The temporal and spatial development of CRF binding sites in the postnatal mouse cerebellum.

This study describes the distribution and relative level of labeling of binding sites for corticotrophin releasing factor (CRF) in the postnatal mouse cerebellum. At birth low levels of labeling are present throughout the cerebellum. However, this labeling is most densely distributed in the caudal and lateral aspects of the cerebellum. By P3 CRF binding sites are present throughout the cerebellum, although the greatest level of labeling is in the posterior lobe of the vermis, especially lobules IX and X; this correlates with the early differential pattern of CRF distribution in cerebellar afferents within these same lobules. At P10, the adult pattern of distribution and level of labeling begins to emerge. The presence of CRF and CRF binding sites at birth, and during postnatal growth, suggests that this peptide could play a role in the regulation of developmental events within the cerebellum.

The distribution of corticotropin-releasing factor (CRF), CRF binding sites and CRF1 receptor mRNA in the mouse cerebellum.

The purpose of the present study is to determine the distribution of CRF containing afferents, and correlate these findings with the distribution of CRF binding sites and the neuronal localization of mRNA for the CRF1 receptor in the cerebellum of a single species, the mouse. Corticotropin releasing factor (CRF) has been localized within climbing fibers and mossy fibers throughout the cerebellar cortex of the mouse using immunohistochemistry. CRF immunoreactive, axonal varicosities also are present within all four of the cerebellar nuclei. 125I-labeled CRF binding sites are evident throughout all three layers of the cerebellar cortex (molecular, Purkinje and granule cell layers), but are not seen within the cerebellar nuclei. In situ hybridization histochemistry was employed using an antisense riboprobe corresponding to the full length sequence of the rat mRNA for the CRF1 receptor. Positive signal is present throughout the cerebellum in Purkinje cells and the granule cell layer. CRF1 receptor mRNA also is expressed within all four of the cerebellar nuclei. Further experiments are required to reconcile the lack of CRF binding sites in the cerebellar nuclei with the positive mRNA receptor expression and the presence of immunoreactive axonal varicosities. In previous physiological experiments, iontophoretic application of CRF enhances spontaneous as well as quisqualate-induced activity of Purkinje cells in slice preparations of the mouse cerebellum. When the results of the anatomical techniques are compared to the physiological data, there is convergent evidence to suggest that CRF influences the firing rate or responsiveness of Purkinje cells directly via release of the peptide from the climbing fiber system and indirectly via the mossy fiber-granule cell-parallel fiber circuit. Taken together, these anatomical and physiological data provide strong evidence to suggest that, in the adult cerebellum, CRF functions as a neuromodulator.

The temporal and spatial development of corticotropin-releasing factor (CRF) binding sites and CRF afferents in the opossum cerebellum.

The intent of this study is to determine the developmental timecourse of the appearance and distribution of corticotropin releasing factor (CRF) binding sites within the developing opossum cerebellum, and to correlate this with the temporal and spatial distribution of CRF-labeled axons. 125I-labeled ovine CRF was used to identify the distribution and temporal expression of CRF binding sites in the opossum cerebellum. By PD8, binding sites are evident over cells in the external granular layer, as well as the subjacent immature Purkinje cell layer, but not over the ventricular layer or the intermediate zone. At PD 8, the intermediate zone, located between the ventricular and immature Purkinje cell layers, contains migrating nuclear, Golgi and Purkinje cells. By PD12, binding sites are present over all layers of the immature cerebellum (the external granular layer, the multitiered Purkinje cell layer, and the intermediate zone of migrating cells), except the ventricular layer. The adult distribution of CRF binding sites is evident by PD30-38 which includes the molecular, Purkinje and internal granule cell layers. The present results provide the first account of the ontogeny of CRF binding sites in the developing cerebellum. The early expression and distribution of CRF receptors, when correlated with the temporal expression and distribution of the peptide, provide additional evidence to support our working hypothesis that CRF functions as a regulator of developmental events which is distinct from its proposed function as a neuromodulator in the mature cerebellum.

Distribution of corticotropin-releasing factor (CRF) binding sites in the opossum cerebellum.

Corticotropin-releasing factor (CRF) has been demonstrated in the hypothalamic-pituitary-adrenal axis and studied in extrahypothalamic sites throughout the brain. Localization of CRF immunoreactivity, CRF mRNA, and CRF receptors within specific brain areas supports an extrahypothalamic function for CRF. Previous reports have revealed the localization of several peptides, including CRF, in the cerebellar cortex and nuclei of the North American opossum (Didelphis marsupialis virginiana); climbing fibers, mossy fibers, and a beaded plexus of axons in the Purkinje cell layer demonstrate CRF immunoreactivity. CRF also is localized within neurons in the inferior olivary complex and other brainstem nuclei which are known to project to the cerebellar cortex. Physiological recordings indicate CRF potentiates the excitatory effects of both aspartate and glutamate, the putative transmitters of the major afferent inputs to the cerebellum. The present study reports that specific CRF binding sites are present in all lobules of the opossum cerebellar cortex, with the greatest density in vermal lobules V through X, the flocculus, and the paraflocculus. The cerebellar nuclei do not appear to be labeled. CRF binding sites are present over all neuronal layers of the cerebellar cortex. The presence of CRF immunoreactivity in climbing fibers, mossy fibers, and a beaded axonal plexus, and CRF binding sites within the cerebellar cortex, as well as the fact that CRF potentiates the excitatory effects of both aspartate and glutamate, indicate that this peptide may function as a neuromodulator in the cerebellum of the North American opossum. An attempt is made to correlate the distribution of CRF-IR neurons and fibers, CRF mRNA, and CRF receptors to the extrahypothalamic function of CRF as it relates to the olivocerebellar pathway.

Distribution of cholecystokinin binding sites in the North American opossum cerebellum.

Previous studies in our laboratory have reported on the differential distribution of several neuropeptides, including the octapeptide cholecystokinin (CCK8), in the cerebellar cortex and nuclei of the North American opossum (Didelphis marsupialis virginiana). The present account reports on the distribution of CCK8 binding sites as determined from serial autoradiographic images of the cerebellum which were labelled by using [125I]Bolton Hunter sulfated CCK8. Evidence for the limited presence of CCK8-like immunoreactivity and CCK8 binding sites in several other species suggests that the distribution of this peptide and its receptor(s) may be species specific. In the opossum, CCK8-like immunoreactivity is present in mossy fiber terminals that distribute throughout the cerebellar cortex; it has a very limited distribution in climbing fibers (King and Bishop (1990) J. Comp. Neurol. 238, 373-384. CCK8 binding sites are present throughout all lobules of the cerebellar cortex and the cerebellar nuclei, which correlates well with the distribution of the peptide. CCK8-like immunoreactivity is located primarily in the granule cell layer, although the greatest density of binding sites is in the molecular layer. The presence of CCK8 is mossy fiber terminals, coupled with the presence of CCK8 binding sites in the cerebellar cortex, and the fact that CCK8 alters the firing rate of Purkinje cells (Madtes et al. (1992) Neurosci. Abstr. 18, 853) indicate this peptide may function as a neuromodulator in the cerebellum of the North American opossum.(ABSTRACT TRUNCATED AT 250 WORDS)

Sensitivity of GABA-displaceable [(3)H]muscimol binding depends upon the composition of the buffering medium.

Synaptosomal membranes were prepared from bovine retinas to measure both high- and low-affinity binding of [(3)H]muscimol which is displaceable by ?-aminobutyric acid (GABA) under various buffering conditions using Tris-citrate (non-physiological) and Krebs-Ringer's bicarbonate (physiological) buffers. The maximum number of high- and low-affinity sites (B(max)) was found when the tissue was prepared and assayed in Tris-citrate; the lowest B(max) occurred when the tissue was prepared and assayed in Krebs. The apparent affinity (K(D)) for the high-affinity site showed a slight dependence on buffering conditions, whereas the K(D) for the low-affinity site did not. These data support the suggestion that physiological ions act to alter the GABA-benzodiazepine receptor complex in vivo. Thus, the correlation of biochemical and electrophysiological data ought to reflect these observations.

Rhizobins, a group of peptides in the free-amino-Acid pool of the soybean-Rhizobium system.

Free-living Rhizobium (according to Bergey's Manual of Systematic Bacteriology, [1984, The Williams & Wilkins Co., Baltimore], Bradyrhizobium) japonicum was found to release a peptide into the nutrient media. Soybean nodules contained this peptide and exuded it into the soil. The name "rhizobin A" is suggested for this peptide. Nodules also contained another peptide, rhizobin B, as well as an unidentified, ninhydrin-positive compound, rhizobin C. The three peptides were confined to the free-amino-acid pool of the soluble fraction and eluted consecutively from a cation-exchange column. Rhizobin A was isolated in a highly purified form; its molecular mass was approximately 1,600 daltons as determined by Sephadex gel filtration and mass spectrometry. The amino-acid composition could be determined only approximately, because a long time was necessary for acid hydrolysis, possibly due to unusual linkages. The rhizobin concentration in soybean nodules continually increased during 50 days of growth, from 2 to approximately 400 mug/g (fresh weight). When combined nitrogen was added to nodulated soybean and subsequently removed, nitrogenase activity, nodulation, and nodule growth first decreased and then recovered. The relative amount of rhizobin A followed a similar pattern. Rhizobins were not detected in the roots, stems, and leaves of nodulated soybean plants. They were present in Lupinus nodules, but absent in alder nodules.

Postnatal development of 3H-GABA-accumulating cells in rabbit retina.

Light and electron microscopic autoradiography demonstrates that 3H-GABA is accumulated by horizontal cells in neonatal rabbit retina but not in the adult. A specific population of horizontal cells appears to be mature at birth and they avidly accumulate 3H-GABA during a 15-minute incubation period in vitro. Uptake into horizontal cells is not observed after the fifth postnatal day; 3H-GABA-accumulating horizontal cell bodies and their processes are the first identifiable components that clearly mark the future location of the outer plexiform layer at birth and as such, may be considered pioneering elements. Our observations raise the interesting possibility that the pioneering horizontal cell may provide structural and/or chemical factors necessary for the subsequent development of the outer plexiform layer of the retina. Labeling patterns of other retinal cells also show varying degrees of change during development. A population of amacrine cells accumulate 3H-GABA at birth. These cells show little change in their morphological or 3H-GABA uptake properties from birth to adulthood. Müller cells show weak accumulation of 3H-GABA at birth. Subsequent to this time, labeling of Müller cells is significantly more robust, resulting in Müller cell domination of retinal autoradiographic patterns in more mature retinas. Every cell body in the ganglion cell layer accumulates 3H-GABA at birth. The number of labeled cells declines during postnatal development, resulting in a very limited adult population. We conclude that the ability of retinal cells to accumulate 3H-GABA does not remain constant during postnatal development; rather each cell population displays a unique maturation sequence that results in a dramatic developmental shift in the number and types of GABA-accumulating cells present in the retina.

GABA receptor binding site "induction" in rabbit retina after nipecotic acid treatment: changes during postnatal development.

The development of the GABA system in the rabbit retina was studied. The number of high- and low-affinity GABA receptor binding sites increased in a sigmoidal manner, with the curve for the low-affinity sites lagging 2-3 days behind that for the high-affinity sites. The KD for both high- (17.5 nM) and low-affinity (138.0 nM) sites remained constant during development. Treatment of isolated eyecups with the uptake blocker nipecotic acid resulted in an increase in the Bmax for high-affinity sites in developing tissue with the maximum sensitivity around eye opening; mature tissue exhibited a decrease in Bmax. In contrast, a gradual decrease in sensitivity to stimulation of the low-affinity sites occurred. These data indicate that the "trophic" action of GABA is limited to the time when the tissue is developing.

Maximal GABA and muscimol binding to high-affinity sites differ in physiological and in non-physiological buffers.

The sensitivity of [(3)H]GABA and [(3)H]muscimol high-affinity binding sites to physiological (Krebs-Ringer's bicarbonate) and non-physiological (Tris-citrate) buffers was examined using synaptosomal membranes from bovine retinas. The maximum number of sites (B(max)) for [(3)H]GABA was present when the tissue was assayed in KRB. With only one exception, this effect was independent of the washing conditions used or a small change in pH. In contrast, [(3)H]muscimol binding sites were maximally present when the tissue was washed in Tris, regardless of the assaying conditions or the small change in pH. Neither [(3)H]GABA nor [(3)H]muscimol was displaced by ( - )baclofen. The apparent dissociation constants (K(d)) of the ligands did not change under any of the conditions tested. These findings demonstrate a fundamental difference between GABA and muscimol binding sites.

Neurotransmitter systems in morphologically undifferentiated human Y-79 retinoblastoma cells: studies of GABAergic, glycinergic, and beta-adrenergic systems.

Elements of three neurotransmitter systems were investigated in morphologically undifferentiated human Y-79 retinoblastoma cells in suspension culture. Specific gamma-aminobutyric acid (GABA) uptake, GABA binding, and glycine binding were absent from these cells, although the cells had been shown to exhibit an active uptake and release of [3H]glycine. Binding and competition studies using both alpha- and beta-adrenergic ligands indicated the presence of a beta-adrenergic receptor. This finding was confirmed by treatment of the cells with beta-agonists in competition with a beta-antagonist and with an alpha-antagonist; the level of cyclic AMP was competitively stimulated. Therefore, human Y-79 cells in suspension culture contain beta-adrenergic receptors, and not glycinergic or GABAergic ones. Thus, the Y-79 cells may be of use in studying the factors involved in developmental regulation of neurotransmitter function.

Metabolism of [3H]nipecotic acid in the rabbit retina.

Nipecotic acid has been demonstrated to block the gamma-aminobutyric acid transport systems. Previous studies have shown that the uptake system is the first transmitter-specific parameter to appear during the development of the rabbit retina. Use of these observations has been made to study the influence on the development of gamma-aminobutyric acid receptors of altering the uptake mechanism by treating newborn pups with nipecotic acid to block GABA transport. The present study of the in vivo metabolism of [3H]nipecotic acid in the CNS measured the changes in the levels of [3H]nipecotic acid in both adult and newborn rabbit retinas after injection of the label into the vitreal chamber. It was found that the effective half-life of [3H]nipecotic acid in the vitreous is about 5 h for adult tissue and 3 h for newborn. In contrast, all retinal fractions retained the label longer, the effective half-lives being about 60 h (adult) and 45 h (newborn). Further, no labeled metabolites of nipecotic acid were detected in either adult or newborn tissue. This study gives evidence that the degradation of nipecotic acid in nervous tissue is minimal and suggests that, although the rate of clearance is faster in neonates, the fate of nipecotic acid in vivo may be similar in both adult and newborn tissues.

Development of muscimol binding sites in chick embryo neural retina in vivo and in vitro: Regulatory effects of cyclic AMP.

We report here the development in the chick embryo retina of binding sites for [(3)H]muscimol, a potent agonist of GABA receptors. In vivo studies were carried out with isolated neural retinas from different stages of development. High-affinity binding sites were absent before embryonic day (E) 8, but increased conspicuously between E10 and E16. Scatchard analysis indicated that this rise was due to an increase in the number of binding sites. Kinetic parameters of the embryonic binding sites were consistent with those typically found for mature muscimol receptors. Measurements of the low-affinity binding site showed a relatively similar developmental pattern although a pronounced decrease in binding to the low-affinity site was observed between E12 and E14. In vitro studies were carried out using glial-free, purified monolayers of retinal neurons, starting at E8. Cultured retinal neurons showed a developmental pattern for high-affinity muscimol binding sites resembling that observed in ovo. These binding sites were susceptible to regulation by cyclic AMP analogues. Increases of 100 to 200% in muscimol binding could be induced by a 24 hr treatment with dibutyryl cyclic AMP, 8-bromo cyclic AMP, or the phosphodiesterase inhibitor IBMX. Scatchard analysis showed that this increase was due to a change in receptor affinity. No effects were found with either butyric acid or with adenosine 5'-monophosphate. These results raise the possibility that cyclic AMP may be involved in the regulation of components of the GABA system.

Chloride ions preferentially mask high-affinity GABA binding sites.

Chloride ions (Cl-) act to regulate the appearance of gamma-aminobutyric acid (GABA) binding sites in synaptic membranes from bovine retinas. Scatchard analysis indicates that the presence of Cl- results in a preferential decrease (85%) in the number of high-affinity binding sites, with no statistically significant change in the affinity (KD) of either the high- or low-affinity sites. The finding that the appearance of this binding site is altered by the presence of Cl- may indicate that Cl- acts, either directly or indirectly, to regulate the appearance, and hence function, of the high-affinity GABA receptor.

GABA as a trophic factor during development.

The process of synaptogenesis has been studied by many investigators to determine the factors which regulate synapse formation. We have used neonatal rabbit retina to investigate the role of the gamma-aminobutyric acid (GABA) neurotransmitter system during development. By utilizing an in vitro incubation treatment of isolated eyecups we found that treatment with nipecotic acid, a GABA uptake blocker, resulted in a 4-fold increase in the amount of specific 3H-muscimol binding. In addition, incubation of the tissue in the presence of the GABA agonists muscimol, 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridine-3-ol (THIP), or GABA itself led to similar increases in specific 3H-muscimol binding. The findings support the conclusion from previous studies that the induction of GABA receptors observed after in vivo treatment of 1-day-old rabbits with nipecotic acid resulted from an increase in the extracellular concentration of GABA. A possible role for GABA in the regulation of GABAergic synapse formation is presented in this report.

Intraocular injections of nipecotic acid produce a preferential block of neuronal 3H-GABA accumulation in adult rabbit retina.

A procedure by which the activity of the retinal GABA uptake system can be manipulated in vivo has been developed. Intraocular injections of nipecotic acid, a proported GABA uptake blocker, were administered to adult rabbits every 48 hours for a two-week period. No behavioral or systemic changes were observed. Injections were well-tolerated with less than 10% loss of the tissue caused by physical damage or injection. Biochemical analyses demonstrated a dose-dependent inhibition of 14C-GABA uptake into retinal tissue. No effect on uptake was observed for saline-treated tissue. Autoradiographic analyses showed that in vivo treatment with nipecotic acid preferentially blocked accumulation of 3H-GABA into the amacrine cell bodies and processes in the inner plexiform layer. This treatment may be especially useful in assessing the functional significance of GABA transport in vivo.

Synaptic interactions in the GABA system during postnatal development in retina.

Using biochemical analyses, we have demonstrated the presence of a high-affinity, sodium- and temperature-dependent uptake system for GABA in the retinas of newborn rabbits. The activity of this system two days after birth is approximately 70 percent of adult values, slowly increasing to adult level by postnatal day 6-8. An intraocular injection nipecotic acid (final concentration = 10 mM) into one-day-old rabbit pups resulted in a 60 percent inhibition in uptake activity. In order to study the possible role of the GABA uptake system in retinal development, we have determined the consequences of blocking GABA uptake with nipecotic acid on the postnatal development of post-synaptic GABA receptors, as measured by 3H-muscimol binding. Nipecotic acid treatment caused a significant increase in receptor binding in retinas prior to eye opening, with the maximal stimulation being one day after the intraocular injection. Our data indicate that the development of GABA receptor sites is influenced by the activity of the GABA uptake system and suggest that GABA may function as a trophic factor in the developing rabbit retina.

[3H]GABA binding in developing rabbit retina.

We have studied the developmental sequence of the GABA system in the rabbit retina using an in vitro binding assay to monitor developmental changes in the post-synaptic receptor. A variety of tissue treatments including perchlorate and Triton X-100 were employed to optimize binding and remove endogenous factors which inhibit binding. Pre-treatment of the tissue with 0.05% Triton X-100 revealed high affinity binding for [3H]GABA which increased in a sigmoidal fashion with the post-natal age of the animal. A constant level of binding, at about 16% of adult levels, was noted until day 8, at which time a rapid increase occurred. At 16 days post-natal, the amount of specific binding reached a plateau near adult levels. Kinetic analysis of the GABA receptor showed an increase in the number of receptors (Bmax) with little or no change in the apparent affinity (KD). Our results suggest that the onset of post-synaptic receptor activity is delayed approximately 1 to 2 days, relative to the pre-synaptic components, and the period of rapid increase in GABA receptor binding coincides with the period of maximum increase in retinal synaptic density.

'Binding' of glycine and gamma-aminobutyric acid to synaptosomal fractions of 6 regions of the feline brain; effects of strychnine.

GABA (6 X 10(-6) M) binding to synaptosome-enriched fractions of cat CNS exhibited a clear rostro-caudal gradient, whereas glycine (6 X 10(-6) M) binding was greatest to particles of cerebellar cortex, and this was followed by medulla approximately equal to caudate nucleus larger than or equal to cerebral cortex larger than or equal to pons larger than corona radiata. Strychnine-SO4 (10(-3) or 10(-4) M) inhibited the binding of GABA and glycine in all brain regions studied; at 10(-5) M this drug inhibited the binding of both GABA and glycine only to particles of the cerebral cortex.

'Binding' of glutamate and asparatate to synaptosomal fractions of six regions of the feline brain.

The order of potency of 'binding' of both glutamate and aspartate to synaptosomal fractions of brain regions was: cerebellar cortex greater than caudate nucleus greater than or equal to cerebral cortex greater than medulla approximately pons greater than corona radiata. Glutamate was bound to a greater extent than aspartate to particles of all regions studied, except for cerebral cortex.