Projections of the vestibular and cerebellar nuclei in Rana pipiens

The efferent projections and cytoarchitecture of the vestibulocerebellar region were examined to determine the nuclear boundaries and potential homologies. The anterior portion of the vestibular complex projects to the ipsilateral oculomotor and trochlear nuclei and is the major source of commissural fibers. Neurons in the rostromedial portions of the complex project to the contralateral trochlear nucleus. Large neurons in the ventrolateral portion of the complex give rise to a bilateral vestibulospinal pathway. Medium-sized neurons in the neuropil and small neurons in the central gray giving rise to bilateral projections to the spinal cord and oculomotor nuclei as well as commissural and ipsilateral cerebellar efferents. Projections from the nucleus of the cerebellum reach the contralateral spinal cord and cerebellar nucleus and there is also a bilateral projection to the ventral rhombencephalic and mesencephalic basal plates. The medial portion of the nucleus gives rise to commissural, ipsilateral mesencephalic and contralateral spinal projections. The lateral portion of the nucleus projects to the contralateral ventral mesencephalon. On the whole, the results of this investigation substantiate the division of the anuran vestibular complex in anurans into nuclei which may be homologous to the superior nucleus and nucleus of Deiters in mammals. The case for distinct descending and medial nuclei is less compelling. Further, it appears possible to divide the nucleus of the cerebellum into medial and lateral components whose connectivity is similar to that of reptiles and to a lesser extent mammals.     

Cholinergic innervation of the optic tectum in the frog Rana pipiens

An immunohistochemical method for choline acetyltransferase (ChAT) identifies presumably cholinergic axons in two retino-receptive laminae in the optic tectum of the frog Rana pipiens. Following eye enucleation there is no loss of immunoreactive axons in the optic tectum. Following unilateral ablation of the nucleus isthmi there is a near-total loss of ChAT-positive axons in the superficial cholinergic lamina contralaterally and in the deeper cholinergic lamina ipsilaterally. Thus, the cholinergic innervation of the tectum appears to derive from the nucleus isthmi. However, ChAT-positive staining of the basal optic nucleus does depend upon an intact retinal input and could derive from either retinal axons or some system trophically dependent on them.     

Some telencephalic connections in the frog, Rana pipiens.

Some afferent, efferent and intrinsic connections of the telencephalon of Rana pipiens were studied using a horseradish peroxidase method. Afferents to the telencephalon from thalamic and brain stem cell groups were demonstrated. These findings, taken together with the results of previous studies, indicate that separate thalamic cell groups project visual, auditory and somatosensory information onto the striatum. A separate thalamic cell group projects to the medial telencephalic wall and probably conveys visual and somatosensory information. These telencephalic afferent systems do not appear to be directly comparable to those of birds and reptiles. Additionally, some telencephalic afferents demonstrated in previous studies using anterograde degeneration techniques were confirmed, and some intratelencephalic connections were identified.     

Retinal ganglion cell morphology in the frog, Rana pipiens

The morphology of retinal ganglion cells in the frog, Rana pipiens, has been examined in retinal flatmounts following backfilling of axons with horseradish peroxidase (HRP). Size and shape of the cell body and of the dendritic arbor, the dendritic branching pattern, and the depth of dendritic arborization within the inner plexiform layer (IPL) were all used to classify these cells. All of the ganglion cells so visualized can be grouped into one of 7 distinct cell classes. Class 1 contains the largest ganglion cells, with a soma size of 323 +/- 5.3 microns2 and dendritic fields of 86,819 +/- 11,817 microns2; the dendrites branch within strata 1 and 2 of the IPL. The second largest cells are class 2, with somas of 245 +/- 19.7 microns2 and dendritic fields of 55,983 +/- 7,392 microns2; the dendrites also branch within strata 1 and 2 of the IPL. Class 3 cells are the next largest class with somas of 211 +/- 11.8 microns2 and dendritic fields of 18,186 +/- 1,394 microns2; there are three varieties of class 3 cells based on the depth of branching of the dendrites: some cells are bistratified, others are tristratified, while still other cells arborize diffusely within the IPL. Class 4 cells are intermediate in size, with somas of 113 +/- 7.4 microns2 and dendrites of 4800 +/- 759 microns2; the dendrites arborize within strata 4 and 5 of the IPL. Class 5 cells have not been quantitatively analyzed because they are heterogeneous in soma and dendritic size. However, class 5 cells all have cell bodies displaced in location into the inner nuclear layer and all have a unique dendritic specialization: they send from 1 to 3 processes into the outer plexiform layer. Class 6 cells are the second smallest cell class with somas of 68.1 +/- 5.13 microns2 and dendritic fields of 888 +/- 182 microns2; the dendrites arborize within strata 3, 4, and 5 of the IP. Class 7 contains the smallest ganglion cells with somas of 62.1 +/- 2.86 microns2 and dendritic fields of 831 +/- 74.2 microns2; the dendrites arborize within strata 3, 4, and 5 of the IPL. The frequency of each cell class is inversely proportional to the size of the dendritic field. Thus, class 7 cells are the most frequent; class 1 cells are the least frequent. Furthermore, each of these 7 classes of ganglion cells has representative cells located in the inner nuclear layer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Retinotopic organization of central optic projections in Rana pipiens

The retinotopic organization of the anuran visual system has been investigated with the method of selective anterograde transport of horseradish peroxidase (HRP) following retinal lesions. The course of optic axons to specific structures was also confirmed by retrograde transport in the optic tract following HRP injections in the tectum and pretectum. As the optic nerve reaches the optic chiasm, the fibers from each of the four retinal quadrants appear as bands with the nasal (n) quadrant entering the chiasmal anterior pole, followed by ventral (v), temporal (t), and dorsal (d) quadrants. The preoptic nucleus is the first structure to be innervated, followed by the suprachiasmatic nucleus; both are innervated directly from fibers in the dorsal part of the optic nerve, which contains fibers from all the retinal quadrants. Each quadrant expands across the dorsoventral extent of the chiasm at the point where it enters. At this level the quadrants are arrayed along the rostrocaudal axis (as they are later in the marginal optic tract) in the sequence n-v-t-d. Optic fibers then spread across the chiasm, the nasal quadrant splits, taking up positions in the rostral and caudal margins of the optic radiation. Following the split in the nasal representation, the optic tract is transformed into topographically arranged sheets in the marginal optic tract. In the other retinorecipient nuclei, the sheet of optic axons is transformed back into the shape of the retinal hemisphere. Topographic maps of this kind display one of two possible orientations: (1) in the tectum and the nucleus lentiformis mesencephali (nLM), the temporal retina is represented in the anterior portion of the nucleus, whereas the nasal quadrant is found in the posterior portion; (2) in the thalamus, the retinotopic map is organized as a mirror-image reversal of that seen in the tectum and nLM (i.e., the nasal pole is anterior, whereas the temporal pole is in the posterior portion of the nucleus). Structures with this type of retinal map include the rostral visual nucleus, the corpus geniculatum, the nucleus of Bellonci, and the posterior thalamic nucleus. A third type of innervation occurs in the nucleus of the basal optic root (nBOR), which is the only mesencephalic visual nucleus not innervated by the marginal optic tract. The basal optic root is formed by the fibers exiting most caudally from the optic chiasm. All the retinal quadrants contribute to the basal optic root, but no evidence of retinotopy was found in nBOR.4+ target nuclei.     

Neurophysiological investigation of the pretectal nucleus lentiformis mesencephali in Rana pipiens

Previous studies have demonstrated that the large-celled, optic pretectal nucleus lentiformis mesencephali (nLM) is essential for horizontal optokinetic nystagmus, yet little is known about its neurophysiology. In the present study, single-unit analysis of nLM utilized a large-field, patterned stimulus presented for 8 directions and 3 velocities of movement. All units localized in nLM were spontaneously active, motion sensitive, with response profiles that ranged from strongly directional and narrowly tuned to asymmetric and broadly tuned. Only about one-third of the units could be classified as directional, and no response bias for horizontal or temporal-to-nasal motion was observed. The majority of directional units showed greatest responsiveness at the lowest stimulus velocity, while the reverse occurred for many broadly tuned units. These low-velocity, highly directional units may be comparable to the 'retinal slip' neurons recently described in the large-celled pretectal nucleus of mammals. Directional information in the nLM of Rana pipiens thus appears to be represented in the activity of a large population of motion-sensitive units which includes both narrowly and broadly tuned individual response profiles. These results are consistent with the population-coding hypothesis recently advanced to account for directional coding in other sensorimotor systems, including primate motor cortex and superior colliculus.     

The pretectal nucleus lentiformis mesencephali of Rana pipiens

The pretectal nucleus lentiformis mesencephali (nLM) of Rana pipiens was investigated with autoradiographic, horseradish peroxidase (HRP), and Golgi techniques. Retinal afferents to nLM originate primarily from the central retina. The primary projection is contralateral with a small ipsilateral component. Following optic nerve transection and HRP impregnation, contralateral retinal afferents show a restricted, dense core of HRP label in the superficial portion of the nucleus with sparser HRP label in the surround. Ipsilateral retinal afferents arborize throughout nLM, except in the dense-core region. Additional afferents to nLM originate from the ipsilateral tectum, the nucleus rotundus, the mesencephalic pretectal gray, the contralateral nLM, and the nucleus of the basal optic root. Afferents from the accessory optic system arborize only in the dense-core region, following HRP injections into the nucleus of the basal optic root, while afferents from the mesencephalic pretectal gray arborize in all parts of nLM except the dense core. Afferents from the tectum and anterior thalamus appear to arborize throughout the nucleus without discernible pattern. The lamination of afferent terminals in nLM was correlated with Nissl-stained cytoarchitectural material in which the majority of large neurons cluster around the dense core of nLM. Three types of neurons occur in nLM: large neurons (25-micron dia.), fusiform neurons (12.5-micron dia.), and stellate neurons (10-micron dia.). Additionally, two cell groups outside nLM which send dendrites into the nucleus were observed: cells of the posterior lateral nucleus and cells of the posterior thalamic pretectal gray. Both large and fusiform neurons project to the deep layers of the optic tectum as well as to the ventral rhombencephalon superficial to the abducens nucleus. While a small number of fusiform neurons project to the nucleus of the basal optic root, the stellate neurons appear to be intrinsic to nLM. The anuran nLM strongly resembles the nucleus of the optic tract in mammals in terms of the site of origin of its retinal afferents, lamination of afferent terminations, its central connections, and its demonstrated involvement in horizontal optokinetic nystagmus.     

Eye-specific segregation requires neural activity in three-eyed Rana pipiens

The addition of a third eye primordium to the forebrain region of a Rana pipiens embryo invariably results in the development of a series of periodic, mutually exclusive eye-specific bands in tectal lobes dually innervated by the host and supernumerary fibers. A number of investigators have proposed that such source-specific segregation arises as a compromise between two mechanisms that are normally involved in retinotectal map formation: one which is dependent on cell surface affinities to align the map and produce a rough retinotopy and a second that "fine tunes" the map by stabilizing adjacent terminals from neighboring retinal ganglion cell bodies at the expense of terminals from non-neighboring cells. In this study we have tested the idea that this second "fine-tuning" mechanism is dependent on neural activity by blocking impulse activity in the optic nerves of three-eyed tadpoles. To assess the requirement for activity on the formation of bands, both normal optic nerves of 17 three-eyed tadpoles were crushed intraorbitally. Two weeks after this operation, the supernumerary retinal projection had debanded and spread to cover the entire tectum in a continuous fashion. By 4 weeks, however, the host optic fibers regenerated back to the tecta and began to form segregated stripes with the fibers from the third eye. Six to 7 weeks after the optic nerve crush the periodic pattern of eye-specific segregation characteristic of dually innervated tecta was again pronounced. When activity in all three optic nerves was eliminated with tetrodotoxin (TTX; embedded in a slow release plastic) during the last 3 weeks of this process, the fibers from the two competing eyes failed to segregate and, instead, formed two completely overlapping, continuous projections across the tectal surface. To test for the requirement of activity in the maintenance of segregation, we also subjected three-eyed tadpoles without optic nerve crush to TTX blockade for 2, 3, and 4 weeks. Animals sacrificed at 2 weeks show overlap of the projections in the rostral tectum but distinct interdigitating stripes in other regions of these lobes. After 3 weeks of blockade, segregation of the projections was less distinct in the central tectum as well. After 4 weeks of TTX blockade the terminals from both eyes spread to form continuous overlapping projections throughout the tectum. Examination of well isolated, individual retinal ganglion cell terminal arbors during this period reveals that they occupy a significantly greater area of tectum following the TTX treatment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neuronal architecture of the dorsal nucleus (Cochlear nucleus) of the frog,Rana pipiens pipiens

The neuronal architecture of the dorsal nucleus of the Northern leopard frog (Rana pipiens pipiens), which is a homolog of the cochlear nucleus of mammals and birds, was investigated. Our study showed that the frog dorsal nucleus contains a number of morphologically distinct cell types that are discernible in terms of the cellular architecture as derived from Nissl-stained material and in terms of the dendritic profile as revealed by horseradish peroxidase-filled single neurons. These cell types are bushy cells, bipolar (or fusiform) cells, octopus cells, stellate cells, giant cells, radiate (or round) cells, and a variety of small cells. The different cell types occupy different regions of the nucleus. Therefore, our results suggest that the dorsal nucleus should no longer be considered to be a uniform nucleus containing a homogeneous population of neurons. Homologies of these cell types with those described in other vertebrate species, including mammals, are proposed. © 1996 Wiley-Liss, Inc.     

A forebrain visual projection in the frog (Rana pipiens)

In the frog (Rana pipiens) using a modification of the Fink-Heimer degeneration stain, a projection was traced from the lateral anterior thalamus via the lateral forebrain bundle to the ipsilateral striatum in the ventrolateral area of the forebrain. Striatal degeneration extended from the anterior commissure to the olfactory bulb. Single-unit microelectrode recording revealed that this area contained visual units that responded to the on and off of light.     

Neurogenesis in the optic tectum of larval Rana pipiens following unilateral enucleation

DNA synthesis and interlayer migrations of cells in the optic tectum of larval Rana pipiens were investigated, using several series of larvae which had been subjected to unilateral enucleation at stage 25, the last embryonic stage. It has been found that DNA synthesis occurs in all cellular layers of the tectum with least activity in peripheral layers. The location of the most active DNA synthesis during the larval period is the same as the location of cell division in the larval tectum, namely, in the layers bordering the ventricle of the optic lobe Unilateral enucleation of stage 25 embryos results in a decrease in DNA synthesis in all cellular layers of the optic tectum contralateral to the operation when compared to the corresponding layers in the ipsilateral tectum. The differences in rate of incorporation of ³H-thymidine between the control and affected lobes become greater during development. Fewer cells are found in each layer of the affected larval tectum than in the corresponding layer of the control tectum. The decrease is greatest in more peripheral layers, whose cells are more intimately associated with the visual circuit than are cells of the deeper layers. Peripheral to layers 1 and 2, the percentages of labeled cells found in each layer are very similar on the two sides, suggesting common factors which control migration. Differences in cell number, therefore, reflect differential cell production rather than differential cell migrations. The distribution of label resulting from ³H-thymidine incorporation at stage III indicates that the distribution of mitotic activity is not uniform in the cephalocaudal axis of the tectum. Greater cell proliferation occurs in the posterior portion of the tectum than in the anterior region throughout larval development. The peripheral control of mitotic divisions in the frog optic tectum remains unknown. The data in the present study, however, support the hypothesis that influences from afferent fibers of the optic tract modify the rates or timing of DNA synthesis in the optic tectum. The data support the notion that the deepest tectal cells respond earliest to the stimulus and these may be ependymal cells which have processes extending to the outer surface of the tectum.     

Musculotopic organization of the hypoglossal nucleus in the grass frog, Rana pipiens

Recent neural tracer studies in several mammalian species have demonstrated a similar musculotopic organization of the hypoglossal motoneurons which innervate individual tongue muscles. The distribution of this musculotopic organization in nonmammalian tetrapods, however, has not received detailed investigation. As part of an ongoing study on the comparative organization of the vertebrate hypoglossal nucleus, the musculotopic organization of the hypoglossal nucleus of Rana pipiens was studied by injection of lectin-conjugated horseradish peroxidase into four distinct tongue muscles and the geniohyoid muscle. Injections into the hyoglossus muscle label neurons in dorsal regions of the hypoglossal nucleus in middle and rostral nucleus levels. Injections into the genioglossus basalis muscle label neurons in ventral and lateral regions of the hypoglossal nucleus in caudal nucleus levels. Injections into the genioglossus medialis muscle label neurons in dorsal regions in caudal levels, throughout the nucleus in middle levels, and in ventral regions in more rostral levels. Injections into the geniohyoid muscle label neurons in the ventral tip of the hypoglossal nucleus and in the ventromedial corner of the medullary gray matter in middle and rostral nucleus levels. These results demonstrate that the organization of the hypoglossal nucleus in Rana pipiens is more complex than previous tracer studies indicated. Similarities in the musculotopic organization of the amphibian and mammalian hypoglossal nuclei suggest an evolutionary conservatism of the motor system controlling tongue movement.     

Multiple classes of photoreceptors and neurons in the frontal organ of Rana pipiens

An attempt has been made to characterize the photoreceptors and neurons of the frontal organ of Rana pipiens and the synaptic contacts of these cells, in order to provide morphological correlates for previous physiological findings. Neurons, glial cells, and photoreceptors with two different appearances can be seen in normal anatomical preparations. The photoreceptors make ribbon synaptic contacts onto both neurons and other photoreceptors. Retrograde transport of either cobalt or horseradish peroxidase through the frontal nerve results in labeling of neurons which are often located near the exit point of the frontal nerve from the frontal organ. Some of the dendritic processes of these neurons ramify among the neurons themselves, while others ramify among the photoreceptors. In addition, the frontal organ contains two photoreceptor types which synaptically contact both neurons and other photoreceptors. In some frontal organs labeled with cobalt (but never in peroxidase-labeled preparations) certain photoreceptors were also labeled. The labeled photoreceptors were probably the result of transcellular cobalt movement from labeled neurons, and not the result of some photoreceptors having axons in the frontal nerve. These results are discussed in conjunction with physiological evidence that chromatic interactions occur within single photoreceptors, each of which contains a single visual pigment with two physiologically active states.     

Biosynthesis and axoplasmic transport of neurophysins in the hypothalamo-neurohypophysial system of Rana pipiens

Biosynthesis, axoplasmic transport, and storage of neurophysin in the amphibian (Rana pipiens) magnocellular peptidergic neurosecretory system were studied, and the results were compared with those reported in mammals. After injection of [35S]cysteine into the preoptic recess, light microscopic autoradiography provides evidence that neurons of the preoptic nucleus (PON) synthesize cysteine-rich proteins. The time course of appearance of these [35S]cysteine-labeled proteins in different regions of the hypothalamo-neurohypophysial system was studied by slab gel autoradiography. [35S]Cysteine-labeled proteins were found in the PON less than 1 hr postinjection, whereas a major labeled protein, tentatively identified as the neurophysin, first appeared in the infundibulum and neural lobe 4 hr after the injection. In addition, the labeled neurophysin persisted in the neural lobe throughout the entire observation period of 5 days. The minimum transport rate for neurophysin was calculated as 0.9 mm/hr (22 mm/day) at 25 degrees C. Two different neurophysins (with isoelectric points (pI) 4.9 +/- 0.1, 4.6 +/- 0.1, and Mr = 23,000, 20,100) may be resolved from the neural lobe extracts by isoelectric focusing and SDS-polyacrylamide gel electrophoresis, respectively. In addition to the neurophysin peaks, two radioactive peaks with pI 5.2 and 5.8 may be detected in the preoptic nucleus and the infundibulum as early as 30 min after [35S]cysteine injection. Preliminary conversion studies suggest a putative precursor role for the pI 5.2 protein. The results indicate that in the amphibian peptidergic neurosecretory system, the synthesis of cysteine-rich neurophysin by the preoptic neurons, the transport through the infundibulum, and the storage in the neural lobe proceed similarily to their mammalian counterparts.     

Control of tectal cell number during larval development in Rana pipiens

Cell production and cell deaths were determined in larval Rana pipiens both in control tecta and in tecta following unilateral eyeball removal in embryos and larvae. Such enucleations produce significantly reduced rates of cell division in the contralateral tecta for virtually the entire larval period (confirming studies with enucleation almost exclusively performed in embryos--Kollros: J. Exp. Zool. 123:153-187, '53, and J. Comp. Neurol. 205:171-178, '82). Significant numbers of cell deaths in all nonependymal tectal cell layers were also observed. Control cell division rates peak at stage X, while cell death peaks are reached in stages XIII-XX. Overall, about 10(6) nonependymal cells are produced in control tecta, and about 350,000 of them die by the end of metamorphosis. Control of cell numbers following enucleation is shown to depend mainly on reductions in cell division rates when the operation occurs early in development and mainly on increases in cell death rates when the operation occurs late in larval life. Such increases in death rates are invariably present within 1 day of the operation whereas the reduced division rates ordinarily require several more days to be seen. The modified rates, both of cell divisions and cell death, are limited to tectal areas to which optic nerve fibers have already extended. Maps of the positions of tectal cell divisions in many larval stages provide the basis for modifying the current dogma that tectal formation occurs as a series of newly formed mediocaudal wedges pushing previously produced wedges rostrolaterad. All such "old" wedges receive substantial cell additions for many stages, with the rate of addition decreasing rostrad earlier than caudad.     

Tectal afferents in Rana pipiens. A reassessment questioning the comparability of HRP-results

Studying afferents to the optic tectum in Rana pipiens by means of retrograde HRP transport, results were obtained, that cast doubt on the reliability of this neuronal tract tracing technique. The tectum was found to receive afferents from e.g. lateral mesencephalic tegmentum, torus semicircularis, contralateral large-celled pretectal nucleus, and ipsilateral thalamic nuclei, which have not previously been demonstrated. In addition, the data obtained lack evidence of projections reported earlier. We suggest, that neuronal projections may be subject to hormone and/or activity dependent changes, that alter the probability of accumulating sufficient amounts of the tracer to become labeled.