Proc. Natl. Acad. Sci. USA Vol. 95, pp. 1888–1893, February 1998 Neurobiology Activity-dependent regulation of Neu differentiation factoryneuregulin expression in rat brain (central nervous systemyneural plasticityytyrosine kinase) RAYA EILAM*, RONIT PINKAS-KRAMARSKI†,BARRY J. RATZKIN‡,MENAHEM SEGAL*, AND YOSEF YARDEN†§ Departments of *Neurobiology and †Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel; and ‡Amgen Center, Thousand Oaks, CA 91320 Communicated by Michael Sela, Weizmann Institute of Science, Rehovot, Israel, December 16, 1997 (received for review September 3, 1997) ABSTRACT Neu differentiation factor (NDFyneuregulin) less, because NDFyneuregulin induces heterodimerization be- is widely expressed in the central and peripheral nervous tween ErbB-3 and ErbB-2, the latter can reconstitute signaling by systems, where it functions as a mediator of the interactions the neuregulins even in cells that do not express ErbB-4 (15, 16). between nerve cells and Schwann, glia, oligodendrocyte, and The NDFyneuregulin family is widely expressed during em- muscle cells, to control cellular proliferation, differentiation, bryonic development in two major locations: the nervous systems and migration. NDF binds to two receptor tyrosine kinases, and the mesenchymal portion of parenchymal organs (10, 17). ErbB-3 and ErbB-4. Here we demonstrate that NDF and its Both a and b isoforms are expressed in mesenchymal cells, where ErbB-4 receptor are highly reactive to changes in ambient they presumably function as mediators of mesenchyme–epithel neuronal activity in the rodent brain in a region-selective interactions. Likewise, the neural isoforms of the neuregulins, manner. Generation of epileptic seizures by using kainic acid, which are mostly of the b isoform, also play a role in cell-to-cell a potent glutamate analog, elevated levels of NDF transcripts interactions. Thus, the ARIA promotes synapse-specific gene in limbic cortical areas, hippocampus, and amygdala. Con- expression, thereby augmenting nerve–muscle interactions (7). comitantly, ErbB-4 mRNA was increased with a similar Other isoforms of NDFyneuregulin promote maturation of as- spatial distribution, but transcription of the other NDF troglia and Schwann cells (8, 18), as well as oligodendrocytes (19), receptor, ErbB-3, did not change. A more moderate stimula- and at the same time inhibit neuronal differentiation (20, 21). tion, forced locomotion, was accompanied by an increase in These observations, together with the abundant expression of NDF transcripts and protein in the hippocampus and in the NDFyneuregulins by brain neurons and astroglia (18, 22), suggest motor cortex. Similar changes were found with ErbB-4, but not that neuregulins are involved in the response of the brain to ErbB-3. Last, a pathway-specific tetanic stimulation of the environmental stimuli. Here we show that brain NDFy perforant path, which produced long-term potentiation, was neuregulin, along with one of its surface receptors, namely followed by induction of NDF expression in the ipsilateral ErbB-4, is induced by three types of stimuli: activation of general dentate gyrus and CA3 area of the hippocampus. Taken excitatory neurotransmission with a glutamate receptor agonist, together, these results indicate that NDF is regulated by locomotion, and tetanic stimulation. These observations imply physiological activity and may play a role in neural plasticity. that the NDFyneuregulin family of ligands plays an important role in activity-dependent brain processes and in neuronal plas- Polypeptide growth factors play a central role in a variety of ticity. environmentally induced structural changes in the cortex and hippocampus of adult brain (1–3). For example, various treat- MATERIALS AND METHODS ments that induce seizure cause an increase in the expression of Materials. A polyclonal rabbit antibody was raised against NGF in hippocampus (4). An even more dramatic and rapid bacterial recombinant NDF-a2 and found to recognize all elevation of brain-derived neurotrophic factor (BDNF) mRNA isoforms of the factor. The antiserum was purified over a was observed after limbic seizures (1). These observations raise column of recombinant rat NDF, and the acid-eluted Ig the question as to whether other polypeptide factors that are fraction was used after dilution (23). Rabbit antibodies to a expressed in the central nervous system are also affected by recombinant extracellular portion of ErbB-3 and an antibody physiological activity. This possibility is especially relevant to a to a synthetic peptide derived from human ErbB-4 were family of soluble factors, variably named Neu differentiation purchased from Santa Cruz Biotechnology. In preliminary factors (NDFs) (5), heregulins (6), acetylcholine receptor induc- experiments we verified that these antisera do not cross-react. ing protein (ARIA) (7), and GGF (8), collectively renamed MK-801 and CPP were from Tocris Neuramin (Bristol, U.K.). neuregulins (9), that has been recently identified as ligands of All other chemicals were from Sigma. ErbB receptor tyrosine kinase. All of the isoforms of NDFy Animals. Adult male Hooded or Wistar rats (3 months old, neuregulin are encoded by a single gene located on human 200–250 g body weight) were used in all experiments. Between chromosome 8 (10). However, alternative splicing generates more 5 and 10 animals were used in each experimental group. than 13 isoforms that differ in their mosaic structure. Most Immunohistochemistry. Following perfusion of the animals important is an intrinsic EGF-like domain that is sufficient for with 2.5% paraformaldehyde, brains were removed and stored receptor binding and has two variants: a and b. Both groups of for 5–10 days in 1% paraformaldehyde. Serial 35-mm-thick isoforms bind directly to two types of receptors, ErbB-3 (11, 12) coronal or sagittal sections were subjected to immunohisto- and ErbB-4 (12, 13). Whereas the cytoplasmic domain of ErbB-4 chemical analysis by using the avidin–biotin peroxidase tech- displays a tyrosine-specific kinase activity, the corresponding nique (Vector Laboratories), as described (24). portion of ErbB-3 is devoid of catalytic function (14). Neverthe- Abbreviations: BDNF, brain-derived neurotrophic factor; CPP, The publication costs of this article were defrayed in part by page charge 3-[(RS)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid; DG, den- tate gyrus; EPSP, excitatory postsynaptic potential; HFS, high- payment. This article must therefore be hereby marked ‘‘advertisement’’ in frequency stimulation; KA, kainic acid; LTP, long term potentiation; accordance with 18 U.S.C. §1734 solely to indicate this fact. NDF, Neu differentiation factor; NMDA, N-methyl-D-aspartate. © 1998 by The National Academy of Sciences 0027-8424y98y951888-6$2.00y0 §To whom reprint requests should be addressed. e-mail: liyarden@ PNAS is available online at http:yywww.pnas.org. weizmann.weizmann.ac.il. 1888 Downloaded by guest on September 26, 2021 Neurobiology: Eilam et al. Proc. Natl. Acad. Sci. USA 95 (1998) 1889 In Situ Hybridization Analysis. NDF-specific primers with the propyl-1-phosphonic acid (CPP) (10 mgykg) was injected 20 min following nucleotide sequences were synthesized: 59-oligonucle- before stimulation, which led to blockade of LTP expression. otide: 59-TGAAGAGCCAGGAGTCAGCTGCAGG-39 and 39- Statistics. Both changes in optical density values obtained oligonucleotide: 59-GGCTCGAGACTCTGAGGACACATA- for mRNA levels and changes in the number of NDF- GG-39. These were used as primers to amplify a 0.3-kb-long immunopositive cells were determined by using a two-way fragment of the rat NDF coding region from cDNA clone 44 (5). ANOVA. The accepted level of significance was 0.05. The amplification procedure was exactly as described before (10), and the resulting DNA was cloned into the Bluescript plasmid RESULTS (Stratagene). T3 and T7 RNA polymerases were used to generate Induction of NDF and ErbB-4 by Epileptic Seizures. As an a 35 [ -( S)thio]UTP-labeled sense and antisense transcripts that initial test of the possibility that transcription of brain NDFy were used as probes. Embedding, sectioning, postfixation, and neuregulin is dynamically regulated we used massive stimulation hybridization were performed as described (25). To prepare of brain activity with the seizure-producing glutamate analog, cRNA probes specific to erbB-3 and to erbB-4, we extracted RNA kainate, which is involved in de novo gene transcription (29, 30). from the cerebellum of a 22-day-old rat by using the guanidinium y KA was injected intraperitoneally, and changes in the expression thiocyanate LiCl method (26). erbB-3- and erbB-4-specific DNA of NDF, as well as its receptors, ErbB-3 and ErbB-4, were sequences were amplified by using the following oligonucleotides: 9 followed. In accordance with previous reports (18, 22), in situ erbB-3-specific primers: 5 -TCCTGGCCGCCCCACATGCAC- hybridization localized NDF transcripts to multiple sites in the AAC 39 [sense; nucleotides 1300–1323 of human erbB-4 (27)] 9 9 brain, including cerebral cortex, thalamus, hypothalamus, and and 5 -GTCACATTTGCCCTCTGCCA-3 (antisense; nucleo- amygdala (Fig. 1). Likewise, transcripts of erbB-3 and erbB-4 tides 1580–1599), and erbB-4-specific primers: 59-TGTGCGTG- were found to be widely expressed in adult rat brain, in agreement CCTGCCCTAGTTCCAAGATGG-39 [sense; nucleotides 945– with previous reports (21, 31): major sites of erbB-4 expression 973 of human erbB-4 (28)] and 59-CCTGTTATCTCTCTGAC- are