Spatiotemporal expression and functional implication of CXCL14 in the developing mice cerebellum

Cerebellar granule neurons migrate from the external granule cell layer (EGL) to the internal granule cell layer (IGL) during postnatal morphogenesis. This migration process through 4 different layers is a complex mechanism which is highly regulated by many secreted proteins. Although chemokines are well-known peptides that trigger cell migration, but with the exception of CXCL12, which is responsible for prenatal EGL formation, their functions have not been thoroughly studied in granule cell migration. In the present study, we examined cerebellar CXCL14 expression in neonatal and adult mice. CXCL14 mRNA was expressed at high levels in adult mouse cerebellum, but the protein was not detected. Nevertheless, Western blotting analysis revealed transient expression of CXCL14 in the cerebellum in early postnatal days (P1, P8), prior to the completion of granule cell migration. Looking at the distribution of CXCL14 by immunohistochemistry revealed a strong immune reactivity at the level of the Purkinje cell layer and molecular layer which was absent in the adult cerebellum. In functional assays, CXCL14 stimulated transwell migration of cultured granule cells and enhanced the spreading rate of neurons from EGL microexplants. Taken together, these results revealed the transient expression of CXCL14 by Purkinje cells in the developing cerebellum and demonstrate the ability of the chemokine to stimulate granule cell migration, suggesting that it must be involved in the postnatal maturation of the cerebellum.     

Growth arrest specific gene 1 is a positive growth regulator for the cerebellum.

Postnatal cerebellum development involves the generation of granule cells and Bergmann glias (BGs). The granule cell precursors are located in the external germinal layer (EGL) and the BG precursors are located in the Purkinje layer (PL). BGs extend their glial fibers into the EGL and facilitate granule cells' inward migration to their final location. Growth arrest specific gene 1 (Gas1) has been implicated in inhibiting cell-cycle progression in cell culture studies (G. Del Sal et al., 1992, Cell 70, 595--607). However, its growth regulatory function in the CNS has not been described. To investigate its role in cerebellar growth, we analyzed the Gas1 mutant mice. At birth, wild-type and mutant mice have cerebella of similar size; however, mature mutant cerebella are less than half the size of wild-type cerebella. Molecular and cellular examinations indicate that Gas1 mutant cerebella have a reduced number of granule cells and BG fibers. We provide direct evidence that Gas1 is required for normal levels of proliferation in the EGL and the PL, but not for their differentiation. Furthermore, we show that Gas1 is specifically and coordinately expressed in both the EGL and the BGs postnatally. These results support Gas1 as a common genetic component in coordinating EGL cell and BG cell proliferation, a link which has not been previously appreciated.     

Developmental neurotoxicity of phenytoin on granule cells and Purkinje cells in mouse cerebellum

Phenytoin (PHT) is a primary antiepileptic drug. Cerebellar malformations in human neonates have been described following intrauterine exposure to PHT. The neonatal period of development in the cerebellum in mice corresponds to the last trimester in humans. To examine the neurotoxic effects of PHT in the developing cerebellum, we administered PHT orally to newborn mice once a day during postnatal days 2-4. We observed many apoptotic cells in the external granular layer (EGL) on postnatal day 5, labeled cells in the EGL still remaining 72 h after labeling with 5-bromo-2'-deoxyuridine, and EGL thicker than that in the control on postnatal day 14. These results showed that PHT induced cell death of external granule cells and inhibited migration of granule cells in cerebella. In specimens immunostained with antibody against inositol 1,4,5-trisphosphate receptor type 1, Purkinje cells in the treated group had poor and immature arbors, and partially showed an irregular arrangement. The motor performance of the treated mice in a rotating rod test was impaired, although there were no changes in muscular strength or in walking pattern at the period of maturity. These findings indicate that PHT induces neurotoxic damage to granule cells and Purkinje cells in the developing cerebellum and impairs selected aspects of motor coordination ability.     

Localization of tissue plasminogen activator mRNA in the developing rat cerebellum and effects of inhibiting tissue plasminogen activator on granule cell migration

Tissue plasminogen activator (tPA) mRNA was localized in the developing cerebellum and the potentials role of tPA in migration of cerebellar granule cells was investigated. Proteolytic assays and Northern blots showed little variation in levels of tPA proteolytic activity or tPA mRNA expression in the developing cerebellum. The distribution of cerebellar tPA mRNA at different ages was visualized by in situ hybridization histochemistry. At postnatal day 7 (P7), most labeled cells were in the internal granule layer or developing white matter, and very few if any premigratory granule cells contained tPA mRNA. Although the molecular layer contained labeled cells at all ages, cell counts indicated that a greater percentage of cells in the molecular layer contained tPA mRNA during adulthood than during the period of granule cell migration. The most striking change in tPA mRNA expression was in Purkinje neurons, most of which began to express tPA mRNA between P7 and P14. The potential role of tPA in granule cell migration was investigated by performing migration assays in cerebellar slice explants in the presence or absence of protease inhibitors. The presence of inhibitors did not affect the distance that granule cells migrated. Data in the present study do not support a role for tPA in granule neuron migration; however, they do indicate that tPA is both spatially and temporally regulated during cerebellar development. Possible functions of tPA in the cerebellum are discussed. © 1995 John Wiley & Sons, Inc.     

Developmental changes in the expression pattern of the JNK activator kinase MUK/DLK/ZPK and active JNK in the mouse cerebellum

JNK is one of the key molecules regulating cell differentiation and migration in a variety of cell types, including cerebral cortical neurons. MUK/DLK/ZPK belongs to the MAP kinase-kinase-kinase class of protein kinases for the JNK pathway and is expressed predominantly in neural tissue. We have determined the expression pattern of MUK/DLK/ZPK and active JNK in the cerebellum at different stages of postnatal development. Quantitative analysis by Western blotting has showed that high expression levels of MUK/DLK/ZPK and active JNK are maintained during the postnatal development of the cerebellum, and that these levels decrease in the adult cerebellum. Immunohistochemical staining has revealed, however, that their distribution in the developing cerebellum is considerably different. Although active JNK is highly concentrated in the premigratory zone of the external germinal layer (EGL), high expression of MUK/DLK/ZPK has been observed in the molecular layer and in the premigratory zone. Neither the active JNK nor MUK protein has been detected in the proliferative zone of the EGL. These observations suggest that during the postnatal development of the cerebellum, the MUK-JNK signaling pathway contributes to the regulation of granule cell differentiation and migration; further, the activity of MUK/DLK/ZPK is tightly regulated by posttranslational mechanisms and by its expression level.This work was supported by a Ishizu Shun memorial scholarship and grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.     

Subclass of astroglia in mouse cerebellum recognized by monoclonal antibody

A monoclonal antibody was detected that distinguishes astrocyte subclasses in mouse cerebellum. This antibody, designated anti-M1, is the product of a hybridoma that arose from the fusion of NS1 myeloma cells and splenocytes derived from a rat immunized with crude membranes from early postnatal mouse cerebella. The distribution and regulation of M1 antigen expression in vivo were examined by indirect immunofluorescence on frozen thin sections of mouse brain. M1 expression shows differing age dependencies within subpopulations of astroglia. M1 is first detectable around postnatal day 7 in white matter astrocytes and persists in this cell type throughout adulthood. By postnatal day 10, M1 is additionally detected in Bergmann glial fibers and in granule layer astrocytes. M1 expression in these latter astrocytic cell types is transient and cannot be detected after the fourth postnatal week. Cerebella of adult neurological mutant weaver mice show abnormal persistence of M1 antigen expression in Bergmann glial fibers. In monolayer cultures of early postnatal cerebella, M1 antigen is detected in a subpopulation of the glial fibrillary acidic protein positive astrocytes. M1 antigen can be detected only in fixed cultured cells which allow intracellular penetration of the antibody. The developmental regulation of M1 expression and the abnormal expression of M1 in weaver mutant cerebella suggest that M1 may be a useful marker for astroglial maturation and differentiation.     

The weaver gene encodes a nonautonomous signal for CNS neuronal differentiation.

In the neurological mutant mouse weaver, CNS precursor cells in the external germinal layer (EGL) of the cerebellar cortex proliferate normally, but fail to differentiate and die in the proliferative zone. To examine the autonomy of expression of the weaver gene, we carried out cell-mixing experiments in vitro. In homotypic, reaggregate cultures, weaver EGL precursor cells expressed the general neuronal markers N-CAM, L1, and MAP2, but failed to express the late neuronal antigens TAG-1 and astrotactin, to extend neurites or to migrate on glial fibers. After reaggregation with wild-type EGL precursor cells, weaver precursor cells extended neurites equivalent in length to wild-type cells, migrated along astroglial fibers, and expressed TAG-1 and astrotactin. Rescue of neurite production was also achieved by the addition of membranes from, but not by medium conditioned by wild-type cells. These findings suggest that the weaver gene acts non-autonomously, encoding a membrane-associated ligand that induces EGL neuronal differentiation.     

Expression of glial antigens C1 and M1 in developing and adult neurologically mutant mice

The distribution of two glial antigens (C1 and M1) has been studied by indi-rect immunofluorescence during postnatal development of the cerebella of normal and neurologically mutant mice (weaver, staggerer, reeler, Purkinje cell degeneration, and wobbler). During the first postnatal week of normal development, C1 antigen is expressed in ependyma, Bergmann glial fibers (BG), and astrocytes of the internal granular layer and white matter. After day 10, C1 antigen is restricted to BG and ependymal cells. During the sec-ond and third week, BG undergo a transient loss of C1 antigen that starts in medioventral areas and spreads in a gradient dorsally and laterally. In reeler, weaver, and staggerer, C1 antigen expression is normal during the first postnatal week, and subsides in BG in a similar spatial gra- dient as described for the normal littermates. However, the loss of C1 anti-gen in BG occurs earlier (first in reeler, then in weaver, and last in staggerer) and is not reversible as it is in normal mice. In Purkinje cell de-generation, C1 antigen expression is diminished in BG after the onset of be-havioral abnormalities. Wobbler is normal with respect to C1 antigen ex-pression at adult ages. M1 antigen is detectable in white matter astrocytes from postnatal day 7 on, and persists in these cells into adulthood. Astrocytes of the internal granular layer and BG express M1 antigen only transiently in normal mice during the second and third weeks. The appearance of M1 antigen in BG occurs in a spatiotemporal gradient, matching the one in which C1 antigen disappears. M1 antigen expression is abnormally maintained in BG of reeler, staggerer, and weaver. In Purkinje cell degeneration, M1 antigen is ex-pressed abnormally at the onset of behavioral abnormalities first in.astro-cytes of the internal granular layer and, with growing age, increasingly also in BG. In wobbler, BG do not express M1 antigen. However, astrocytes of the granular layer are abnormally M1 antigen-positive.     

A neuroectoderm-associated ganglioside participates in fibronectin receptor-mediated adhesion of germinal cells to fibronectin

Previous studies with a rat neural cell line have shown that the D1.1 ganglioside, an O-acetylated derivative of GD3, is involved in cellular adhesion to fibronectin. In vivo, D1.1 is present on germinal cells of the developing rat central nervous system, but not on postmitotic cells that migrate away from the germinal zones. These observations suggest that D1.1 could participate in adhesive interactions with germinal zones and that the loss of D1.1 could be involved in the decision to being migration. In support of this hypothesis, immunofluorescence histochemistry shows that both fibronectin and fibronectin receptor are colocalized with D1.1 in the ventricular zones of the embryonic rat brain and in the external granule cell layer of the postnatal cerebellum. Dishes coated with monoclonal antibody against D1.1 were used to isolate D1.1-positive germinal cells from Embryonic Day 14 cerebrum and from Postnatal Day 6 cerebellum. These cells are able to adhere to fibronectin-coated dishes by a mechanism that is inhibitable by a synthetic hexapeptide containing the arg-gly-asp cell recognition sequence of fibronectin. Adhesion is also partially inhibited by antibody against fibronectin receptor and is slowed by anti-D1.1 antibody, implicating both the receptor and the ganglioside in the adhesion process. During 3 days in culture these D1.1-positive, fibronectin receptor-positive cells exhibit a neuronal phenotype, as judged by morphology and staining with tetanus toxin. This further confirms the neuroepithelial origin of the cells. The cells do not synthesize detectable amounts of fibronectin, thus leaving unidentified the source of the fibronectin seen in the germinal zones in tissue sections. Immunoprecipitation experiments show that the fibronectin receptors present on these cells are heterodimers. Under nonreducing conditions, the immunoprecipitates contain an alpha-subunit of 150-160 kDa and a beta-subunit of 115-125 kDa.     

Developmental expression and intracellular location of P400 protein characteristic of Purkinje cells in the mouse cerebellum

The developmental expression and intracellular localization of a cerebellum-characteristic 250-kDa glycoprotein, P400 protein, were studied by immunohistochemical and immunoblot methods using a monoclonal antibody against P400 protein. In the cerebellum of normal mouse, the expression of P400 protein increased from Postnatal Day 3 to Day 21. This enhancement of P400 protein expression occurred only in the Purkinje cells and proceeded with the growth of their dendritic arborization. Electron microscopic analysis indicated that P400 protein is present at the plasma membrane, the endoplasmic reticulum, and the postsynaptic densities of Purkinje cells. Immunohistochemistry of the cerebella of neurological mutant mice indicated that the Purkinje cells of reeler, weaver, and pcd mutant mice retain the ability to produce a large amount of P400 protein. However, the Purkinje cells of staggerer mutant mouse proved to be incapable of enhanced P400 protein expression. These results indicate that P400 protein is a Purkinje cell-characteristic plasma membrane-associated glycoprotein, which is also present at the postsynaptic density and endoplasmic reticulum and that the expression of P400 protein in Purkinje cells is closely associated with the growth of their dendritic arborization.     

Type 2 and type 3 inositol 1,4,5-trisphosphate (IP3) receptors promote the differentiation of granule cell precursors in the postnatal cerebellum

During postnatal development of the cerebellum, granule cell precursors (GCPs) proliferate in the external granular layer (EGL), exit the cell cycle, differentiate, and migrate from the EGL to the internal granular layer. In the present study, we report that type 2 and 3 inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R2 and IP₃R3) regulate the differentiation of GCPs after postnatal day 12 (P12). 5-Bromodeoxyuridine labeling experiments revealed that in mutant mice lacking both of these receptors (double mutants) a greater number of GCPs remain undifferentiated after P12. Consequently, the EGL of the double mutants is thicker than that of control mice at this age and thereafter. In addition, granule cells remain in the EGL of the double mutants at P21, an age when migration has concluded in wild-type mice. Whereas differentiation of GCPs was reduced in the double mutants, the absence of IP₃R2 and IP₃R3 did not affect the doubling time of GCPs. We conclude that intracellular calcium release via IP₃R2s and IP₃R3s promotes the differentiation of GCPs within a specific interval of postnatal development in the cerebellum.     

Deficits in motor coordination with aberrant cerebellar development in mice lacking testicular orphan nuclear receptor 4

Since testicular orphan nuclear receptor 4 (TR4) was cloned, its physiological function has remained largely unknown. Throughout postnatal development, TR4-knockout (TR4-/-) mice exhibited behavioral deficits in motor coordination, suggesting impaired cerebellar function. Histological examination of the postnatal TR4-/- cerebellum revealed gross abnormalities in foliation; specifically, lobule VII in the anterior vermis was missing. Further analyses demonstrated that the laminations of the TR4-/- cerebellar cortex were changed, including reductions in the thickness of the molecular layer and the internal granule layer, as well as delayed disappearance of the external granule cell layer (EGL). These lamination irregularities may result from interference with granule cell proliferation within the EGL, delayed inward migration of postmitotic granule cells, and a higher incidence of apoptotis. In addition, abnormal development of Purkinje cells was observed in the postnatal TR4-/- cerebellum, as evidenced by aberrant dendritic arborization and reduced calbindin staining intensity. Expression of Pax-6, Sonic Hedgehog (Shh), astrotactin (Astn), reelin, and Cdk-5, genes correlated with the morphological development of the cerebellum, is reduced in the developing TR4-/- cerebellum. Together, our findings suggest that TR4 is required for normal cerebellar development.     

Deficits in cerebellar granule cell development and social interactions in CD47 knockout mice

CD47 is involved in neurite differentiation in cultured neurons, but the function of CD47 in brain development is largely unknown. We determined that CD47 mRNA was robustly expressed in the developing cerebellum, especially in granule cells. CD47 protein was mainly expressed in the inner layer of the external granule layer (EGL), molecular layer, and internal granule layer (IGL), where granule cells individually become postmitotic and migrate, leading to neurite fasciculation. At postnatal day 8 (P8), CD47 knockout mice exhibited an increased number of proliferating granule cells in the EGL, whereas the CD47 agonist peptide 4N1K increased the number of postmitotic cells in primary granule cells. Knocking out the CD47 gene and anti‐CD47 antibody impaired the radial migration of granule cells from the EGL to the IGL individually in mice and slice cultures. In primary granule cells, knocking out CD47 reduced the number of axonal collaterals and dendritic branches; by contrast, overexpressing CD47 or 4N1K treatment increased the axonal length and numbers of axonal collaterals and dendritic branches. Furthermore, the length of the fissure between Lobules VI and VII was decreased in CD47 knockout mice at P21 and at 14 wk after birth. Lastly, CD47 knockout mice exhibited increased social interaction at P21 and depressive‐like behaviors at 10 wk after birth. Our study revealed that the cell adhesion molecule CD47 participates in multiple phases of granule cell development, including proliferation, migration, and neurite differentiation implying that aberrations of CD47 are risk factors that cause abnormalities in cerebellar development and atypical behaviors.© 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 463–484, 2015     

Temporal regulation of cerebellar EGL migration through a switch in cellular responsiveness to the meninges

We have studied the temporal and spatial control of cell migration from the external germinal layer (EGL) in the mammalian cerebellum as a model for cortical migration. Our results have demonstrated that embryonic EGL cells do not migrate into internal layers because they respond to a diffusible attractant in the meninges, the nonneural tissues covering the nervous system, and to a repellent in the neuroepithelium. Two developmental changes are important for postnatal EGL migration: the disappearance of the repellent in the inner layers and a switch in cellular responsiveness of EGL cells so that the postnatal EGL cells respond to the repellent, but not the attractant in the meninges. Besides revealing the signaling role of meninges in cortical development, our study suggests that an active mechanism is required to prevent cell migration, and that mechanisms of cell migration should be studied even in the absence of apparent changes in cell positions. We propose a model for the developmental control of neuronal migration in the cerebellar cortex.     

2-Deoxyglucose Incorporation in the Cerebellum of Weaver and Nervous Mutant Mice

Abstract: The 2-deoxyglucose autoradiographic method has been used to study activity in cerebellum of the weaver and nervous mutant mice. Patterns of 2-deoxyglucose incorporation into the cerebral hemispheres from weaver and nervous strains did not differ significantly from those of the controls. In the normal cerebellum, 2-deoxyglucose incorporation was maximal in the granular layer, where mossy fibers form synapses with the dendrites of granule cells. In the cerebellum of nervous mice, which lacks Purkinje cells, the incorporation of the 2-deoxyglucose was maximal in the granular layer, but the incorporation into the molecular layer appeared less than in the control. The incorporation into the cerebellum from weaver, which lacks granule cells, was much higher than that of the control, the maximal incorporation being found in the Purkinje cell layer and in cell masses located in the white matter. These data suggest that the heterologous synapses that mossy fibers or climbing fibers form with the cells in the Purkinje cell layer and the cells in the white matter in the weaver cerebellum are functional.     

Impaired Structural and Functional Development of Cerebellum Following Gestational Exposure of Deltamethrin in Rats: Role of Reelin

Reelin is an extracellular matrix molecule that is involved in the normal development of the cerebellar lamination, Bergmann glial fibres alignment, Purkinje cell monolayer arrangement and granule cell migration. In this study, we have examined the effects of maternal exposure of deltamethrin (DLT), a type II pyrethroid insecticide, on the structural and functional development of rat cerebellum during postnatal life. DLT (0.75 mg/kg body weight, intraperitoneally dissolved in dimethylsulphoxide) was administered in timed pregnant rats during two different gestational time periods, i.e. gestational days of 7–10 and 11–14, respectively. In DLT exposed rats, a significant overexpression of reelin was observed in the cells of the external granule cell layer (EGL) and internal granule cell layer along with an ectopic expression of reelin in the EGL as well as in the migrating granule cells just below the EGL, revealing an arrest of granule cell migration in this zone. Mis-orientation and hypertrophy of the Bergmann glial fibres further hampered the journey of the granule cells to their final destination. Possibly reelin overexpression also caused misalignment of the Purkinje cells and inhibited the neurite growth leading to a significant decrease in the spine density, main dendritic length and width of the dendritic arbour. Thus, it is proposed that the DLT exerts its neurotoxic effects possibly via the intracellular accumulation and low release of reelin leading to an impaired granule cell and Purkinje cell migration, inhibition of neurite outgrowth and reduced spine density. Such impaired cerebellar development leads to motor coordination deficits.     

A subpial, transitory germinal zone forms chains of neuronal precursors in the rabbit cerebellum

Protracted neurogenesis occurs at different postnatal stages in different brain locations, whereby leading to site-specific adult neurogenesis in some cases. No spontaneous genesis of neurons occurs in the cerebellum after the postnatal genesis of granule cells from the external germinal layer (EGL), a transitory actively proliferating zone which is thought to be exhausted before puberty. Here, we show the protracted genesis of newly generated neuronal precursors in the cerebellar cortex of young rabbits, persisting beyond puberty. Neuroblasts generated within an actively proliferating subpial layer thus extending the postnatal EGL are arranged to form thousands of tangential chains reminiscent of those responsible for cell migration in the forebrain subventricular zone. These subpial chains cover the whole cerebellar surface from the 2nd to the 5th month of life, then disappearing after puberty. In addition, we describe the appearance of similar groups of cells at the end of granule cell genesis in the mouse cerebellum, here limited to the short period of EGL exhaustion (4-5 days). These results show common features do exist in the postnatal reorganization of secondary germinal layers of brain and cerebellum at specific stages, parallel to differences in the slowing down of cerebellar neurogenesis among mammalian species.     

Histological changes during development of the cerebellum in the chick embryo exposed to a static magnetic field

Few studies have been performed to evaluate the ultrastructural changes that exposure to static magnetic fields (SMF) can cause to the processes of cell migration and differentiation in the cerebellum during development. Thus, we have studied the development of the cerebellum in the chick embryo (n = 144) under a uniform SMF (20 mT). All of our observations were done on folium VIc of Larsell's classification. The cerebella of chick embryos, which were exposed solely on day 6 of incubation and sacrificed at day 13 of incubation [short exposure (S)1; n = 24], showed an external granular layer (EGL) that was less dense than the EGL in the control group (n = 24). The molecular layer (ML) exhibited a low number of migratory neuroblastic elements. Moreover, the internal granular layer (IGL) was immature, with the cellular elements less abundant and more dispersed than in controls. In chick embryos exposed on day 6 of incubation and sacrificed at day 17 (S2; n = 24), the outstanding feature was the regeneration of the different layers of the cerebellar cortex. The cerebellar cortex of chick embryos exposed continuously to an identical field from the beginning of the incubation up to day 13 [long exposure (L)1; n = 24] or day 17 (L2; n = 24) of incubation showed a higher number of alterations than that of group S1. Electron microscopy confirmed the findings from light microscopy and, at the same time, showed clear signs of cell degeneration and delay in the process of neuronal differentiation. This was more apparent in groups L1 (100%) and L2 (100%) than in groups S1 (95.4%) and S2 (65.2%). In conclusion, the present study showed that SMF can induce irreversible developmental effects on the processes of cell migration and differentiation of the chick cerebellar cortex. Bioelectromagnetics 18:36–46, 1997. © 1997 Wiley-Liss, Inc.     

Brain nucleic acids and protein in various neurological mutant mice

A study was made to compare alterations in the cerebral contents of nucleic acids and protein of several mouse strains affected by different neurological mutations: jimpy, msd, quaking, reeler, weaver, and dwarf. In normal and affected jimpy and msd mice the brain components analyzed were very similar. On the other hand, the cerebral hemispheres of quaking mice showed significant decreases in total RNA and DNA, when compared with those of normal littermates. In the affected reeler and weaver mice, total protein, RNA, and DNA in the cerebellum differed markedly from controls. Protein decreased slightly, whereas nucleic acids showed no significant variation in the cerebral hemispheres of the same mutants. The cerebella and cerebral hemispheres of affected dwarf mice had wet weights and total protein contents that were about 20% lower than those of their controls; DNA did not vary significantly in the various brain regions analyzed. The decrease of DNA we report in reeler and weaver mutant cerebellum in toto quantifies the lack of cell number, in contrast to histological studies which give only semiquantitative information.