Mouse fibroblast growth factor 10: cDNA cloning, protein characterization, and regulation of mRNA expression

Fibroblast growth factor 7 (FGF-7) or keratinocyte growth factor (KGF), is a potent and specific mitogen for epithelial cells. We have recently identified a novel human FGF-7 homologue, named FGF-10. To study the expression of this new FGF family member and its regulation in wound repair, we cloned the mouse FGF-10 (mFGF-10) cDNA. The encoded protein is 92% identical to human FGF-10 and 91% identical to rat FGF-10. When expressed in mammalian 293 cells, the mFGF-10 protein was glycosylated but remained cell- or extracellular matrix-associated. Upon addition of heparin, mFGF-10 protein was released into the media. mRNA encoding mFGF-10 was relatively abundant in lung, skin, brain and heart. In the skin, both FGF-7 and mFGF-10 were expressed in the dermal, but not the epidermal compartment. In contrast to FGF-7, mFGF-10 expression was not induced during cutaneous wound repair. In cultured fibroblasts, expression of mFGF-10 was strongly repressed by transforming growth factor beta and tumor necrosis factor alpha, whereas epidermal growth factor and interleukin-1beta had no effect. These results demonstrate a differential regulation of mFGF-10 and FGF-7 expression in vitro and during the wound healing process.     

Structure and expression of human fibroblast growth factor-10

We isolated the cDNA encoding a novel member of the human fibroblast growth factor (FGF) family from the lung. The cDNA encodes a protein of 208 amino acids with high sequence homology (95.6%) to rat FGF-10, indicating that the protein is human FGF-10. Human FGF-10 as well as rat FGF-10 has a hydrophobic amino terminus ( approximately 40 amino acids), which may serve as a signal sequence. The apparent evolutionary relationships of human FGFs indicate that FGF-10 is closest to FGF-7. Chromosomal localization of the human FGF-10 gene was examined by in situ hybridization. The gene was found to map to the 5p12-p13 region. Human FGF-10 (amino acids 40 to 208 with a methionine residue at the amino terminus) was produced in Escherichia coli and purified from the cell lysate. Recombinant human FGF-10 (approximately 19 kDa) showed mitogenic activity for fetal rat keratinizing epidermal cells, but essentially no activity for NIH/3T3 cells, fibroblasts. The specificity of mitogenic activity of FGF-10 is similar to that of FGF-7 but distinct from that of bFGF. In structure and biological activity, FGF-10 is similar to FGF-7.     

Expression of fibroblast growth factor family during postnatal skeletal muscle hypertrophy

The potential role of the fibroblast growth factor (FGF) family during stretch-induced postnatal skeletal muscle hypertrophy was analyzed by using an avian wing-weighting model. After 2 or 11 days of weighted stretch, anterior latissimus dorsi (ALD) muscles were, on average, 34 (P < 0.01) and 85% (P < 0.01) larger, respectively, than unweighted ALD control muscles. By using quantitative RT-PCR, FGF-1 mRNA expression was found to be significantly decreased in ALD muscles stretched for 2 or 11 days. In contrast, FGF-4 and FGF-10 mRNA expression was significantly increased 2 days after initiation of stretch. FGF-2, FGF-10, fibroblast growth factor receptor 1, and FREK mRNA expression was significantly increased at 11 days poststretch. Increases in FGF-2 and FGF-4 protein could be detected throughout the myofiber periphery after 11 days of stretch. On a cellular level, FGF-2 and FGF-4 proteins were differentially localized. This differential expression pattern and protein localization of the FGF family in response to stretch-induced hypertrophy suggest distinct roles for individual FGFs during the postnatal hypertrophy process.     

Characterization of recombinant human fibroblast growth factor (FGF)-10 reveals functional similarities with keratinocyte growth factor (FGF-7)

A newly identified member of the fibroblast growth factor (FGF) family, designated FGF-10, is expressed during development and preferentially in adult lung. The predicted FGF-10 protein is most related to keratinocyte growth factor (KGF, or FGF-7). The latter is unique among FGFs in that it binds and signals only through the FGF receptor (FGFR2b) isoform KGF receptor (KGFR) expressed specifically by epithelial cells. In order to examine the biological and biochemical properties of human FGF-10, we isolated the cDNA and expressed its encoded protein in bacteria. The recombinant protein (rFGF-10) was a potent mitogen for Balb/MK mouse epidermal keratinocytes with activity detectable at 0.1 nM and maximal at around 5 nM. Within this concentration range, FGF-10 did not stimulate DNA synthesis in NIH/3T3 mouse fibroblasts. rFGF-10 bound the KGFR with high affinity comparable to that of KGF, and did not bind detectably to either the FGFR1c (Flg) or FGFR2c (Bek) receptor isoforms. The mitogenic activity of FGF-10 could be distinguished from that of KGF by its different sensitivity to heparin and lack of neutralization by a KGF monoclonal antibody. These results indicate that FGF-10 and KGF have similar receptor binding properties and target cell specificities, but are differentially regulated by components of the extracellular matrix.     

Identification and characterization of a novel member of the fibroblast growth factor family

A new member of the fibroblast growth factor (FGF) family, FGF-13, has been molecularly cloned as a result of high throughput sequencing of a human ovarian cancer cell library. The open reading frame of the novel human gene (1419 bp) encodes for a protein of 216 a.a. with a molecular weight of 22 kDa. The FGF-13 sequence contains an amino-terminal hydrophobic region of 23 a.a. characteristic of a signal secretion sequence. FGF-13 is most homologous, 70% similarity at the amino acid level, to FGF-8. Northern hybridization analysis demonstrated prominent expression of FGF-13 in human foetal and adult brain, particularly in the cerebellum and cortex. In proliferation studies with BaF3 cells, FGF-13 preferentially activates cell clones expressing either FGF receptor variant, 3-IIIc or 4. The signal transduction pathways of FGF-13 and FGF-2 were compared in rat hippocampal astrocytes. The two FGFs induce an equivalent level of tyrosine phosphorylation of mitogen-activated protein kinase (MAPK) and c-raf activation. However, FGF-13 is more effective than FGF-2 in inducing the phosphorylation of phospholipase C-gamma (PLC-gamma). Treatment of neuronal cultures from rat embryonic cortex with FGF-13 increases the number of glutamic acid decarboxylase immunopositive neurons, the level of high-affinity gamma-aminobutyric acid (GABA) uptake, and choline acetyltransferase enzyme activity. The GABAergic neuronal response to FGF-13 treatment is rapid with a significant increase occurring within 72 h. We have identified a novel member of the FGF family that is expressed in the central nervous system (CNS) and increases the number as well as the level of phenotypic differentiation of cortical neurons in vitro.     

FGF-18, a novel member of the fibroblast growth factor family, stimulates hepatic and intestinal proliferation

The fibroblast growth factors (FGFs) play key roles in controlling tissue growth, morphogenesis, and repair in animals. We have cloned a novel member of the FGF family, designated FGF-18, that is expressed primarily in the lungs and kidneys and at lower levels in the heart, testes, spleen, skeletal muscle, and brain. Sequence comparison indicates that FGF-18 is highly conserved between humans and mice and is most homologous to FGF-8 among the FGF family members. FGF-18 has a typical signal sequence and was glycosylated and secreted when it was transfected into 293-EBNA cells. Recombinant murine FGF-18 protein (rMuFGF-18) stimulated proliferation in the fibroblast cell line NIH 3T3 in vitro in a heparan sulfate-dependent manner. To examine its biological activity in vivo, rMuFGF-18 was injected into normal mice and ectopically overexpressed in transgenic mice by using a liver-specific promoter. Injection of rMuFGF-18 induced proliferation in a wide variety of tissues, including tissues of both epithelial and mesenchymal origin. The two tissues which appeared to be the primary targets of FGF-18 were the liver and small intestine, both of which exhibited histologic evidence of proliferation and showed significant gains in organ weight following 7 (sometimes 3) days of FGF-18 treatment. Transgenic mice that overexpressed FGF-18 in the liver also exhibited an increase in liver weight and hepatocellular proliferation. These results suggest that FGF-18 is a pleiotropic growth factor that stimulates proliferation in a number of tissues, most notably the liver and small intestine.     

Itraconazole oral solution versus clotrimazole troches for the treatment of oropharyngeal candidiasis in immunocompromised patients

The development of the nervous system appears to be under the control of multiple growth factors, neurotrophins and cytokines, which may be expressed either continuously or transiently throughout defined stages of cellular generation, proliferation or differentiation. Fibroblast growth factor (FGF) cytokines and their receptors are abundantly expressed in the embryonic nervous system but their localization at autonomic levels in the fetal spinal cord has not yet been detailed. Immunoreactivity to FGF-2, probably the best characterized member of the FGF family (FGF-1 to FGF-10) and of one of its high affinity receptors, FGFR-1, was found in autonomic neurons at embryonic day E14, the peak day of generation and proliferation in the common ventral motoneuron pool. It was also continuously present throughout the investigated subsequent stages (E15 to postnatal day P30). Immunogold electron microscopy revealed the cytoplasmic localization of FGF-2 and FGFR-1 in intermediolateral neurons, the major group of sympathetic preganglionic neurons in the spinal cord. In these neurons, immunocytochemistry from E14 onwards showed the co-distribution of both markers at the period of axonal outgrowth to peripheral targets, e.g. the adrenal medulla. Our findings suggest autocrine and/or paracrine actions of FGF-2 for sympathetic preganglionic development but do not support its role as a target-derived neurotrophic factor for autonomic neuron development.     

Tissue culture assays using Caco-2 cell line differentiate virulent from non-virulent Listeria monocytogenes strains

OBJECTIVE: Fibroblast growth factor 9 (FGF-9) is a relatively new member of the FGF family isolated from the conditioned medium of a human glioblastoma cell line as a secreting type factor that exhibits a growth-stimulating effect on primary glial cells. To elucidate the roles of FGF-9 in human brain tumors, the expression and biological activities of FGF-9 were studied using culture cells and surgically obtained tumor specimens. METHODS: Measurement of FGF-9 and basic FGF in conditioned media of cell cultures was performed by using a sandwich enzyme immunoassay. The mitogenic effect of FGF-9 was evaluated by cell growth studies. FGF-9 expression in vivo was demonstrated by immunohistochemistry. RESULTS: One of 4 glioma cell lines and 4 of 16 human meningiomas examined actually secreted detectable amounts of FGF-9 proteins. In comparison, basic FGF production was detected from 3 of 4 glioma cell lines and 11 of 16 human meningiomas. Similarly to basic FGF, recombinant human FGF-9 significantly stimulated the in vitro cell proliferation in three of four glioma cell lines investigated in a dose-dependent manner. A time course growth study using U87 MG cells revealed an accelerated growth stimulation by FGF-9 after Day 4. The growth stimulatory activity was also shown in three of four human meningiomas studied. Moderate to strong immunoreactivity for FGF-9 was observed in 40 (82%) of 49 human brain tumors examined irrespective of origin, tumor type, grade of malignancy, or whether initial or recurrent. In contrast, strong immunostaining was localized in neurons in the normal human cerebral cortex. CONCLUSION: The present findings suggest that FGF-9 may be involved in the biology of human brain tumors with a possible importance in tumor cell growth. Whether the growth factor is more generally involved in oncogenesis of human tumors awaits further investigation.     

Distribution of fibroblast growth factor (FGF)-2 and FGF receptor-1 messenger RNA expression and protein presence in the mid-trimester human fetus

Fibroblast growth factors (FGF) are known to have key roles in embryonic growth and morphogenesis, but their presence and contributions to fetal development are unclear. In particular, little information exists as to the relevance of FGF and their specific receptors to human fetal development. We studied the anatomical distribution of messenger RNA encoding FGF-2 and one of its high affinity receptors, FGFR1, using in situ hybridization in a variety of human fetal tissues in early second trimester. Corresponding protein distributions were determined by immunohistochemistry. Both FGF-2 and FGFR1 mRNA and proteins were found to be present in every organ and tissue examined, but with defined cellular localizations. In skeletal muscle, both FGF-2 and FGFR1 mRNA and peptides were present in differentiated fibers, and both co-localized to proliferating chondrocytes of the epiphyseal growth plate. FGF-2 and FGFR1 mRNA and peptides were also present within cardiac or gastrointestinal smooth muscle. Within the gastrointestinal tract FGF-2 mRNA and peptide were located in the submucosal tissue, whereas FGFR1 was expressed within the overlying mucosa. Similarly, in skin, FGF-2 was expressed within the dermis whereas FGFR1 mRNA and peptide were most apparent in the stratum germinativum of the epidermis. In kidney and lung, FGFR1 mRNA was located in the tubular and alveolar epithelia respectively, whereas FGF-2 was expressed in both epithelial and mesenchymal cell populations. Both growth factor and receptor were widespread in both neuroblasts and glioblasts in the cerebral cortex of the brain. Immunoreactivity for FGF-2 and FGFR1 was seen in all vascular endothelial cells of major vessels and capillaries. Within the skin, kidney, lung, and intestine FGF-2 immunoreactivity was found in basement membranes underlying epithelia, and was associated with the extracellular matrix and plasma membranes of many cell types. The results show that FGF-2 and one of its receptors are widely expressed anatomically in the mid-trimester human fetus.     

Distribution and putative roles of fibroblast growth factor-2 isoforms in corneal endothelial modulation

PURPOSE: Corneal endothelial modulation factor (CEMF) released by inflammatory cells induces de novo synthesis of fibroblast growth factor (FGF)-2, which is a morphogen and a potent mitogen of corneal endothelial cells (CECs). Four isoforms of FGF-2 have been found in the nucleus, cytoplasm, or extracellular matrix (ECM) in different cell lines. In the present study, the profiles of the isoforms of FGF-2 that are induced by CEMF were investigated, and whether the differential localization of the isoforms of FGF-2 plays a role in CECs proliferation and subsequent modulation was examined. METHODS: Nuclear, cytoplasmic, and ECM fractions of normal and modulated CECs were separated, and FGF-2 isoforms were further purified by heparin-Sepharose column. The molecular sizes of the isoforms were determined by immunoblot analysis, using a specific antibody directed against FGF-2. Cell proliferation was determined by cell counting. Cellular localization of FGF-2 was determined by immunofluorescence staining during different stages of cell growth. RESULTS: To confirm that CEMF modulated CECs under the conditions used in this study, its effect on cell proliferation and cell shape was determined: CEMF-treated cells showed enhanced cell proliferation profiles and fibroblastlike morphology. In rapidly growing normal CECs, FGF-2 was predominantly present in the nucleus. As the cells reached confluence, the staining potential in the nucleus was markedly reduced. Cytoplasmic staining of FGF-2 was barely detectable, regardless of cell stages. In CEMF-modulated cells, the rapidly growing cells showed strong staining of FGF-2 in the nucleus, whereas cytoplasmic and ECM staining was weak. When modulated cells reached confluence, the staining of FGF-2 in the nuclei remained strong, whereas ECM staining was significantly increased. Immunoblot analysis of the subcellular fraction showed that the 24-kDa FGF-2 was predominantly present in the nucleus, whereas the 18-kDa form was the major molecule in cytoplasmic and ECM fractions in normal and modulated cells. CONCLUSIONS: These findings indicate that 24-kDa nuclear FGF-2 may be involved in cell proliferation in growing CECs. The persistent nuclear localization and simultaneous ECM localization of FGF-2 are induced by CEMF, and these FGF-2 isoforms seem to play a role in cell proliferation and modulation.     

Corneal scarring in the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study: baseline prevalence and repeatability of detection

Fibroblast growth factor-16 (FGF-16) is the most recent member of the FGF family to be cloned. Since the biologic activity of rat FGF-16 (rFGF-16) was unknown, and this protein has no apparent signal sequence, we transformed its entire cDNA into Escherichia coli for high-level expression and further characterization of this novel protein. An attempt was made to purify the expressed protein from the supernatant of mechanically lysed cells using sequential cation-exchange chromatography. This resulted in a gradual loss of the protein as precipitate throughout the purification process. In addition to precipitation during purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the partially purified materials showed a cluster of protein bands around 20k to 29k. Sequence analysis of the major bands indicated that two N-terminal truncations had occurred, during E. coli fermentation, purification, or both. The largest truncation resulted in the removal of the 34 N-terminal amino acids, including the initiation codon methionine. We cloned d34 rFGF-16, expressed the gene in E. coli, and developed a purification process for this form. Even with this truncated form, precipitation was a problem. We were largely able to overcome this problem, however, by including EDTA throughout the purification process. We have characterized the structure of purified d34 rFGF-16 extensively using circular dichroism, Fourier transform infrared spectroscopy, and sedimentation velocity analysis. These studies revealed that the protein has a distinct tertiary structure, consists primarily of beta-strands, has a weak tendency to self-associate, and is fairly extended. We then performed biologic assays which showed that d34 rFGF-16 induces oligodendrocyte proliferation in vitro, and induces hepatocellular proliferation and increased liver weight in vivo. In summary, FGF-16, a novel FGF family member, has both unique structural and biological properties.     

The glycine box: a determinant of specificity for fibroblast growth factor

Acidic fibroblast growth factor (FGF-1), keratinocyte growth factor (FGF-7), and FGF-10 are homologues with distinct specificity. In the presence of heparin, FGF-1 binds and activates in vitro all FGFR subtypes, while FGF-7 exhibits absolute specificity for the IIIb splice variant of FGFR2. FGF-10 exhibits a similar specificity but also binds the FGFR1IIIb isoform. Neither FGF-7 nor FGF-10 will bind to IIIc isoforms of FGFR. Molecular models of FGF, heparin, and the FGFR ectodomain suggested that sequences between beta-strands 10 and 12 of FGF may be important for the interaction of FGF with the heparin-FGFR ectodomain duplex. Site-directed mutants of FGF-7 and FGF-10 were prepared to test whether this domain might underlie failure of FGF-7 and FGF-10 to bind to the FGFRIIIc isoforms. Constructions with substitution of FGF-1 sequences spanning the entire C-terminus encoded in exon 3 or only C-terminal sequences spanning beta-strands 10 through 12 conferred ability on FGF-7 to bind to and activate FGFRIIIc without a significant loss in binding to or activation of FGFR2IIIb. A series of twelve different substitutions of shorter segments of FGF-1 sequences into the C-terminal portion of FGF-7 or FGF-10 revealed that substitution of GSCKRG for GIPVRG or the tri-peptide sequence KKN for NQK just N-terminal to it conferred dual activities on both the FGF-7 and FGF-10 backbones. The results suggest that the combined sequence domain, which we call the FGF glycine box (G-box), is a major determinant for the specificity of the binding of FGF to heparan sulfate-FGFR duplexes.     

Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property

Glia-activating factor (GAF) is a novel heparin-binding growth factor purified from the culture supernatant of a human glioma cell line. It shows a spectrum of activity slightly different from those of other known growth factors. We have isolated the cDNA which encodes human GAF. A homology search revealed that GAF would be the ninth member of the FGF family, and we therefore call it FGF-9. The human FGF-9 cDNA cloned by using oligonucleotide probes encoded a polypeptide consisting of 208 amino acids. Sequence similarity to other members of the FGF family was estimated to be around 30%. Two cysteine residues and other consensus sequences in family members were also well conserved in the FGF-9 sequence. FGF-9 was found to have no typical signal sequence in its N terminus like those in acidic FGF and basic FGF. Acidic FGF and basic FGF are known not to be secreted from cells in a conventional manner. However, FGF-9 was found to be secreted from cells after synthesis despite its lack of a typical signal sequence. It could be detected exclusively in the culture medium of cDNA-transfected COS cells. The amino acid sequence of proteins purified from culture supernatant of the CHO cell line, which was cDNA transfected and selected as a high producer of FGF-9, showed that no peptides were cleaved from the N terminus except the initiation methionine. The rat FGF-9 cDNA was also cloned, and the structural analysis indicated that the PGF-9 gene is highly conserved. Expression of the FGF-9 gene could be detected in the brain and kidney of the adult rat. Restricted gene expression in organs and the unique secretion nature of the protein suggest that FGF-9 plays a physiological role which differs from those of well-characterized acidic FGF and basic FGF.     

Clumsiness in children: a defect of kinaesthetic perception?

The distribution of type I interleukin-1 receptor (IL-1R1) mRNA in the rat brain was examined by in situ hybridization technique. IL-1R1 mRNA was expressed in several brain regions including the anterior olfactory nucleus, medial thalamic nucleus, posterior thalamic nucleus, basolateral amygdaloid nucleus, ventromedial hypothalamic nucleus, arcuate nucleus, median eminence, mesencephalic trigeminal nucleus, motor trigeminal nucleus, facial nucleus and Purkinje cells of the cerebellum. Furthermore, we identified neuronal expression of IL-1R1 mRNA using simultaneous detection (double in situ hybridization) of IL-1R1 mRNA with neuron specific enolase mRNA. In addition to the expression in neuronal cells, IL-1R1 mRNA was also expressed on the vascular walls and the epithelial cells of the choroid plexus and the ventricles. These findings suggest the possibility that IL-1 produces its multiple effects on the central nervous system through the actions not only on neuronal cells but also on endothelial and epithelial cells.     

Growth factor regulation of cell growth and proliferation in the nervous system. A new intracrine nuclear mechanism

This article discusses a novel intracrine mechanism of growth-factor action in the nervous system whereby fibroblast growth factor-2 (FGF-2) and its receptor accumulate in the cell nucleus and act as mediators in the control of cell growth and proliferation. In human and rat brain the levels and subcellular localization of FGF-2 differ between quiescent and reactive astrocytes. Quiescent cells express a low level of FGF-2, which is located predominantly within the cytoplasm. In reactive astrocytes, the expression of FGF-2 increases and the proteins are found in both the cytoplasm and nucleus. In glioma tumors, FGF-2 is overexpressed in the nuclei of neoplastic cells. Similar changes in FGF-2 expression and localization are found in vitro. The nuclear accumulation of FGF-2 reflects a transient activation of the FGF-2 gene by potentially novel transactivating factors interacting with an upstream regulatory promoter region. In parallel with FGF-2, the nuclei of astrocytes contain the high-affinity FGF-2 receptor, FGFR1. Nuclear FGFR1 is full length, retains kinase activity, and is localized within the nuclear interior in association with the nuclear matrix. Transfection of either FGF-2 or FGFR1 into cells that do not normally express these proteins results in their nuclear accumulation and concomitant increases in cell proliferation. A similar regulation of nuclear FGF-2 and FGFR1 is observed in neural crestderived adrenal medullary cells and of FGF-2 in the nuclei of cerebellar neurons. Thus, the regulation of the nuclear content of FGF-2 and FGFR1 could serve as a novel mechanism controlling growth and proliferation of glial and neuronal cells.     

Differential binding of fibroblast growth factor-2 and -7 to basement membrane heparan sulfate: comparison of normal and abnormal human tissues

Fibroblast growth factors (FGFs) play multiple roles during development and in adult tissues as paracrine regulators of growth and differentiation. FGFs signal through transmembrane receptor tyrosine kinases, but heparan sulfate is also required for signaling by members of the FGF family. In addition, heparan sulfate may be involved in determining tissue distribution of FGFs. Using biotinylated FGF-2 and FGF-7 (KGF) as probes, we have identified specific interactions between FGFs and heparan sulfates in human tissues. Both FGF species bind to tissue mast cells and to epithelial cell membranes. Binding to basement membrane heparan sulfate is tissue source dependent and specific. Although FGF-2 strongly binds to basement membrane heparan sulfate in skin and most other tissue sites examined, FGF-7 fails to bind to basement membrane heparan sulfate in most locations. However, in subendothelial matrix in blood vessels and in the basement membrane of a papillary renal cell carcinoma, strong FGF-7 binding is seen. In summary, distinct and specific affinities of heparan sulfates for different FGFs were identified that may affect growth factor activation and local distribution. Heparan sulfate may have a gatekeeper function to either restrict or permit diffusion of heparin-binding growth factors across the basement membrane.     

Immunohistochemical localization of metabotropic glutamate receptors, mGluR7a and mGluR7b, in the central nervous system of the adult rat and mouse: a light and electron microscopic study

The distributions of two alternative splicing variants of metabotropic glutamate receptor mGluR7, mGluR7a and mGluR7b, were examined immunohistochemically in the rat and mouse by using variant-specific antibodies raised against C-terminal portions of rat mGluR7a and human mGluR7b. Many regions throughout the central nervous system (CNS) showed mGluR7-like immunoreactivities (LI). The distribution patterns of mGluR7-LI in the rat were substantially the same as those in the mouse, although some species differences were observed in a few regions. Intense mGluR7a-LI was seen in the main and accessory olfactory bulbs, anterior olfactory nucleus, islands of Calleja, superficial layers of the olfactory tubercle, piriform cortex and entorhinal cortex, periamygdaloid cortex, amygdalohippocampal area, hippocampus, layer I of the neocortical regions, globus pallidus, superficial layers of the superior colliculus, locus coeruleus, and superficial layers of the medullary and spinal dorsal horns. The distribution of mGluR7b was more restricted. It was intense in the islands of Calleja, substantia innominata, hippocampus, ventral pallidum, and globus pallidus. The medial habenular nucleus also showed intense mGluR7a-LI in the rat but not in the mouse. For both mGluR7a- and mGluR7b-LI, localization in the active zones of presynaptic axon terminals was confirmed electron microscopically at synapses of both the asymmetrical and symmetrical types. It is noteworthy that mGluR7a-LI is seen preferentially in relay nuclei of the sensory pathways and that both mGluR7a- and mGluR7b-LI are observed not only in presumed glutamatergic axon terminals, but also in non-glutamatergic axon terminals including presumed inhibitory ones. Thus, mGluR7 may play roles not only as an autoreceptor in glutamatergic axon terminals, but also as a presynaptic heteroreceptor in non-glutamatergic axon terminals in various CNS regions.