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Hippocampal damage and kainic acid injection induce a rapid increase in mRNA for BDNF and NGF in the rat brain
EXPERIMENTAL
NEUROLOGY
114,35-43 (1991)
Hippocampal Damage and Kainic Acid Injection Induce a Rapid Increase in mRNA for BDNF and NGF in the Rat Brain Department of medical ~hern~t~, Laboratory of ~o~eu~ar Ne~rob~olo~ and ~De~artme~t of Pharmacology, Iiurolinska Institute, Box 60400 S-10401 Stockholm. Sweden
tions in the nervous system is largely derived from extensive studies of p-nerve growth factor (NGF), a wellcharacterized neurotrophic factor required for the development and maintenance of peripheral sensory and sympathetic neurons (30). More recently, NGF has also been detected in the brain, where the highest levels of its mRNA have been found in hippocampus and cerebral cortex (15,26,42,48), areas receiving major cholinergic projections from the basal forebrain. Subsequently, in situ hybridization has been used to demonstrate that within hippocampus, NGF mRNA is expressed in neurons, implying that NGF exerts a trophic effect in the brain through neuron-to-neuron interactions (2, 9, 37, 39, 49). Basal forebrain cholinergic neurons respond to exogenously added NGF by increasing the level of choline acetyltransferase (17,24,34,35) the key enzyme in the synthesis of acetylcholine, and can be prevented from degeneration after transection of the septohippocampal pathway by either chronic infusion of exogenous NGF (18, 27, 50) or by implantation of genetically modified cells producing recombinant NGF (40, 45). Furthermore, implantation of recombinant NGF-producing cells into cholinergically denervated cerebral cortex results in a marked increase in the survival of, and fiber outgrowth from, grafts of fetal basal forebrain neurons (6). In addition, intracerebral infusion of NGF in aged rats reduces basal forebrain cholinergic neuron atrophy and results in an improved spatial memory in behaviorally impared rats (10). Thus, ample evidence exsist that NGF supports basal forebrain cholinergic neurons. In similarity to NGF, BDNF supports neuronal survival both in vitro and in viuo (3,22) and the amino acid sequence predicted from the recently isolated porcine BDNF gene shows striking amino acid similarities to NGF (29). This structural similarity suggests that NGF and BDNF are members of a family of neurotrophic factors. Taking advantage of the amino acid similarity between NGF and BDNF, molecular clones for a third member of the NGF family, named hippocampus-derived neurotrophic factor (I-IDNF) (8), neurotrophin-3 (23, 32, 41) or NGF-2 (25) have recently been isolated.
In situ hybridization and Northern blots were used to study expression of mRNAs for members of the nerve growth factor family in the rat brain following an excitatory stimulus. One hour after a unilateral needle insertion or saline injection into the dorsal hippocampus, the level of brain-derived neurotrophic factor (BDNF) mRNA increased markedly in granular neurons of the dentate gyrus and in the piriform cortex ipsilateral to the injection. The same treatment also increased the level of NGF mRNA in granular neurons of the ipsilatera1 dentate gyrus. The rapid increase in BDNF and NGF mRNA after a needle insertion or injection of saline was transient and preceded by an increase in c-fos mRNA in the same brain regions. In contrast to a needle insertion per se or a saline injection, 7 h after a unilateral injection of kainic acid into the dorsal hippocampus, the level of BDNF mRNA was dramatically increased in the ipsilateral hippocampus, as well as in the ipsilateral fron~parietal, piriform and perihinal cortex, the amygdaloid complex, claustrum, and ventromedial hypothalamus. A less pronounced increase was also seen in these brain areas on the eontralateral side. Northern blots revealed that the level of BDNF mRNA increased 5- and 40-fold in the contra- and ipsilateral hippocampus, respectively, compared to sham-operated control animals. In contrast to BDNF and NGF, the level of hippocampus-derived neurotrophic factor/ neurotrohin-3 (HDNF/NT-3) mRNA was not altered by either needle insertion or injection of saline or kainic acid. These results indicate that hippocampal damage differentially induces expression of the NGF family of neurotrophie factors in the brain and suggest further that BDNF mRNA expression in the brain is markedly enhanced by an increased synaptic activity. o issi Academic
Press,
Inc.
INTRODUCTION Development and maintenance of neurons of the vertebrate nervous system depend on the presence of neurotrophic factors produced in the target areas of the sensitive neurons. The concept of neurotrophic interac-
35 All
Copyright Q 1991 rights of reproduction
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36
BALLARiN
Recently the NGF family has been shown to also include a fourth member, named neurotrophin-4 (NT-4) (16). In the brain, BDNF mRNA is widely distributed with the highest level in hippocampus (E&21,47) whereas the distribution of NT-3 mRNA is remarkably restricted being mostly confined to hippocampus (823). In hippocampus, INT-3, BDNF, or NGF mRNA-expressing neurons are located in a partially nonoverlapping arrangement (9, 37). NT-3 mRNA-expressing neurons, mostly with a pyramidal morphology, are concentrated in medial CA1 and in CA2 (8,9,37), whereas BDNF mRNAexpressing neurons, also of pyramidal morphology, are most abundant in CA3 and in the hilar region of the dentate gyrus (E&9,22,37,47). Neurons expressing high levels of NGF mRNA are also found in the hilar region, while only scattered NGF mRNA-expressing neurons are found in CAl, CA2, and CA3 (9, 13, 14, 37). NGF, BDNF, and NT-3 are maximally expressed in the brain at different times of development with a peak of NT-3 mRNA shortly after birth, followed by maximal expression of BDNF and NGF mRNA 2 and 3 weeks after birth, respectively (8, 33). The peak of NT-3 mRNA expression shortly after birth appears to be due to a high and transient expression of NT-3 mRNA in neurons of the developing cingulate cortex (11). In similarity to NT-3, BDNF mRNA has also been shown to be transiently expressed in many brain areas during early postnatal brain development, notably in the brain stem (12). The temporal and spatial expression of the three mRNAs suggests that their genes are strictly regulated during brain development and that the factors may support partially different neurons in the early postnatal and adult brain. In the present study we show that expression of both BDNF and NGF mRNA is rapidly and transiently induced in the hippocampus after a needle insertion into that area and that 7 h after kainic acid injection, the expression of BDNF mRNA is dramatically increased in neurons of both hippocampus and more distant brain areas. This distant effect is presumably caused by a paroxysmal discharge initiated by the drug, suggesting that increased synaptic activity can regulate expression of BDNF mRNA. EXPERIMENTAL
PROCEDURES
Kainic Acid Injections Male Sprague-Dawley rats (180-220 g) were anesthetized with a mixture of air and halotane and placed in a David Kopf stereotaxic frame. One group of rats (n = 24) were unilaterally injected with 1 ~1 of kainic acid (0.5 WgIpl in saline, pH 7.4) into the right dorsal hippocampus (coordinates: rostra1 3.3, lateral 2.8, ventral 4.8 from Bregma (36). Another group of rats were injected with 1~1 of saline (n = 40) or with the needle alone (n =
ET AL.
5). In addition, a group of sham-operated animals (n = 18) was used as a control. After the injection, the skin was sutured and the animals were left for the indicated times. In Situ Hybridization Tissue sections of dorsal hippocampus were cut on a cryostat (Leitz, F.R.G.) in 16-pm sections and thawed onto poly-L-lysine-coated slides (50 pg/ml). After fixation with 10% formalin, the sections were processed for in situ hybridization with deoxyadenosine[cu-[35S-thio]triphosphate] 3’-end-labeled probes as previously described (6). To detect BDNF-specific mRNA, a 50-mer oligonucleotide complementary to rat BDNF mRNA, corresponding to nucleotides 748-798 in pig BDNF (29), was used. For NGF-specific mRNA, a 50-mer oligonucleotides complementary to nucleotides 869 to 919 of rat NGF (49) was used. To detect NT-3-specific mRNA, a 50-mer oligonucleotides complementary to nucleotides 667 to 717 in rat INT-3 mRNA was used (8). These three oligonucleotide probes show 56 to 68% nucleotide homology to each other and are specific for their respective mRNAs as previously demonstrated (9). For c-fosspecific mRNA, a 48-mer oligonucleotide complementary to nucleotides 543 to 591 of rat c-fos (5) was used. To determine the intensity of labeling over individual cells the number of silver grains was counted manually over cells. Five different sections from four animals in each experimental paradigm were included in the analysis. The in situ hybridization revealed identical results in all animals within each experimental group studied. RNA Blot Analysis Frozen tissue samples were homogenized in 4 M guanidine isothiocyanate, 0.1 A4 &mercaptoethanol, 0.025 M sodium citrate (pH 7.0) and homogenized three times for 15 s with a Polytron. Each homogenate was layered over a 4-ml cushion of 5.7 M CsCl in 0.025 M sodium citrate (pH 5.5) and centrifuged at 15°C in a Beckman SW41 rotor at 35,000 rpm for 16 h (4). Poly(A)+ RNA was purifiedby oligo (dT)-cellulose chromatography (l), and the recovery of RNA was quantified spectrophotometrically before use in RNA blot analysis. Poly(A)+ RNA (15 pg) from each sample was electrophoresed in a 1% agarose gel containing 0.7% formaldehyde, followed by transfer to a nitrocellulose filter. The filter was hybridized to a 185-bp PCR fragment from rat BDNF corresponding to amino acids 183-239 of pig BDNF (29). The fragment was labeled with [a-32P]dCTP by nick-translation to a specific activity of approximately 5 X 10’ cpmlpg. Hybridization was carried out as described (7, 9) followed by washing at high stringency and exposure to Kodak XAR-5 film at -70°C. Appropriate exposures of the autoradiograms
REGULATION
were quantified meter.
using
a Shimadzu
OF
CS-9000
NGF
AND
densito-
RESULTS
A Transient Increase in the Level of BDNF and NGF mRNA following a Needle Insertion into the Hippocampus The short-term effects on the expression of mRNAs for the NGF family following a unilateral single injection of the excitotoxin kainic acid into the dorsal hippocampus was studied by in situ hybridization. The levels of BDNF, NGF, and NT-3 mRNAs in sham-operated animals, used as controls, were similar to those seen in animals 7 h after a saline injection shown in Fig. 1. One hour after injection, the level of BDNF mRNA increased markedly ipsilateral to the injection in the dentate gyrus, throughout the pyramidal cell layer of the hippocampus, as well as in piriform cortex (Fig. 1B). The level of NGF mRNA also increased on the ipsilatera1 side although this increase was confined to the dentate gyrus (Fig. 1F). For both BDNF and NGF, the increase in dentate gyrus was due to a larger number of granular neurons expressing detectable amounts of either of the two mRNAs, as well as an increased intensity of labeling over individual neurons. Similarly, the elevation in BDNF mRNA expression in the pyramidal cell layer and in piriform cortex were also due to a larger number of neurons expressing detectable amounts of BDNF mRNA as well as a higher intensity of labeling over individual neurons. In contrast to NGF and BDNF, the level of NT-3 mRNA was not affected by this treatment (Fig. 1J). Three different controls were used in the experiments: (i) sham-operated animals where the skull was opened with no needle insertion, (ii) unilateral injection of saline into the dorsal hippocampus, and (iii) insertion of the needle in the same place without injection. Unexpectedly, 1 h after saline injection, as well as 1 h after needle insertion, the levels of BDNF and NGF mRNA also increased in the ipsilateral hippocampus compared to sham-operated, control animals (Figs. 1A and 1E). Sham-operated animals showed no change in BDNF or NGF mRNA compared to nontreated animals and the mRNA levels for all three factors in these animals were similar to the ones seen in animals 7 h after a saline injection as shown in Figs. lC, lG, and 1K. None of the treatments had an effect on the level of NT-3 mRNA (Figs. 11 and 1J). The increase in BDNF and NGF mRNA caused by saline injection compared to shamoperated animals was first seen 15 and 30 min after the injection, respectively (Fig. 2). For both mRNAs the increase was maximal 1 h after the insertion (Fig. 2) and 7 h later, the levels of both BDNF and NGF mRNA were the same as in sham-operated animals. Hybridization of
BDNF
mRNA
EXPRESSION
37
adjacent sections to a c-fos mRNA-specific probe revealed elevated c-fos mRNA levels in the ipsilateral hippocampus already 5 min after the injection with a maxima1 level 30 min later. At this time point, elevated levels of c-fos mRNA were also seen in frontoparietal, perirhinal, and piriform cortex. The level of c-fos mRNA declined at 60 min and had essentially returned to control levels 2 h after the injection (Fig. 2). Kainic Acid Mediated Increase in the Level of BDNF mRNA Seven hours after a single injection of kainic acid in the dorsal hippocampus, the level of BDNF mRNA was dramatically increased in the ipsilateral hippocampus compared to the saline-injected control (compare Figs. 1C and 1D). Moreover, this marked increase was also found in the internal and external pyramidal layers of frontoparietal and perirhinal cortex, piriform cortex, amygdaloid complex, claustrum, and ventromedial hypothalamus. A large increase was also seen in the contralateral hippocampus and piriform cortex. The same treatment did not significantly change the level of NGF mRNA in the ipsilateral hippocampus compared to saline controls (compare Figs. 1G and 1H). In the case of NT-3, a small decrease was seen in the ipsilateral dentate gyrus both after saline or kainic acid injection (Figs. 1K and 1L). The dramatic increase in the level of BDNF mRNA was due to a large increase in the number of neurons expressing detectable levels of BDNF mRNA, as well as a marked increase in the level of BDNF mRNA in individual neurons (Fig. 3). In fact, in all regions with increased BDNF mRNA levels most, if not all, neurons expressed high levels of BDNF mRNA after the kainic acid injection compared to only a minority in saline-injected animals (compare Figs. 3A to 3B, 3C to 3D, and 3E to 3F). In addition, a grain count over BDNF mRNA-expressing neurons revealed an approximate 7fold increase in the number of grains over individual granular neurons in the dentate gyrus compared to saline-injected control (Fig. 4). A 6- and 3-fold increase in the number of grains were also found over neurons from the CA1 region of the hippocampus and piriform cortex, respectively (Fig. 4). Levels of BDNF mRNA Measured by Northern Blot Analysis In agreement with the results of the in situ hybridization, the level of BDNF mRNA in the hippocampus, measured by Northern blot analysis, was clearly increased both 1 and 7 h after injection of kainic acid compared to sham-operated animals (Fig. 5). Densitometric scanning of the autoradiograms revealed that the level of BDNF mRNA was elevated approximately 2- and 6fold on the contralateral and ipsilateral side, respec-
saline lh
FIG. 1. Expression of members of the NGF family of neurotrophic factors after intrahippocampal injections of saline or kainic acid. Adult Sprague-Dawley rats were injected into the CA3 region of the hippocampus with either saline (A, E, I, C, G, K) or kainic acid (B, F, J, D, H, L). The brains were sectioned either 1 h (A, B, E, F, I, J) or 7 h (C, D, G, H, K, L) after the injections. The sections were then hybridized to oligonucleotide probes specific for BDNF (A-D), NGF (E-H), or NT-3 (I-L). Bar in A is 3 mm and is the same for all panels. Note the increased level of BDNF mRNA in the ipsilateral (right side) hippocampus 1 h after injection of either saline (A) or kainic acid (B). Also, note the increase in NGF mRNA in the ipsilateral dentate gyrus 1 h after injection of either saline (E) or kainic acid (F). A marked increase in the level of BDNF mRNA is seen in several different brain regions (see text) 7 h after injection of kainic acid (D) compared to injection with saline (C). The increase is most pronounced on the ipsilateral side but is also seen on the contralateral side. Shown in the figure are dark-field photomicrographs of X-ray films exposed for 5 days. This exposure time is shorter than previously used to localize cells in the brain expressing members of the NGF family (9) and reveals relatively low signals in control animals but was chosen in order to visualize the increase in mRNA levels 1 h after saline injection or 7 h after kainic acid injection. The levels of BDNF, NGF, and NT-3 mRNAs in sham-operated animals (not shown) were similar to those seen in animals 7 h after saline injection. Abbreviations: DG, dentate gyrus; Pir, piriform cortex; CAl, CA2, CA3, pyramidal areas of the hippocampus.
tively, 1 h after the injection compared to sham-operated animals. Seven hours after the kainic acid injection, the levels of BDNF mRNA were increased approximately 5- and 40-fold on the contralateral and ipsilateral side, respectively. In agreement with previous studies (8, 29, 32), two BDNF mRNAs of 1.4 and 4.0 kb were seen, and the levels of both mRNA species increased to the same extent by the kainic acid injection. DISCUSSION
In the present study we show that the levels of both BDNF and NGF mRNA are increased in the hippocam-
pus 1 h after injection of saline or kainic acid into the dorsal hippocampus, compared to sham-operated animals. In the case of BDNF, a clear increase was also seen in the piriform cortex. On the other hand, the level of NT-3 mRNA, a third member of the NGF-family, was not changed by this treatment. Limbic seizures introduced by unilateral electrolytic lesion in the hilar region of the dentate gyrus have been shown to increase the level of NGF mRNA in granular neurons of the dentate gyrus and at later times after the lesion, as well as in neocortical and olfactory forebrain neurons (14). Our results show that the same increase in the hippocampus is obtained 1 h after an intrahippocampal injection of saline, kainic acid, or just a needle
insertion. Thus, the increase seen 1 h after kainic acid injection appears not to be caused by the excitotoxin but rather by the needle insertion per se. The increase in the levels of BDNF or NGFmRNA after saline injection or needle insertion was clearly transient with maximal levels 1 h after insertion. The cavity created by the injection of fluid into the dentate gyrus has been shown to cause an axotomy of the granule cells (46). Our results show that expression of BDNF and NGF increase rapidly in the hippocampus after injection of fluid into the hippocampus and suggest that this increase could be an early response to a mechanical damage and neuronal injury. Of interest is also the finding that expression of NGF mRNA is induced in the hypothalamus of aggressive male mice (44) suggesting that changes in NGF expression may not only occur in response to mechanically induced alterations in the brain but can also be caused by more subtle physiological changes induced by an external stimulus. The rapid increase in BDNF and NGF mRNA after a saline injection into the hippocampus was preceded by an increase in c-fos mRNA. A lesion of the sciatic nerve has been shown to cause a rapid increase in NGF expression in Schwann cells (20,31). The increase in NGF mRNA reaches a maximal level after 12 h and is preceded by a rapid and transient increase in c-fos and c-jun mRNAs (20). The transcription factor AP-1, a
5 min FIG. 2. Time-course sections were prepared hippocampus. Adjacent
15min for the induction from sham-operated sections were then
heterodimer between c-fos and c-jun (38), binds to a AP-1 site within the first intron of the NGF gene (19). This suggests that in the periphery the transcriptor factor AP-1 is involved in the induction of NGF mRNA expression in the lesioned nerve. It remains to be established if the c-fos and c-jun proteins (AP-1) also play a role in the induction of NGF and BDNF mRNA expression following an intrahippocampal saline injection. In contrast to the rapid and transient increase seen after a needle insertion alone, injection of kainic acid into the dorsal hippocampus resulted in a more persistent increase in the level of BDNF mRNA. This increase was not only found in the hippocampus but was also seen in several other brain regions, including the internal and external pyramidal layer of frontoparietal and perihinal cortex, piriform cortex, amygdaloid complex, claustrum, and ventromedial hypothalamus. The effect was more pronounced ipsilateral to the lesion but was also seen on the contralateral side. In agreement with the in situ hybridization results, Northern blot analysis showed 5- and 40-fold higher levels of BDNF mRNA on the contralateral and ipsilateral hippocampus, respectively, 7 h after kainic acid injection compared to the saline control. Recently, Zafra et al. (51) showed, by Northern blot analysis, markedly increased levels of BDNF and NGF mRNA in hippocampus and cerebral cortex after a systemic injection of kainic acid.
30min of c-fos, BDNF, and NGF mRNA following adult rats or from animals at the indicated hybridized with the indicated probes.
60min
120 min
saline injection into the dorsal hippocampus. Tissue times after a saline injection into the CA3 region of the
40
BALLARiN
ET
AL.
FIG. 3. Bright-field illumination showing BDNF mRNA-expressing neurons 7 h after injection of saline or kainic acid. BDNF mRNA-expressing neurons in the dentate gyrus (DG), CA1 region of the hippocampus (CAl), or piriform cortex (Pir), 7 h after injection of saline (A, C, E) or kainic acid (B, D, F). Note that 7 h after the kainic acid injection, the number of granular neurons expressing high levels of BDNF mRNA is greatly increased compared to the saline control (compare A and B). The same effects are seen in CA1 pyramidal neurons of the hippocampus (compare C and D) as well as in neurons of the piriform cortex (compare E and F). Scale bar in A is 20 pm and is the same for ail other panels. All panels shown are at coordinate -2.8 with respect to Bregma.
The same authors also provided pharmacolo~cal evidence that this effect was mediated through an excitatory amino acid receptor of the non-NMDA type. Intracerebroventricular injection of kainic acid was also recently shown to cause a marked increase in the level of NGF mRNA in the dentate gyrus and this effect was maximal at 2-3 h after the injection (13). The increased levels of mRNA in the hippocampus following a systemic injection was maximal at 3 h, and by 7 h after the injection the increase in NGF mRNA was significantly reduced (51). This may explain the fact that we did not, by in situ hybridization, detect a significant increase in the level of NGF mRNA in the hipp~ampus 7 h after the intrahippocampal injection of kainic acid. Kainic acid is an analog of the excitatory neurotransmitter glutamate and it has been reported that after intrahippocampal administration of the dose used in
this study, signs of neuronal degeneration are seen 4 h after the injection in the CA3 and CA1 pyramidal cells (28). Neuronal degeneration in areas distal to the injection site is less apparent and occurs at later times (28). Under the experimental conditions used in this study, evidence have been presented that the distant effect of kainic acid is due to paroxysmal discharge caused by the drug (43). Thus, it is likely that the increase in BDNF mRNA expression seen in distant regions is due to a spreading of synchronous neuronal discharges. In agreement with this possibility, the regions of the brain showing elevated levels of BDNF mRNA were the same distant regions that have previously been shown to be primarily affected by an intrahippocampal injection of kainic acid (28). The same areas also showed increased levels of c-fos mRNA 7 h after kainic acid injection (data not shown). In this context it is interesting to note
REGULATION
OF
NGF
AND
BDNF
mRNA
41
EXPRESSION
that these brain areas are the same regions as have been previously shown to express BDNF mRNA under normal physiological conditions (8,9,22,37,47). No significant effect was seen with regard to NT-3 mRNA either 1 or 7 h after injection of kainic acid. If this is due to differences in transcriptional regulation of NT-3 and
101
FIG. 5. Levels of BDNF mRNA in the hippocampus after injection of kainic acid. Fifteen micrograms of polyadenylated RNA isolated from the ipsilateral and contralateral hippocampus, 1 or 7 h after injection of kainic acid or from sham-operated animals, was electrophoresed in a formaldehyde-containing agarose gel. The gel was blotted and hybridized to the BDNF-radiolabeled probe, followed by washing and exposure to X-ray film. Five hippocampi were pooled for each RNA preparation and two independent measurement, with similar results, were performed. The 1eveI of BDNF mRNA in animals 7 h after saline injection (not shown) was similar to the level in sham-operated animals. Abbreviations: ipsi, ipsilateral side to the injection; contra, contralateral side to the injection.
n. shem
sal
1
1
KA
1
sal
7
KA
7
CA1
: sham
sal
1
KA
1
sal
7
Pir
(A
7
r
BDNF or merely reflects a different responsiveness of NT-3 mRNA-expressing neurons to kainic acid remains to be elucidated. The dramatic increase in BDNF mRNA expression in many brain regions known to express BDNF mRNA, and also known to undergo a paroxysmal discharge after an intrahippocampal injection of kainic acid, suggests that BDNF mRNA expression in the brain can be upregulated by an increased synaptic activity. Furthermore, the rapid and transient increase in NGF and BDNF mRNA following a needle insertion into the hippocampus could indicate that neuronal damage and trauma also increase the expression of these neurotrophic factors in the hippocampus. ACKNOWLEDGMENTS
KA
1
s-4
7
KA
7
FIG. 4. Number of grains over BDNF mRNA-expressing neurons after injection of saline or kainic acid. Sections including dentate gyrus (DG), CA1 region of the hippocampus (CAl), or piriform cortex (Pir), 1 and 7 h after injection of saline (Sal) or kainic acid (KA) were hybridized to the BDNF mRNA-specific probe. After the hybridization, the number of grains over individual neurons were calculated on emulsion autoradiographs from the sections. Tissue sections from sham-operated animals (sham) were processed in the same way. All sections were hybridized in parallel and the values (mean + SEM) were calculated using five different sections from four animals of each experimental paradigm. The number of grains over individual neurons in sham-operated animals was arbitrarily set at 1.0.
This work was supported by the search Council, Petrus and Augusta (University of Colorado) AG 04418, Research Council (8653), and funds M.B. was supported by a C.I.R.I.T. de Catalunya (Spain) and P.E. by Council.
Swedish National Science ReHedlunds Stiftelse, U.S. Grants NS 09199, the Swedish Medical from the Karolinska Institute. fellowship from the General&at the Swedish Medical Research
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21. HOFER, M., S. R. PAGLIUSI, A. HOHN, J. LEIBROCK, AND Y. A. BARDE. 1990. Regional distribution of brain-derived neurotrophic factor mRNA in the adult mouse brain. EMBO J. 9: 24592464. 22. HOFER, M. M., AND Y.-A. BARDE. 1988. Brain-derived neurotrophic factor prevents neuronal death in vivo. Nature 331: 261262. 23. HOHN, A., J. LEIBROCK, K. BAILEY, AND Y. A. BARDE. 1990. Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family. Nature 344: 339-341. 24. HONEGGER, P., AND D. LENOIR. 1982. Nerve growth factor (NGF) stimulation of cholinergic telencephalic neurons in aggregating cell cultures. Brain Res. 256: 229-238. 25. KAISHO, Y., K. YOSHIMIJRA, AND N. K. 1990. Cloning andexpression of a cDNA encoding a novel human neurotrophic factor. FEBS 266: 181-191. 26. KORSCHING, S., G. AUBURGER, R. HE~MANN, J. SCOTT, AND H. THOENEN. 1985. Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation. EMBO J. 4: 1389-1393. 27. KROMER, L. F. 1981. Nerve growth factor treatment after brain injury prevents neuronal death. Science 235: 214-216. 28. K~HLER, C. 1983. Exitotoxins. (Fuxe, K., Roberts, P., and Schwartz, R., Eds.), Wenner-Gren Center International Symposium series. Vol. 39, pp. 99-111. 29. LEIBROCK, J., F. LOTTSPEICH, A. HOHN, M. HOFER, B. HENGERER, P. MASIAKOWSKI, H. THOENEN, AND Y. A. BAF~DE.1989. Molecular cloning and expression of brain-derived neurotrophic factor. Nature 341: 312. 30. LEVI-M• NTALCINI, R. 1981. The nerve growth factor 35 years later. Science 237: 1154-1162. 31. LINDHOLM, D., R. HEUMANN, M. MEYER, AND H. THOENEN. 1981. Interleukin-1 regulates synthesis of nerve growth factor in non-neuronal cells of rat sciatic nerve. Nature 330: 658-659. 32. MAISONPIERRE, P. C., L. BELLUSCIO, B. FRIEDMAN, R. F. ALDERSON, W. S. J, M. E. FORTH, R. M. LINDSAY, AND Y. G. D. 1990. NT-3, BDNF and NGF in the developing rat nervous system: Parallel as well as reciprocal patterns of expression. Neuron 5: 501-509. 33. MAISONPIERRE, P. C., L. BELLUSCIO, S. SQUINTO, N. Y. IP, M. E. FORTH, R. M. LINDSAY, AND G. D. YANCOPOLOUS. 1990. Neurotrophin-3: A neurotrophic factor related to NGF and BDNF. Science 247: 1446-1451. 34. MART~Z, H. J., C. F. DREYFUS, G. M. JONAKAIT, AND I. B. BLACK. 1981. Nerve growth factor selectively increases cholinergic markers but not neuropeptides in rat basal forebrain in culture. 412: 295-301. 35. MOBLEY, W. C., J. L. RUTKOWSKI, G. I. TENNEKOON, K. BuCHANAN, AND M. V. JOHNSTON. 1985. Choline acetyltransferase activity in striatum of neonatal rats increased by nerve growth factor. 229: 284-281. 36. PAXINOS, S., AND C. WATSON. 1982. The Rat Brain in Stereotaxic Coordinates. Academic Press, Australia. 37. PHILLIPS, H. S., J. M. HAINS, G. R. LAF~AMEE, A. ROSENTHAL,
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39.
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AND J. W. WINSLOW. 1990. Widespread expression of BDNF but not NT-3 by target areas of basal forebrain choline@ neurons, Science250:290-294. RAUSCHER, F., D. R. COHEN, T. CURRAN, T. J. Bos, P. K. VOGT, D. BOHMANN, R. TJIAN, AND B. J. FRANZA. 1988. Fos-associated protein p39 is the product of the jun proto-oncogene. Science 240: 1010-1016. RENNERT, R. D., AND G. HEIRICH. 1986. Nerve growth factor mRNA in the brain: Localization by in situ hyb~~zation. Biothem. Bio~~ys. Res. Commu~. 138: 813-818. ROSENBERG, M., T. FRIEDMANN, R. C. ROBERTSON, M. TusZYNSKI, J. A. WOLF, X. 0. BREAKEFIELD, AND F. H. GAGE. 1988. Grafting genetically modified ceils to the damaged brain: Restorative effects of NGF expression. Science 242: 157551578. ROSENTHAL, A., G. D. V., T. NGUYEN, M. LEWIS, A. SHIH, G. R. LARAMEE, K. NIKOLICS, AND J. W. WINSLOW. 1990. Primary structure and biological activity of a novel human neurotrophic factor. Neuron 4: 767-773. SHELTON, D. L., AND L. F. REICHARDT. 1986. Studies on the expression of @nerve growth factor (NGF) in the central nervous system: level and regional distribution of NGF mRNA suggests that NGF function as a trophic factor for several distinct populations of neurons. Froc. Natl. Acad. Sci. USA 83: 27142718. SLOVITER, R. S., AND B. P. DAMIANO. 1981. Sustained electricaf stimulation of the perforant path duplicates kainate-induced electrophysiological effects and hippocampal damage in rats. Neurosci.
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44. SPILLANTINI, M. G., L. ALOE, E. ALLEVA, S. R. DE, M. GOEDERT, AND M. R. LEVI. 1989. Nerve growth factor mRNA and protein
MRNA EXPRESSION increase in hypothalamus Natl.
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51.
Acad.
Sci. USA
43 in a mouse model of aggression. Proc.
86: 8555-8559.
STR~MMBERG, I., C. WETMORE, T. EBENDAL, P. ERNFORS, H. PERSSON, AND L. OLSON. 1990. Rescue of basal forebrain cholinergic neurons after implantation of genetically modified cells producing recombinant NGF. J. Neurosci. Re.r. 25: 405-411. VIETJE, B. P., AND J. WELLS. 1989. Selective lesions of granule cells by fluid injections into the dentate gyrus. Exp. Neural. 106: 275-282. WETMORE, C., P. E~Fo~, H. PERSSON, AND L. OLSON. 1989. Localization of brain-derived neurotrophic factor mRNA to neurons in the brain by in situ hybridization. Exp. Neural. 109: 141-152. WHITTEMORE, S. R., T. EBENDAL, L. LXRKFORS, L. OLSON, A. SEIGER, I. STROMBERG, AND H. PERSSON. 1986. Developmental and regional expression of nerve growth factor messenger RNA and protein in the rat central nervous system. Proc. Natl. Acad. Sci. CJSA 83: 817-821. WHI~EMORE, S. R., P. L. FRIEDMAN, D. LARHAMMAR, H. PERSSON, C. M. GONZALEZ, AND V. R. HOLETS. 1988. Rat beta-nerve growth factor sequence and site of synthesis in the adult hippocampus. J. Neurosci. Res. 20: 403-410. WILLOWS, L. R., S. VARON, G. PETERSON, K. WI~R~, W. FISCHER, A. BJ~RKL~D, AND F. H. GAGE. 1986. Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria fornix transection. Proc. Natl. Acad. Sci. USA 83:9231-9235. ZAFRA, F., B. HENGERER, J. LEIBROCK, H. THOENEN, AND D. LINDHOLM. 1990. Activity dependent regulation of BDNF and NGF mRNAs in the rat hippocampus is mediated by nonNMDA glutamate receptors. EMBO J. 9: 3545-3550.
NEUROLOGY
114,35-43 (1991)
Hippocampal Damage and Kainic Acid Injection Induce a Rapid Increase in mRNA for BDNF and NGF in the Rat Brain Department of medical ~hern~t~, Laboratory of ~o~eu~ar Ne~rob~olo~ and ~De~artme~t of Pharmacology, Iiurolinska Institute, Box 60400 S-10401 Stockholm. Sweden
tions in the nervous system is largely derived from extensive studies of p-nerve growth factor (NGF), a wellcharacterized neurotrophic factor required for the development and maintenance of peripheral sensory and sympathetic neurons (30). More recently, NGF has also been detected in the brain, where the highest levels of its mRNA have been found in hippocampus and cerebral cortex (15,26,42,48), areas receiving major cholinergic projections from the basal forebrain. Subsequently, in situ hybridization has been used to demonstrate that within hippocampus, NGF mRNA is expressed in neurons, implying that NGF exerts a trophic effect in the brain through neuron-to-neuron interactions (2, 9, 37, 39, 49). Basal forebrain cholinergic neurons respond to exogenously added NGF by increasing the level of choline acetyltransferase (17,24,34,35) the key enzyme in the synthesis of acetylcholine, and can be prevented from degeneration after transection of the septohippocampal pathway by either chronic infusion of exogenous NGF (18, 27, 50) or by implantation of genetically modified cells producing recombinant NGF (40, 45). Furthermore, implantation of recombinant NGF-producing cells into cholinergically denervated cerebral cortex results in a marked increase in the survival of, and fiber outgrowth from, grafts of fetal basal forebrain neurons (6). In addition, intracerebral infusion of NGF in aged rats reduces basal forebrain cholinergic neuron atrophy and results in an improved spatial memory in behaviorally impared rats (10). Thus, ample evidence exsist that NGF supports basal forebrain cholinergic neurons. In similarity to NGF, BDNF supports neuronal survival both in vitro and in viuo (3,22) and the amino acid sequence predicted from the recently isolated porcine BDNF gene shows striking amino acid similarities to NGF (29). This structural similarity suggests that NGF and BDNF are members of a family of neurotrophic factors. Taking advantage of the amino acid similarity between NGF and BDNF, molecular clones for a third member of the NGF family, named hippocampus-derived neurotrophic factor (I-IDNF) (8), neurotrophin-3 (23, 32, 41) or NGF-2 (25) have recently been isolated.
In situ hybridization and Northern blots were used to study expression of mRNAs for members of the nerve growth factor family in the rat brain following an excitatory stimulus. One hour after a unilateral needle insertion or saline injection into the dorsal hippocampus, the level of brain-derived neurotrophic factor (BDNF) mRNA increased markedly in granular neurons of the dentate gyrus and in the piriform cortex ipsilateral to the injection. The same treatment also increased the level of NGF mRNA in granular neurons of the ipsilatera1 dentate gyrus. The rapid increase in BDNF and NGF mRNA after a needle insertion or injection of saline was transient and preceded by an increase in c-fos mRNA in the same brain regions. In contrast to a needle insertion per se or a saline injection, 7 h after a unilateral injection of kainic acid into the dorsal hippocampus, the level of BDNF mRNA was dramatically increased in the ipsilateral hippocampus, as well as in the ipsilateral fron~parietal, piriform and perihinal cortex, the amygdaloid complex, claustrum, and ventromedial hypothalamus. A less pronounced increase was also seen in these brain areas on the eontralateral side. Northern blots revealed that the level of BDNF mRNA increased 5- and 40-fold in the contra- and ipsilateral hippocampus, respectively, compared to sham-operated control animals. In contrast to BDNF and NGF, the level of hippocampus-derived neurotrophic factor/ neurotrohin-3 (HDNF/NT-3) mRNA was not altered by either needle insertion or injection of saline or kainic acid. These results indicate that hippocampal damage differentially induces expression of the NGF family of neurotrophie factors in the brain and suggest further that BDNF mRNA expression in the brain is markedly enhanced by an increased synaptic activity. o issi Academic
Press,
Inc.
INTRODUCTION Development and maintenance of neurons of the vertebrate nervous system depend on the presence of neurotrophic factors produced in the target areas of the sensitive neurons. The concept of neurotrophic interac-
35 All
Copyright Q 1991 rights of reproduction
0014-4ssw91 $3.00 by Academic Press, Inc. in any form reserved.
36
BALLARiN
Recently the NGF family has been shown to also include a fourth member, named neurotrophin-4 (NT-4) (16). In the brain, BDNF mRNA is widely distributed with the highest level in hippocampus (E&21,47) whereas the distribution of NT-3 mRNA is remarkably restricted being mostly confined to hippocampus (823). In hippocampus, INT-3, BDNF, or NGF mRNA-expressing neurons are located in a partially nonoverlapping arrangement (9, 37). NT-3 mRNA-expressing neurons, mostly with a pyramidal morphology, are concentrated in medial CA1 and in CA2 (8,9,37), whereas BDNF mRNAexpressing neurons, also of pyramidal morphology, are most abundant in CA3 and in the hilar region of the dentate gyrus (E&9,22,37,47). Neurons expressing high levels of NGF mRNA are also found in the hilar region, while only scattered NGF mRNA-expressing neurons are found in CAl, CA2, and CA3 (9, 13, 14, 37). NGF, BDNF, and NT-3 are maximally expressed in the brain at different times of development with a peak of NT-3 mRNA shortly after birth, followed by maximal expression of BDNF and NGF mRNA 2 and 3 weeks after birth, respectively (8, 33). The peak of NT-3 mRNA expression shortly after birth appears to be due to a high and transient expression of NT-3 mRNA in neurons of the developing cingulate cortex (11). In similarity to NT-3, BDNF mRNA has also been shown to be transiently expressed in many brain areas during early postnatal brain development, notably in the brain stem (12). The temporal and spatial expression of the three mRNAs suggests that their genes are strictly regulated during brain development and that the factors may support partially different neurons in the early postnatal and adult brain. In the present study we show that expression of both BDNF and NGF mRNA is rapidly and transiently induced in the hippocampus after a needle insertion into that area and that 7 h after kainic acid injection, the expression of BDNF mRNA is dramatically increased in neurons of both hippocampus and more distant brain areas. This distant effect is presumably caused by a paroxysmal discharge initiated by the drug, suggesting that increased synaptic activity can regulate expression of BDNF mRNA. EXPERIMENTAL
PROCEDURES
Kainic Acid Injections Male Sprague-Dawley rats (180-220 g) were anesthetized with a mixture of air and halotane and placed in a David Kopf stereotaxic frame. One group of rats (n = 24) were unilaterally injected with 1 ~1 of kainic acid (0.5 WgIpl in saline, pH 7.4) into the right dorsal hippocampus (coordinates: rostra1 3.3, lateral 2.8, ventral 4.8 from Bregma (36). Another group of rats were injected with 1~1 of saline (n = 40) or with the needle alone (n =
ET AL.
5). In addition, a group of sham-operated animals (n = 18) was used as a control. After the injection, the skin was sutured and the animals were left for the indicated times. In Situ Hybridization Tissue sections of dorsal hippocampus were cut on a cryostat (Leitz, F.R.G.) in 16-pm sections and thawed onto poly-L-lysine-coated slides (50 pg/ml). After fixation with 10% formalin, the sections were processed for in situ hybridization with deoxyadenosine[cu-[35S-thio]triphosphate] 3’-end-labeled probes as previously described (6). To detect BDNF-specific mRNA, a 50-mer oligonucleotide complementary to rat BDNF mRNA, corresponding to nucleotides 748-798 in pig BDNF (29), was used. For NGF-specific mRNA, a 50-mer oligonucleotides complementary to nucleotides 869 to 919 of rat NGF (49) was used. To detect NT-3-specific mRNA, a 50-mer oligonucleotides complementary to nucleotides 667 to 717 in rat INT-3 mRNA was used (8). These three oligonucleotide probes show 56 to 68% nucleotide homology to each other and are specific for their respective mRNAs as previously demonstrated (9). For c-fosspecific mRNA, a 48-mer oligonucleotide complementary to nucleotides 543 to 591 of rat c-fos (5) was used. To determine the intensity of labeling over individual cells the number of silver grains was counted manually over cells. Five different sections from four animals in each experimental paradigm were included in the analysis. The in situ hybridization revealed identical results in all animals within each experimental group studied. RNA Blot Analysis Frozen tissue samples were homogenized in 4 M guanidine isothiocyanate, 0.1 A4 &mercaptoethanol, 0.025 M sodium citrate (pH 7.0) and homogenized three times for 15 s with a Polytron. Each homogenate was layered over a 4-ml cushion of 5.7 M CsCl in 0.025 M sodium citrate (pH 5.5) and centrifuged at 15°C in a Beckman SW41 rotor at 35,000 rpm for 16 h (4). Poly(A)+ RNA was purifiedby oligo (dT)-cellulose chromatography (l), and the recovery of RNA was quantified spectrophotometrically before use in RNA blot analysis. Poly(A)+ RNA (15 pg) from each sample was electrophoresed in a 1% agarose gel containing 0.7% formaldehyde, followed by transfer to a nitrocellulose filter. The filter was hybridized to a 185-bp PCR fragment from rat BDNF corresponding to amino acids 183-239 of pig BDNF (29). The fragment was labeled with [a-32P]dCTP by nick-translation to a specific activity of approximately 5 X 10’ cpmlpg. Hybridization was carried out as described (7, 9) followed by washing at high stringency and exposure to Kodak XAR-5 film at -70°C. Appropriate exposures of the autoradiograms
REGULATION
were quantified meter.
using
a Shimadzu
OF
CS-9000
NGF
AND
densito-
RESULTS
A Transient Increase in the Level of BDNF and NGF mRNA following a Needle Insertion into the Hippocampus The short-term effects on the expression of mRNAs for the NGF family following a unilateral single injection of the excitotoxin kainic acid into the dorsal hippocampus was studied by in situ hybridization. The levels of BDNF, NGF, and NT-3 mRNAs in sham-operated animals, used as controls, were similar to those seen in animals 7 h after a saline injection shown in Fig. 1. One hour after injection, the level of BDNF mRNA increased markedly ipsilateral to the injection in the dentate gyrus, throughout the pyramidal cell layer of the hippocampus, as well as in piriform cortex (Fig. 1B). The level of NGF mRNA also increased on the ipsilatera1 side although this increase was confined to the dentate gyrus (Fig. 1F). For both BDNF and NGF, the increase in dentate gyrus was due to a larger number of granular neurons expressing detectable amounts of either of the two mRNAs, as well as an increased intensity of labeling over individual neurons. Similarly, the elevation in BDNF mRNA expression in the pyramidal cell layer and in piriform cortex were also due to a larger number of neurons expressing detectable amounts of BDNF mRNA as well as a higher intensity of labeling over individual neurons. In contrast to NGF and BDNF, the level of NT-3 mRNA was not affected by this treatment (Fig. 1J). Three different controls were used in the experiments: (i) sham-operated animals where the skull was opened with no needle insertion, (ii) unilateral injection of saline into the dorsal hippocampus, and (iii) insertion of the needle in the same place without injection. Unexpectedly, 1 h after saline injection, as well as 1 h after needle insertion, the levels of BDNF and NGF mRNA also increased in the ipsilateral hippocampus compared to sham-operated, control animals (Figs. 1A and 1E). Sham-operated animals showed no change in BDNF or NGF mRNA compared to nontreated animals and the mRNA levels for all three factors in these animals were similar to the ones seen in animals 7 h after a saline injection as shown in Figs. lC, lG, and 1K. None of the treatments had an effect on the level of NT-3 mRNA (Figs. 11 and 1J). The increase in BDNF and NGF mRNA caused by saline injection compared to shamoperated animals was first seen 15 and 30 min after the injection, respectively (Fig. 2). For both mRNAs the increase was maximal 1 h after the insertion (Fig. 2) and 7 h later, the levels of both BDNF and NGF mRNA were the same as in sham-operated animals. Hybridization of
BDNF
mRNA
EXPRESSION
37
adjacent sections to a c-fos mRNA-specific probe revealed elevated c-fos mRNA levels in the ipsilateral hippocampus already 5 min after the injection with a maxima1 level 30 min later. At this time point, elevated levels of c-fos mRNA were also seen in frontoparietal, perirhinal, and piriform cortex. The level of c-fos mRNA declined at 60 min and had essentially returned to control levels 2 h after the injection (Fig. 2). Kainic Acid Mediated Increase in the Level of BDNF mRNA Seven hours after a single injection of kainic acid in the dorsal hippocampus, the level of BDNF mRNA was dramatically increased in the ipsilateral hippocampus compared to the saline-injected control (compare Figs. 1C and 1D). Moreover, this marked increase was also found in the internal and external pyramidal layers of frontoparietal and perirhinal cortex, piriform cortex, amygdaloid complex, claustrum, and ventromedial hypothalamus. A large increase was also seen in the contralateral hippocampus and piriform cortex. The same treatment did not significantly change the level of NGF mRNA in the ipsilateral hippocampus compared to saline controls (compare Figs. 1G and 1H). In the case of NT-3, a small decrease was seen in the ipsilateral dentate gyrus both after saline or kainic acid injection (Figs. 1K and 1L). The dramatic increase in the level of BDNF mRNA was due to a large increase in the number of neurons expressing detectable levels of BDNF mRNA, as well as a marked increase in the level of BDNF mRNA in individual neurons (Fig. 3). In fact, in all regions with increased BDNF mRNA levels most, if not all, neurons expressed high levels of BDNF mRNA after the kainic acid injection compared to only a minority in saline-injected animals (compare Figs. 3A to 3B, 3C to 3D, and 3E to 3F). In addition, a grain count over BDNF mRNA-expressing neurons revealed an approximate 7fold increase in the number of grains over individual granular neurons in the dentate gyrus compared to saline-injected control (Fig. 4). A 6- and 3-fold increase in the number of grains were also found over neurons from the CA1 region of the hippocampus and piriform cortex, respectively (Fig. 4). Levels of BDNF mRNA Measured by Northern Blot Analysis In agreement with the results of the in situ hybridization, the level of BDNF mRNA in the hippocampus, measured by Northern blot analysis, was clearly increased both 1 and 7 h after injection of kainic acid compared to sham-operated animals (Fig. 5). Densitometric scanning of the autoradiograms revealed that the level of BDNF mRNA was elevated approximately 2- and 6fold on the contralateral and ipsilateral side, respec-
saline lh
FIG. 1. Expression of members of the NGF family of neurotrophic factors after intrahippocampal injections of saline or kainic acid. Adult Sprague-Dawley rats were injected into the CA3 region of the hippocampus with either saline (A, E, I, C, G, K) or kainic acid (B, F, J, D, H, L). The brains were sectioned either 1 h (A, B, E, F, I, J) or 7 h (C, D, G, H, K, L) after the injections. The sections were then hybridized to oligonucleotide probes specific for BDNF (A-D), NGF (E-H), or NT-3 (I-L). Bar in A is 3 mm and is the same for all panels. Note the increased level of BDNF mRNA in the ipsilateral (right side) hippocampus 1 h after injection of either saline (A) or kainic acid (B). Also, note the increase in NGF mRNA in the ipsilateral dentate gyrus 1 h after injection of either saline (E) or kainic acid (F). A marked increase in the level of BDNF mRNA is seen in several different brain regions (see text) 7 h after injection of kainic acid (D) compared to injection with saline (C). The increase is most pronounced on the ipsilateral side but is also seen on the contralateral side. Shown in the figure are dark-field photomicrographs of X-ray films exposed for 5 days. This exposure time is shorter than previously used to localize cells in the brain expressing members of the NGF family (9) and reveals relatively low signals in control animals but was chosen in order to visualize the increase in mRNA levels 1 h after saline injection or 7 h after kainic acid injection. The levels of BDNF, NGF, and NT-3 mRNAs in sham-operated animals (not shown) were similar to those seen in animals 7 h after saline injection. Abbreviations: DG, dentate gyrus; Pir, piriform cortex; CAl, CA2, CA3, pyramidal areas of the hippocampus.
tively, 1 h after the injection compared to sham-operated animals. Seven hours after the kainic acid injection, the levels of BDNF mRNA were increased approximately 5- and 40-fold on the contralateral and ipsilateral side, respectively. In agreement with previous studies (8, 29, 32), two BDNF mRNAs of 1.4 and 4.0 kb were seen, and the levels of both mRNA species increased to the same extent by the kainic acid injection. DISCUSSION
In the present study we show that the levels of both BDNF and NGF mRNA are increased in the hippocam-
pus 1 h after injection of saline or kainic acid into the dorsal hippocampus, compared to sham-operated animals. In the case of BDNF, a clear increase was also seen in the piriform cortex. On the other hand, the level of NT-3 mRNA, a third member of the NGF-family, was not changed by this treatment. Limbic seizures introduced by unilateral electrolytic lesion in the hilar region of the dentate gyrus have been shown to increase the level of NGF mRNA in granular neurons of the dentate gyrus and at later times after the lesion, as well as in neocortical and olfactory forebrain neurons (14). Our results show that the same increase in the hippocampus is obtained 1 h after an intrahippocampal injection of saline, kainic acid, or just a needle
insertion. Thus, the increase seen 1 h after kainic acid injection appears not to be caused by the excitotoxin but rather by the needle insertion per se. The increase in the levels of BDNF or NGFmRNA after saline injection or needle insertion was clearly transient with maximal levels 1 h after insertion. The cavity created by the injection of fluid into the dentate gyrus has been shown to cause an axotomy of the granule cells (46). Our results show that expression of BDNF and NGF increase rapidly in the hippocampus after injection of fluid into the hippocampus and suggest that this increase could be an early response to a mechanical damage and neuronal injury. Of interest is also the finding that expression of NGF mRNA is induced in the hypothalamus of aggressive male mice (44) suggesting that changes in NGF expression may not only occur in response to mechanically induced alterations in the brain but can also be caused by more subtle physiological changes induced by an external stimulus. The rapid increase in BDNF and NGF mRNA after a saline injection into the hippocampus was preceded by an increase in c-fos mRNA. A lesion of the sciatic nerve has been shown to cause a rapid increase in NGF expression in Schwann cells (20,31). The increase in NGF mRNA reaches a maximal level after 12 h and is preceded by a rapid and transient increase in c-fos and c-jun mRNAs (20). The transcription factor AP-1, a
5 min FIG. 2. Time-course sections were prepared hippocampus. Adjacent
15min for the induction from sham-operated sections were then
heterodimer between c-fos and c-jun (38), binds to a AP-1 site within the first intron of the NGF gene (19). This suggests that in the periphery the transcriptor factor AP-1 is involved in the induction of NGF mRNA expression in the lesioned nerve. It remains to be established if the c-fos and c-jun proteins (AP-1) also play a role in the induction of NGF and BDNF mRNA expression following an intrahippocampal saline injection. In contrast to the rapid and transient increase seen after a needle insertion alone, injection of kainic acid into the dorsal hippocampus resulted in a more persistent increase in the level of BDNF mRNA. This increase was not only found in the hippocampus but was also seen in several other brain regions, including the internal and external pyramidal layer of frontoparietal and perihinal cortex, piriform cortex, amygdaloid complex, claustrum, and ventromedial hypothalamus. The effect was more pronounced ipsilateral to the lesion but was also seen on the contralateral side. In agreement with the in situ hybridization results, Northern blot analysis showed 5- and 40-fold higher levels of BDNF mRNA on the contralateral and ipsilateral hippocampus, respectively, 7 h after kainic acid injection compared to the saline control. Recently, Zafra et al. (51) showed, by Northern blot analysis, markedly increased levels of BDNF and NGF mRNA in hippocampus and cerebral cortex after a systemic injection of kainic acid.
30min of c-fos, BDNF, and NGF mRNA following adult rats or from animals at the indicated hybridized with the indicated probes.
60min
120 min
saline injection into the dorsal hippocampus. Tissue times after a saline injection into the CA3 region of the
40
BALLARiN
ET
AL.
FIG. 3. Bright-field illumination showing BDNF mRNA-expressing neurons 7 h after injection of saline or kainic acid. BDNF mRNA-expressing neurons in the dentate gyrus (DG), CA1 region of the hippocampus (CAl), or piriform cortex (Pir), 7 h after injection of saline (A, C, E) or kainic acid (B, D, F). Note that 7 h after the kainic acid injection, the number of granular neurons expressing high levels of BDNF mRNA is greatly increased compared to the saline control (compare A and B). The same effects are seen in CA1 pyramidal neurons of the hippocampus (compare C and D) as well as in neurons of the piriform cortex (compare E and F). Scale bar in A is 20 pm and is the same for ail other panels. All panels shown are at coordinate -2.8 with respect to Bregma.
The same authors also provided pharmacolo~cal evidence that this effect was mediated through an excitatory amino acid receptor of the non-NMDA type. Intracerebroventricular injection of kainic acid was also recently shown to cause a marked increase in the level of NGF mRNA in the dentate gyrus and this effect was maximal at 2-3 h after the injection (13). The increased levels of mRNA in the hippocampus following a systemic injection was maximal at 3 h, and by 7 h after the injection the increase in NGF mRNA was significantly reduced (51). This may explain the fact that we did not, by in situ hybridization, detect a significant increase in the level of NGF mRNA in the hipp~ampus 7 h after the intrahippocampal injection of kainic acid. Kainic acid is an analog of the excitatory neurotransmitter glutamate and it has been reported that after intrahippocampal administration of the dose used in
this study, signs of neuronal degeneration are seen 4 h after the injection in the CA3 and CA1 pyramidal cells (28). Neuronal degeneration in areas distal to the injection site is less apparent and occurs at later times (28). Under the experimental conditions used in this study, evidence have been presented that the distant effect of kainic acid is due to paroxysmal discharge caused by the drug (43). Thus, it is likely that the increase in BDNF mRNA expression seen in distant regions is due to a spreading of synchronous neuronal discharges. In agreement with this possibility, the regions of the brain showing elevated levels of BDNF mRNA were the same distant regions that have previously been shown to be primarily affected by an intrahippocampal injection of kainic acid (28). The same areas also showed increased levels of c-fos mRNA 7 h after kainic acid injection (data not shown). In this context it is interesting to note
REGULATION
OF
NGF
AND
BDNF
mRNA
41
EXPRESSION
that these brain areas are the same regions as have been previously shown to express BDNF mRNA under normal physiological conditions (8,9,22,37,47). No significant effect was seen with regard to NT-3 mRNA either 1 or 7 h after injection of kainic acid. If this is due to differences in transcriptional regulation of NT-3 and
101
FIG. 5. Levels of BDNF mRNA in the hippocampus after injection of kainic acid. Fifteen micrograms of polyadenylated RNA isolated from the ipsilateral and contralateral hippocampus, 1 or 7 h after injection of kainic acid or from sham-operated animals, was electrophoresed in a formaldehyde-containing agarose gel. The gel was blotted and hybridized to the BDNF-radiolabeled probe, followed by washing and exposure to X-ray film. Five hippocampi were pooled for each RNA preparation and two independent measurement, with similar results, were performed. The 1eveI of BDNF mRNA in animals 7 h after saline injection (not shown) was similar to the level in sham-operated animals. Abbreviations: ipsi, ipsilateral side to the injection; contra, contralateral side to the injection.
n. shem
sal
1
1
KA
1
sal
7
KA
7
CA1
: sham
sal
1
KA
1
sal
7
Pir
(A
7
r
BDNF or merely reflects a different responsiveness of NT-3 mRNA-expressing neurons to kainic acid remains to be elucidated. The dramatic increase in BDNF mRNA expression in many brain regions known to express BDNF mRNA, and also known to undergo a paroxysmal discharge after an intrahippocampal injection of kainic acid, suggests that BDNF mRNA expression in the brain can be upregulated by an increased synaptic activity. Furthermore, the rapid and transient increase in NGF and BDNF mRNA following a needle insertion into the hippocampus could indicate that neuronal damage and trauma also increase the expression of these neurotrophic factors in the hippocampus. ACKNOWLEDGMENTS
KA
1
s-4
7
KA
7
FIG. 4. Number of grains over BDNF mRNA-expressing neurons after injection of saline or kainic acid. Sections including dentate gyrus (DG), CA1 region of the hippocampus (CAl), or piriform cortex (Pir), 1 and 7 h after injection of saline (Sal) or kainic acid (KA) were hybridized to the BDNF mRNA-specific probe. After the hybridization, the number of grains over individual neurons were calculated on emulsion autoradiographs from the sections. Tissue sections from sham-operated animals (sham) were processed in the same way. All sections were hybridized in parallel and the values (mean + SEM) were calculated using five different sections from four animals of each experimental paradigm. The number of grains over individual neurons in sham-operated animals was arbitrarily set at 1.0.
This work was supported by the search Council, Petrus and Augusta (University of Colorado) AG 04418, Research Council (8653), and funds M.B. was supported by a C.I.R.I.T. de Catalunya (Spain) and P.E. by Council.
Swedish National Science ReHedlunds Stiftelse, U.S. Grants NS 09199, the Swedish Medical from the Karolinska Institute. fellowship from the General&at the Swedish Medical Research
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