J 9:2459-2464. speciesin astrocytes was in many respectsdifferent. The obser- Hollmann M, Hartley M, Heinemann S (199 1) Ca2+ permeability of KA-AMPA-gated glutamate receptor channels depends on subunit vations made in astrocyte cultures may be of importance in composition. Science 252:85 l-853. pathophysiological situations. Thus, after brain injury, there is Isackson PJ. Huntsman MM. Murrav KD. Gall CM (199 1) BDNF an increasein the expressionof growth factors, such as FGF, mRNA expression is increased in-adult’ rat forebrain after limbic IL-l, and TGF-P (Giulian and Lachman, 1985; Finklestein et seizures: temporal patterns of induction distinct from NGF. 61937-948. al., 1988; Lindholm et al., 1992b), all of which regulate NGF Lindholm D, Heumann R, Hengerer B, Thoenen H (1988) Interleukin expression. In agreementwith the present data, the expression 1 increases stability and transcription of mRNA encoding nerve growth of NGF but not of BDNF is increasedafter a stab injury to the factor in cultured rat fibroblasts. J Biol Chem 263: 16348-1635 1. cerebral cortex (Lindholm et al., 1992b). Lindholm D, Hengerer B, Zafra F, Thoenen H (1990) Transforming growth factor-0 1 stimulates expression of nerve growth factor in the rat CNS. Neuroreport 1:9-12. 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