Frog Brain Research Articles

Differential Inhibitory Control of Semicircular Canal Nerve Afferent-Evoked Inputs in Second-Order Vestibular Neurons by Glycinergic and GABAergic Circuits

Labyrinthine nerve-evoked monosynaptic excitatory postsynaptic potentials (EPSPs) in second-order vestibular neurons (2 degrees VN) sum with disynaptic inhibitory postsynaptic potentials (IPSPs) that originate from the thickest afferent fibers of the same nerve branch and are mediated by neurons in the ipsilateral vestibular nucleus. Pharmacological properties of the inhibition and the interaction with the afferent excitation were studied by recording monosynaptic responses of phasic and tonic 2 degrees VN in an isolated frog brain after electrical stimulation of individual semicircular canal nerves. Specific transmitter antagonists revealed glycine and GABA(A) receptor-mediated IPSPs with a disynaptic onset only in phasic but not in tonic 2 degrees VN. Compared with GABAergic IPSPs, glycinergic responses in phasic 2 degrees VN have larger amplitudes and a longer duration and reduce early and late components of the afferent nerve-evoked subthreshold activation and spike discharge. The difference in profile of the disynaptic glycinergic and GABAergic inhibition is compatible with the larger number of glycinergic as opposed to GABAergic terminal-like structures on 2 degrees VN. The increase in monosynaptic excitation after a block of the disynaptic inhibition in phasic 2 degrees VN is in part mediated by a N-methyl-d-aspartate receptor-activated component. Although inhibitory inputs were superimposed on monosynaptic EPSPs in tonic 2 degrees VN as well, the much longer latency of these IPSPs excludes a control by short-latency inhibitory feed-forward side-loops as observed in phasic 2 degrees VN. The differential synaptic organization of the inhibitory control of labyrinthine afferent signals in phasic and tonic 2 degrees VN is consistent with the different intrinsic signal processing modes of the two neuronal types and suggests a co-adaptation of intrinsic membrane properties and emerging network properties.

Evolutionarily Conserved Glucocorticoid Regulation of Corticotropin-Releasing Factor Expression

Glucocorticoids (GCs) exert feedback regulation on corticotropin-releasing factor (CRF) neurons in mammals. The nature of GC actions is cell-type specific, being either inhibitory (e.g. paraventricular nucleus) or stimulatory (e.g. amygdala and bed nucleus of the stria terminalis). Nothing is known about differential regulation of CRF gene expression by GCs in nonmammalian vertebrates. We studied the actions of GCs on CRF expression in discrete brain regions of the frog Xenopus laevis. Treatment with corticosterone (CORT) decreased, whereas the corticosteroid synthesis inhibitor metyrapone increased CRF expression in the anterior preoptic area (homolog of the mammalian paraventricular nucleus), as measured by CRF primary transcript, mRNA, and CRF immunoreactivity (ir) (by immunocytochemistry). By contrast to the preoptic area, CORT increased CRF-ir in the medial amygdala and bed nucleus of the stria terminalis, whereas metyrapone decreased CRF-ir in the medial amygdala. CRF-ir and glucocorticoid receptor-ir were colocalized in cells in the frog brain. In transient transfection assays in PC-12 cells, GCs decreased forskolin-induced activation of the frog CRF promoters. Treatment with CORT also reduced CRF promoter activity in transfected tadpole brain in vivo. Frog glucocorticoid receptor bound with high-affinity in vitro to regions in the proximal promoters of frog CRF genes that are homologous with the human CRF gene. Our findings suggest that the neural cell-type specificity and molecular mechanisms of GC-dependent regulation of CRF are phylogenetically ancient, and that the limbic pathways mediating behavioral and physiological responses to stressors were likely present in the earliest land-dwelling vertebrates.

Xenopus tropicalis as a Test System for Developmental and Reproductive Toxicity

The usefulness of Xenopus tropicalis as a model species to investigate endocrine disruption and developmental reproductive toxicity was assessed. In our test system tadpoles were exposed to test substances from shortly after hatching until metamorphosis, including the period of gonadal differentiation. Effects on the sex hormone and thyroid hormone axes were evidenced as skewed sex ratios, malformations of reproductive organs, altered cytochrome (CYP19) (aromatase) activity, and gene expression in gonads and brain, as well as changed thyroid histology and time to metamorphosis. Reproductive toxicity was evaluated at sexual maturity. Male-to-female sex reversal was implied at concentrations as low as 6 pM (1.8 ng/L) ethynylestradiol (EE2), which is comparable to EE2 levels observed in the environment. EE2-exposed males that were not sex reversed had significantly reduced fertility and a reduced amount of spermatozoa in testes compared with control males. This indicates that reproduction in wild frogs might be impaired by estrogenic environmental pollutants. Aromatase activity in brain and testes of adult frogs was not affected by larval EE2 exposure. Preliminary results indicate that exposure to the environmentally relevant pharmaceutical clotrimazole modulated aromatase activity in brain and gonads during sex differentiation, which warrants further investigation. The susceptibility to estrogen-induced sex reversal of X. tropicalis was comparable to that of other frog species and fish. Similarities between the reproductive effects in X. tropicalis and those reported in fish, birds, and mammals after developmental exposure to estrogens make X. tropicalis promising model for research on endocrine disruption and developmental reproductive toxicity.

Aromatization in the neural target tissues of the female frog, Rana esculenta: seasonal studies

It is well known that certain actions of androgen are mediated through in situ brain aromatization to estrogen. This study was undertaken to investigate seasonally the androgen utilization in the neural target tissues of the female frog, Rana esculenta, through a quantification of aromatase activity and estrogen receptors (ER). Aromatase activity was detected in the microsomal fraction of the brain and co-localization with ER was only in the cytoplasmatic area. Western analysis of aromatase revealed one immunoreactive band with a molecular weight of approximately 56 kDa. ER were present seasonally in both the cytosol and the nuclear brain extracts. Data on aromatase enzyme activity and relative levels of aromatase protein are discussed and more detail on the mechanisms involved in frog brain regulating estrogen synthesis supposed. Further studies are in progress to cloning the sex steroid-synthesizing enzyme CYP19 in the neural target tissues of Rana esculenta.

Analog Integrated Circuit for Motion Detection with Simple-Shape Recognition Based on Frog Vision System

We proposed in this research a novel two-dimensional network based on the frog visual system, with a motion detection function and a newly developed simple-shape recognition function, for use in object discrimination by integrated circuits. Specifically, the network mimics the signal processing of the small-field cell in a frog brain, consisting of the tectum and thalamus, which generates signals of the motion and simple shape of an object. The proposed network is constructed from simple analog complementary metal oxide semiconductor (CMOS) circuits; a test chip of the proposed network was fabricated with a 1.2 μm CMOS process. Measurements on the chip clarified that the proposed network can generate signals of the moving direction, velocity, and simple shape, as well as perform information processing of the small-field cell. Results with the simulation program with integrated circuit emphasis (SPICE) showed that the analog circuits used in the network have low power consumption. Applications of the proposed network are expected to realize advanced vision chips with functions such as object discrimination and target tracking.

Expression of Lhx9 Isoforms in the Developing Gonads of Rana rugosa

Lhx9 is a LIM-homeodomain (HD) transcription factor. Transcripts of the Lhx9 gene are present in the urogenital ridges of mouse embryos. In Lhx9-deficient mice, a discrete gonad is not formed (Birk et al., 2000). To date, however, the role of Lhx9 in gonad formation has not been studied in animals other than chicks and mice. Thus, this study was aimed at determining whether or not Lhx9 is expressed in the developing gonads of amphibians. We first isolated cDNAs of Lhx9 and four isoforms, named Lhx9alpha, beta, gamma and delta, from the frog Rana rugosa. All the isoforms lacked the last 14 amino acids of the HD (the DNA-binding motif). RT-PCR analysis revealed that Lhx9 and Lhx9alpha were highly expressed in the ovary, testis, brain and heart of adult frogs. However, neither Lhx9 nor any of the four isoforms was expressed in the pancreas and kidney. When their expression in the developing gonads of R. rugosa was examined by RT-PCR, transcripts of Lhx9 and Lhx9alpha were detected with no sexual dimorphism during sex determination. Lhx9gamma and Lhx9delta were also expressed, but at a low level. In contrast, the Lhx9beta transcript was hardly detected. In addition, in situ hybridization analysis showed that R. rugosa Lhx9 was expressed in somatic cells in the developing gonad and mesonephros far before sex determination. Taken together, Lhx9 and its four isoforms are probably involved in organogenesis in R. rugosa. All the isoforms may function as an endogenous dominant-negative form of Lhx9 in this species.

Selective asymmetry in a conserved forebrain to midbrain projection

How the left and right sides of the brain acquire anatomical and functional specializations is not well understood. The zebrafish has proven to be a useful model to explore the genetic basis of neuroanatomical asymmetry in the developing forebrain. The dorsal diencephalon or epithalamus consists of the asymmetric pineal complex and adjacent paired nuclei, the left and right medial habenulae, which in zebrafish larvae, exhibit differences in their size, neuropil density and patterns of gene expression. In all vertebrates, axons from the medial habenular nuclei project within a prominent fiber bundle, the fasciculus retroflexus, to a shared midbrain target, the interpeduncular nucleus of the ventral tegmentum. However, in zebrafish, projections from the left habenula innervate the dorsal and ventral regions of the target nucleus, whereas right habenular efferents project only to the ventral region. A similar dorsoventral difference in habenular connectivity is found in another teleost species, the highly derived southern flounder, Paralichthys lethostima. In this flatfish, directional asymmetry of the habenular projection appears to be independent of the left-right morphology and orientation that an individual adopts post-metamorphosis. Comparative anterograde labeling of the brains of salamanders, frogs and mice reveals that axons emanating from the left and right medial habenulae do not project to different domains, but rather, they traverse the target nucleus in a complementary mirror image pattern. Thus, although the habenulo-interpeduncular conduction system is highly conserved in the vertebrate brain, the stereotypic dorsoventral topography of left-right connections appears to be a feature that is specific to teleosts.

Pings the Body Electric (and Means It Too). Focus on “Interval Coding. I. Burst Interspike Intervals as Indicators of Stimulus Intensity” and “Interval Coding. II. Dendrite-Dependent Mechanisms”

Structure/function questions are at the forefront of research in biological sensory systems. These questions are very elegantly addressed by Oswald et al. and Doiron et al. in a pair of articles on signal representation in electric fish in this issue of Journal of Neurophysiology (p. 2731–2743 and

Direction selectivity in rabbit retina

Direction selectivity in rabbit retina

Immunohistochemical localization and biological activity of the steroidogenic enzyme cytochrome P450 17α‐hydroxylase/C17, 20‐lyase (P450C17) in the frog brain and pituitary

It is now clearly established that the brain has the capability of synthesizing various biologically active steroids including 17-hydroxypregnenolone (17OH-Delta(5)P), 17-hydroxyprogesterone (17OH-P), dehydroepiandrosterone (DHEA) and androstenedione (Delta(4)). However, the presence, distribution and activity of cytochrome P450 17alpha-hydroxylase/C17, 20-lyase (P450(C17)), a key enzyme required for the conversion of pregnenolone (Delta(5)P) and progesterone (P) into these steroids, are poorly documented. Here, we show that P450(C17)-like immunoreactivity is widely distributed in the frog brain and pituitary. Prominent populations of P450(C17)-containing cells were observed in a number nuclei of the telencephalon, diencephalon, mesencephalon and metencephalon, as well as in the pars distalis and pars intermedia of the pituitary. In the brain, P450(C17)-like immunoreactivity was almost exclusively located in neurons. In several hypothalamic nuclei, P450(C17)-positive cell bodies also contained 3beta-hydroxysteroid dehydrogenase-like immunoreactivity. Incubation of telencephalon, diencephalon, mesencephalon, metencephalon or pituitary explants with [(3)H]Delta(5)P resulted in the formation of several tritiated steroids including 17OH-Delta(5)P, 17OH-P, DHEA and Delta(4). De novo synthesis of C(21) 17-hydroxysteroids and C(19) ketosteroids was reduced in a concentration-dependent manner by ketoconazole, a P450(C17) inhibitor. This is the first detailed immunohistochemical mapping of P450(C17) in the brain and pituitary of any vertebrate. Altogether, the present data provide evidence that CNS neurons and pituitary cells can synthesize androgens.

Proliferative activity in the frog brain: A PCNA-immunohistochemistry analysis

By means proliferating cell nuclear antigen (PCNA) immunohistochemistry, we have provided a detailed neuroanatomical mapping of proliferative activity during development and adulthood in the frog ( Rana esculenta) brain. Western blot analysis confirmed the presence of this protein in brain extracts from adults and tadpoles. Proliferative activity was observed in the ventricular and subventricular zones throughout the brain. The present study provides details as to which of the morphologically distinguishable brain region(s) has a long-lasting proliferative activity and in which region this activity undergoes a progressive decrease during development. In the subventricular zones of the third ventricle, PCNA-labeled cells were particularly abundant in the magnocellular preoptic nucleus and the ventromedial thalamic nucleus. It was observed that proliferation zones are present practically in all major subdivisions of the forebrain, midbrain and hindbrain, including the cerebellum in which PCNA-labeled cells were located in the outer granular layer and the inner molecular layer. The habenulae, epiphysis and isthmic nuclei never showed the presence of PCNA-immunoreactive nuclei. The widespread proliferative activity implies that the frog brain has a great potential for neurogenesis/gliogenesis not only during larval development but also in the adulthood.

Temperature and humidity alter prolactin receptor expression in the skin of toad (Bufo bankorensis and Bufo melanostictus)

Bufo bankorensis and Bufo melanostictus, the only two species of Bufonidae genus in Taiwan, live in habitats that differ in altitude and humidity. This study tested the hypothesis that prolactin receptor (PRLR) expression responds to environmental change. Western blot analysis showed that the PRLR protein was widely distributed in brain, lung, liver, kidney, dorsal skin and ventral skin of toads. The level PRLR protein was elevated in the dorsal skin of the two toad species treated with dry or wet conditions for 14 days. The increase in PRLR of dorsal skin in B. bankorensis was higher than that in B. melanostictus. This experimental result suggests that B. bankorensis secretes more mucus to reduce water evaporation from its thinner cuticle than B. melanostictus. The expression of PRLR protein was increased in the lung of B. bankorensis and decreased in the lung of B. melanostictus. Moreover, PRLR protein levels were increased in the kidneys in the two species toad, likely due to reduction in water lost through lung and urine. The two toad species were subjected to varying temperatures (25 degrees C, 15 degrees C and 10 degrees C) for 14 days. The lowest PRLR protein expression was observed at 10 degrees C. Comparison of the decreasing trend in PRLR protein levels demonstrated that the variation in B. bankorensis was significantly higher than that in B. melanostictus. Comparisons of variation in PRLR protein expression in the two species under different environments suggest that B. bankorensis is more adaptable to different environments than B. melanostictus.

Expression of PACAP Receptors in the Frog Brain during Development

This study describes the expression of PAC1 and VPAC1 receptor (PAC1-R and VPAC1-R) mRNAs in the brain of frog (Rana esculenta) during development. PAC1-R mRNA was detected in the periventricular and subependymal layers of the thalamus and epithalamus and in the ependymal layer of the mesencephalon and rhombencephalon (stage 20), in the amygdala, in the habenular complex, in the periventricular nucleus of the hypothalamus, and in the ventral cerebellum (stage 30). VPAC1-R mRNA expression was observed only at stage 20, in the floor of the hypothalamus. These results suggest that, in amphibians, pituitary adenylate cyclase-activating polypeptide (PACAP) may play a role in brain development.

How Much the Eye Tells the Brain

In the classic "What the frog's eye tells the frog's brain," Lettvin and colleagues showed that different types of retinal ganglion cell send specific kinds of information. For example, one type responds best to a dark, convex form moving centripetally (a fly). Here we consider a complementary question: how much information does the retina send and how is it apportioned among different cell types? Recording from guinea pig retina on a multi-electrode array and presenting various types of motion in natural scenes, we measured information rates for seven types of ganglion cell. Mean rates varied across cell types (6-13 bits . s(-1)) more than across stimuli. Sluggish cells transmitted information at lower rates than brisk cells, but because of trade-offs between noise and temporal correlation, all types had the same coding efficiency. Calculating the proportions of each cell type from receptive field size and coverage factor, we conclude (assuming independence) that the approximately 10(5) ganglion cells transmit on the order of 875,000 bits . s(-1). Because sluggish cells are equally efficient but more numerous, they account for most of the information. With approximately 10(6) ganglion cells, the human retina would transmit data at roughly the rate of an Ethernet connection.

Vasotocin and mesotocin stimulate neurosteroid biosynthesis in the frog brain. Possible involvement of a V1a receptor

Vasotocin and mesotocin stimulate neurosteroid biosynthesis in the frog brain. Possible involvement of a V1a receptor

The amphibian globin gene repertoire as revealed by the Xenopus genome

The draft genome sequence of the Western clawed frog Xenopus (Silurana) tropicalis facilitates the identification, expression analysis and phylogenetic classification of the amphibian globin gene repertoire. Frog and mammalian neuroglobin display about 67% protein sequence identity, with the expected predominant expression in frog brain and eye. Frog and mammalian cytoglobins share about 69% of their amino acids, but the frog protein lacks the mammalian-type extension at the C-terminus. Like in mammals, X. tropicalis cytoglobin is expressed in many organs including neural tissue. Neuroglobin and cytoglobin genomic regions are syntenically conserved in all vertebrate classes. Frog and fish globin X show only 57% amino acid identity, but gene synteny analysis confirms orthology. The expression pattern of X. laevis globin X differs from that in fish, with a prominent expression in the eye and weak expression in most other examined tissues. Globin X is possibly present as two paralogous copies in X. tropicalis, with one copy showing transition stages of non-functionalization. The amphibian genome contains a previously unknown globin type (tentatively named ‘globin Y’) which is expressed in a broad range of tissues and is distantly related to the cytoglobin lineage. The globin Y gene is linked to a cluster of larval and adult hemoglobin α and β genes which contains substantially more paralogous hemoglobin gene copies than previously published. Database and gene synteny analyses confirm the absence of a myoglobin gene in X. tropicalis.

Analog Integrated Circuit for Detection of an Approaching Object with Simple-Shape Recognition Based on Lower Animal Vision

A network for the detection of an approaching object with simple-shape recognition is proposed based on lower animal vision. The locust can detect an approaching object through a simple process in the descending contralateral movement detector (DCMD) in the locust brain, by which the approach velocity and direction of the object is determined. The frog can recognize simple shapes through a simple process in the tectum and thalamus in the frog brain. The proposed network is constructed of simple analog complementary metal oxide semiconductor (CMOS) circuits. The integrated circuit of the proposed network is fabricated with the 1.2 μm CMOS process. Measured results for the proposed circuit indicate that the approach velocity and direction of an object can be detected by the output current of the analog circuit based on the DCMD response. The shape of moving objects having simple shapes, such as circles, squares, triangles and rectangles, was recognized using the proposed frog-visual-system-based circuit.

A Two-Dimensional Network of Analog Circuits for Motion Detection Based on the Frog Visual System

A two-dimensional network for motion detection constructed of simple analog circuits was proposed and designed based on the frog visual system. In the frog visual system, the two-dimensional motion of a moving object can be detected by performing simple information processing in the tectum and thalamus of the frog brain. The measured results of the test chip fabricated by a 1.2 μm complementary metal oxide semiconductor (CMOS) process confirmed the correct operation of the basic circuits in the network. The results obtained with the simulation program with integrated circuit emphasis (SPICE) showed that the proposed network can detect the motion direction and velocity of a moving object. Thus, a chip for two-dimensional motion detection was realized using the proposed network.

Ontogeny of the somatostatin variant [Pro2,Met13]somatostatin-14 in the brain, pituitary, and sensory organs of the frogRana esculenta

Two forms of somatostatin are expressed in the frog brain, i.e., somatostatin-14 (SS1) and the [Pro(2), Met(13)]somatostatin-14 variant (SS2). We have previously described the ontogeny of SS1-immunoreactive cells in the brain of Rana esculenta. In the present study, we have investigated the distribution of prepro-SS2 (PSS2)-expressing neurons in the brain of the same species during development by using antibodies directed against the N-flanking region of SS2 (PSS2(54-66)). Immunoreactive perikarya first appeared in the ventral hypothalamus at stages IV-VII. Subsequently, positive neurons were seen in the nucleus of the diagonal band of Broca, the anterior preoptic area, the posterior tuberculum (stages VIII-XII), as well as the dorsal (stages XIII-XV) and medial (stages XIX-XX) periventricular preoptic nucleus. At metamorphic climax and in newly metamorphosed frogs, positive perikarya were found in the striatum and in the interpeduncular nucleus. PSS2(54-66)-immunoreactive fibers were already widely distributed during the first stages of development, indicating that SS2 may act as a neuromodulator and/or neurotransmitter during ontogeny. The presence of PSS2(54-66)-positive nerve fibers in olfactory structures suggests that, in tadpoles, SS2 may be involved in the processing of olfactory information. The occurrence of PSS2(54-66)-like immunoreactivity in taste buds, and in the olfactory and vomeronasal organs indicates that SS2 may mediate the unconditioned and reinforcing properties of natural chemicals. Finally, the intenseexpression of PSS2(54-66)-like immunoreactivity in melanotrope cells of the pituitary suggests that SS2 may diffuse toward the pars distalis to regulate the activity of adenohypophysial cells during tadpole development.

Distribution of hyaluronan in the central nervous system of the frog

The qualitative and quantitative distribution pattern of hyaluronan (HA), a component of the extracellular matrix (ECM), was studied in the frog central nervous system by using a highly specific HA probe and digital image analysis. HA reaction was observed in both the white and the gray matter, showing a very intense staining around the perikarya and dendrites in the perineuronal net (PN). In the telencephalon, strong reaction was found in different parts of the olfactory system, in the pallium, and in the amygdala. In the diencephalon, intensive staining was found in the nucleus of Bellonci, the dorsal habenula, the lateral and central thalamic nuclei, and the subependymal zone of the third ventricle. In the mesencephalon, layers of optic tectum displayed different intensities, with the strongest reaction in layers B, D, F, 3, and 5. Other structures of the mesencephalon showed regional differences. The PN was especially intensively stained around the perikarya of the toral nuclei, the oculomotor and trochlear nuclei, and the basal optic nucleus. In the rhombencephalon, the granular layer of cerebellum, the vestibulocochlear nuclei, the superior olive, the spinal tract of the trigeminal nerve, and parts of the reticular formation showed the most intense reaction in the PN. In the spinal cord, considerable HA staining was found in the white matter and around the perikarya of motoneurons. The present study is the first description of the HA-positive areas of frog brain and spinal cord demonstrating the heterogeneity of HA distribution in the frog central nervous system.