Serological evidence of Neospora caninum in alpacas from eastern Australia.

BACKGROUND: The aim of this study was to investigate if there was any serological evidence of Neospora caninum in alpaca populations in south-eastern Australia. METHODS AND RESULTS: Serum samples from 100 alpacas were collected from four farms. All serum samples were screened for N. caninum antibodies using a commercially available competitive ELISA. Of the 100 alpacas sampled, 3 were suspect seropositive for N. caninum. CONCLUSION: There is natural N. caninum seroprevalence in alpacas in south-eastern Australia; however, it remains undetermined whether or not this infection is currently contributing to reproductive failure in alpacas in Australia.

Best ethical practices for clinicians and laboratories in the provision of noninvasive prenatal testing.

OBJECTIVE: The goal of this study is to provide an ethical framework for clinicians and companies providing noninvasive prenatal testing using cell-free fetal DNA or whole fetal cells. METHOD: In collaboration with a National Institutes of Health-supported research ethics consultation committee together with feedback from an interdisciplinary group of clinicians, members of industry, legal experts, and genetic counselors, we developed a set of best practices for the provision of noninvasive prenatal genetic testing. RESULTS: Principal recommendations include the amendment of current informed consent procedures to include attention to the noninvasive nature of new testing and the potential for a broader range of results earlier in the pregnancy. We strongly recommend that tests should only be provided through licensed medical providers and not directly to consumers. CONCLUSION: Prenatal tests, including new methods using cell-free fetal DNA, are not currently regulated by government agencies, and limited professional guidance is available. In the absence of regulation, companies and clinicians should cooperate to adopt responsible best ethical practices in the provision of these tests.

Fine root biomass and turnover of two fast-growing poplar genotypes in a short-rotation coppice culture.

BACKGROUND AND AIMS: The quantification of root dynamics remains a major challenge in ecological research because root sampling is laborious and prone to error due to unavoidable disturbance of the delicate soil-root interface. The objective of the present study was to quantify the distribution of the biomass and turnover of roots of poplars (Populus) and associated understory vegetation during the second growing season of a high-density short rotation coppice culture. METHODS: Roots were manually picked from soil samples collected with a soil core from narrow (75 cm apart) and wide rows (150 cm apart) of the double-row planting system from two genetically contrasting poplar genotypes. Several methods of estimating root production and turnover were compared. RESULTS: Poplar fine root biomass was higher in the narrow rows than in the wide rows. In spite of genetic differences in above-ground biomass, annual fine root productivity was similar for both genotypes (ca. 44 g DM m-2 year-1). Weed root biomass was equally distributed over the ground surface, and root productivity was more than two times higher compared to poplar fine roots (ca. 109 g DM m-2 year-1). CONCLUSIONS: Early in SRC plantation development, weeds result in significant root competition to the crop tree poplars, but may confer certain ecosystem services such as carbon input to soil and retention of available soil N until the trees fully occupy the site.

Stimulation of the inferior olivary complex alters the distribution of the type 1 corticotropin releasing factor receptor in the adult rat cerebellar cortex.

In a previous study, it was shown that populations of climbing fibers, derived from the inferior olivary complex (IOC) contain the peptide corticotropin releasing factor (CRF) and that the expression of this peptide in climbing fibers could be modulated by the level of activity in olivary afferents. The intent of this study was to determine if there was comparable plasticity in the distribution of the type 1 CRF receptor (CRF-R1) in the cerebellum of the rat. Our results indicate that CRF-R1 was localized primarily to Purkinje cell somata and their primary dendrites and granule cells. In addition, scattered immunolabeling was present over the somata of Golgi cells, basket cells and stellate cells, as well as Bergmann glial cells and their processes. IOC stimulation for 30 min at 1 Hz increased CRF-R1 expression in molecular layer interneurons and processes of Bergmann glial cells. Little to no effect on CRF receptor distribution was observed in Purkinje cells, granule cells, or Golgi cells. IOC stimulation at 5 Hz however, increased CRF-R1 expression in the processes of Bergmann glial cells while decreasing its expression in basket, stellate and, to some extent, in Purkinje cells. The present results suggest that there is activity-dependent plasticity in CRF-R1 expression that must be considered in defining the mechanism by which the CRF family of peptides modulates activity in cerebellar circuits. The present results also suggest that the primary targets of CRF released from climbing fibers are Bergmann glial cells and interneurons in the molecular layer. Further, interneurons responded with a decrease in receptor expression following more intense levels of stimulation suggesting the possibility of internalization of the receptor. In contrast, Bergmann glial cells showed an increased expression in receptor expression. These data suggest that CRF released from climbing fibers may modulate the physiological properties of basket and stellate cells as well as having a heretofore unidentified and potentially unique effect on Bergmann glia.

Presynaptic localization of a truncated isoform of the type 2 corticotropin releasing factor receptor in the cerebellum.

It is now well established that corticotropin releasing factor is present in two major excitatory afferent systems to the cerebellum, namely climbing fibers and mossy fibers. Two major classes of corticotropin releasing factor receptors, each with unique binding characteristics, have been identified as type 1 and type 2. In this study we used an antibody made to the n-terminus of the type 2 corticotropin releasing factor receptor. Characterization of this antibody showed that it strongly labeled a protein with a molecular weight of 16-32 kDa and only faintly labels a 62-83 kDa protein. The lower molecular weight protein corresponds to the weight of a recently described truncated isoform of this receptor that is designated corticotropin releasing factor-type 2alpha-truncated isoform. We carried out transfection paradigms using corticotropin releasing factor-type 2alpha-truncated isoform constructs and confirmed that the antibody recognized the truncated isoform of the type 2 corticotropin releasing factor receptor. Further, light and electron microscopic studies were carried out in mice and rats to define the distribution of the truncated receptor. Immunoreactivity is evident in the basal region of many, but not all Purkinje cell bodies and their initial axonal segments, as well as the initial axonal segments of isolated Golgi cells, and cerebellar nuclear neurons. In addition, punctate elements in the molecular layer were immunolabeled. The localization of the receptor to the initial segment of Purkinje cells was confirmed with electron microscopy. Further, the punctate labeling in the molecular layer was localized to parallel fibers and their terminals. In conclusion, evidence has been presented to show that distinct isoforms of the corticotropin releasing factor receptor are present in the cerebellum. The complex interactions between corticotropin releasing factor and other members of the corticotropin releasing factor family of peptides with both pre- and postsynaptic receptors support a growing concept that corticotropin releasing factor plays an important role in modulating activity in cerebellar circuits and ultimately in controlling motor behavior.

Fine root chemistry and decomposition in model communities of north-temperate tree species show little response to elevated atmospheric CO2 and varying soil resource availability.

Rising atmospheric [CO2] has the potential to alter soil carbon (C) cycling by increasing the content of recalcitrant constituents in plant litter, thereby decreasing rates of decomposition. Because fine root turnover constitutes a large fraction of annual NPP, changes in fine root decomposition are especially important. These responses will likely be affected by soil resource availability and the life history characteristics of the dominant tree species. We evaluated the effects of elevated atmospheric [CO2] and soil resource availability on the production and chemistry, mycorrhizal colonization, and decomposition of fine roots in an early- and late-successional tree species that are economically and ecologically important in north temperate forests. Open-top chambers were used to expose young trembling aspen (Populus tremuloides) and sugar maple (Acer saccharum) trees to ambient (36 Pa) and elevated (56 Pa) atmospheric CO2. Soil resource availability was composed of two treatments that bracketed the range found in the Upper Lake States, USA. After 2.5 years of growth, sugar maple had greater fine root standing crop due to relatively greater allocation to fine roots (30% of total root biomass) relative to aspen (7% total root biomass). Relative to the low soil resources treatment, aspen fine root biomass increased 76% with increased soil resource availability, but only under elevated [CO2]. Sugar maple fine root biomass increased 26% with increased soil resource availability (relative to the low soil resources treatment), and showed little response to elevated [CO2]. Concentrations of N and soluble phenolics, and C/N ratio in roots were similar for the two species, but aspen had slightly higher lignin and lower condensed tannins contents compared to sugar maple. As predicted by source-sink models of carbon allocation, pooled constituents (C/N ratio, soluble phenolics) increased in response to increased relative carbon availability (elevated [CO2]/low soil resource availability), however, biosynthetically distinct compounds (lignin, starch, condensed tannins) did not always respond as predicted. We found that mycorrhizal colonization of fine roots was not strongly affected by atmospheric [CO2] or soil resource availability, as indicated by root ergosterol contents. Overall, absolute changes in root chemical composition in response to increases in C and soil resource availability were small and had no effect on soil fungal biomass or specific rates of fine root decomposition. We conclude that root contributions to soil carbon cycling will mainly be influenced by fine root production and turnover responses to rising atmospheric [CO2], rather than changes in substrate chemistry.

Soil carbon dioxide partial pressure and dissolved inorganic carbonate chemistry under elevated carbon dioxide and ozone.

Global emissions of atmospheric CO(2) and tropospheric O(3) are rising and expected to impact large areas of the Earth's forests. While CO(2) stimulates net primary production, O(3) reduces photosynthesis, altering plant C allocation and reducing ecosystem C storage. The effects of multiple air pollutants can alter belowground C allocation, leading to changes in the partial pressure of CO(2) (pCO(2)) in the soil , chemistry of dissolved inorganic carbonate (DIC) and the rate of mineral weathering. As this system represents a linkage between the long- and short-term C cycles and sequestration of atmospheric CO(2), changes in atmospheric chemistry that affect net primary production may alter the fate of C in these ecosystems. To date, little is known about the combined effects of elevated CO(2) and O(3) on the inorganic C cycle in forest systems. Free air CO(2) and O(3) enrichment (FACE) technology was used at the Aspen FACE project in Rhinelander, Wisconsin to understand how elevated atmospheric CO(2) and O(3) interact to alter pCO(2) and DIC concentrations in the soil. Ambient and elevated CO(2) levels were 360+/-16 and 542+/-81 microl l(-1), respectively; ambient and elevated O(3) levels were 33+/-14 and 49+/-24 nl l(-1), respectively. Measured concentrations of soil CO(2) and calculated concentrations of DIC increased over the growing season by 14 and 22%, respectively, under elevated atmospheric CO(2) and were unaffected by elevated tropospheric O(3). The increased concentration of DIC altered inorganic carbonate chemistry by increasing system total alkalinity by 210%, likely due to enhanced chemical weathering. The study also demonstrated the close coupling between the seasonal delta(13)C of soil pCO(2) and DIC, as a mixing model showed that new atmospheric CO(2) accounted for approximately 90% of the C leaving the system as DIC. This study illustrates the potential of using stable isotopic techniques and FACE technology to examine long- and short-term ecosystem C sequestration.

Evidence for an axonal localization of the type 2 corticotropin-releasing factor receptor during postnatal development of the mouse cerebellum.

Previous studies have described the embryonic and postnatal development of CRF, as well as the type 1 CRF receptor in the mouse cerebellum. The present immunohistochemical study localizes the cellular distribution of the type 2 CRF receptor (CRF-R2) during postnatal development of the mouse cerebellum. Western blot analysis indicates that the antibody used in this analysis recognizes both a full-length and a truncated isoform of the type 2 receptor. We propose that each isoform has a unique cellular distribution. In the present study, the postnatal (P) development (P0-P14) and cellular localization of CRF-R2 in different cell types was analyzed using PAP and double-label fluorescent immunohistochemistry; cell-specific antibodies were used to identify cells expressing CRF-R2 at different stages of postnatal development. At P0, CRF-R2 immunoreactivity was localized within the somata of Purkinje cells and migrating GABAergic interneurons. CRF-R2 was first observed in the initial axonal segments of some Purkinje cells at P5, and was evident in many Purkinje cell axon hillocks at P8. Punctate immunoreactivity is present in the molecular layer by P5 and is interpreted to be immunolabeled parallel fibers. Between P8 and P14, CRF-R2 immunostaining is present in the initial axonal segments of Golgi cells, within the internal granule cell layer. Finally, CRF-R2 is present in both radial glia in the molecular layer as well as in astrocytes in the white matter and internal granule cell layer from P5 to P14. The present results suggest that CRF-R2, both the truncated and the full-length isoforms, are present in the developing cerebellum, each with a unique cellular distribution. The immunohistochemical evidence indicates that the truncated isoform of the type 2 CRF receptor is in the axons of several different types of cerebellar cortical neurons, and suggests that CRF could play a role in cerebellar development by modulating the release of transmitters from excitatory and/or inhibitory interneurons, which in turn could directly alter the maturation of cerebellar circuits. In contrast, the binding of a ligand to the full-length isoform of CRF-R2 or to CRF-R1, both in a postsynaptic location, may have a more direct effect on regulating the responsiveness of these cells to growth factors or neurotransmitters released from afferent axons by regulating permeability of ion channels or altering second messenger systems.

Cellular localization of corticotropin releasing factor receptors in the adult mouse cerebellum.

Corticotropin releasing factor is a 41 amino acid peptide that is present in afferent systems that project to the cerebellum. In the adult, this peptide modulates the activity of Purkinje cells by enhancing their responsiveness to excitatory amino acids. Two different types of corticotropin releasing factor receptors, designated type 1 and type 2, have been identified. The purpose of this study is to use immunohistochemistry to identify which corticotropin releasing factor receptors are present in the cerebellum of the adult mouse and to determine their cellular distribution. Receptor type 1 immunostaining is present throughout all lobules of the cerebellar cortex. Distinct labeling is present over the somas of most, if not all, Purkinje cells as well as the primary dendrites of Purkinje cells located at the base of vermal folia. In vermal lobules V, VI, VIII and IX numerous glial fibrillary acidic protein immunoreactive processes, oriented radially in the molecular layer, also are immunoreactive for receptor type 1. In the granule cell layer, scattered type 1 immunoreactive puncta are present throughout most cerebellar lobules. Receptor type 2 immunoreactive puncta are present throughout the molecular layer in all lobules. In addition, scattered basket and/or stellate cells, identified with a GABA antibody, are immunopositive for the type 2 receptor. In the Purkinje cell layer, the type 2 receptor immunolabeling is confined to the basal pole of the Purkinje cell including the initial axonal segment. In the granule cell layer, labeling is present over large cell bodies, and their initial axonal segments. These are likely to be Golgi cells, based on their co-staining with GABA. Finally, numerous elongated processes within the white matter, which are likely to be axons, also are type 2 immunoreactive. These data indicate that both types of corticotropin releasing factor receptor are present in the mouse cerebellum. However, the unique distribution of the two types of receptor strongly suggests a differential role for corticotropin releasing factor in modulating the activity of neurons, axons and glial cells via cell-specific ligand-receptor interactions.

Corticotropin releasing factor induces proliferation of cerebellar astrocytes.

In the adult cerebellum, corticotropin releasing factor (CRF), that is localized in climbing fibers, mossy fibers, and a fine varicose plexus along the Purkinje cell layer, modulates the responsiveness of Purkinje cells to excitatory amino acids. During development, CRF has been detected in the primitive cerebellar anlage as early as embryonic day (E)10, and is continuously expressed throughout embryonic and postnatal cerebellar ontogeny. To investigate a possible trophic role for CRF during cerebellar development, cerebellar culture studies using E18 mouse embryos were carried out. In our culture paradigm, that used serum-free defined medium to suppress cell proliferation, CRF induced proliferation of cells in a dose-dependent manner in a range of concentrations between 0.1-10 microM. The proliferating cells were identified as astrocytes based on their expression of vimentin and GFAP. BrdU incorporation studies supported the proposed mitogenic effect of CRF on developing astrocytes. The mitogenic effects of CRF seemed to be primarily on immature astrocytes determined by their differential expression of vimentin and GFAP. Astrocytes at more advanced stages of development, as determined by the extent of process outgrowth and GFAP expression, incorporated less BrdU compared to immature astrocytes. CRF receptors were localized in astrocytes, and the proliferation of astrocytes induced by CRF was inhibited by astressin, a competitive CRF receptor antagonist. In conclusion, CRF induces proliferation of astrocytes derived from the developing cerebellum, that suggests a gliotrophic role for CRF during cerebellar development.

Localization of gp130 in the developing and adult mouse cerebellum.

Interleukin-6 (IL-6) type cytokines show functional redundancy in the immune, hematopoietic, and nervous system, which is believed to result from sharing of the signal transducing receptor gp130. IL-6 type cytokines and their binding receptors have been localized in the adult cerebellum. However, the cellular localization and developmental regulation of gp130 in the cerebellum have not been determined. In the present study the expression pattern of gp130 in the developing and adult mouse cerebellum was investigated. At embryonic day (E)15 and E17, gp130 immunoreactivity is present primarily in fiber bundles that course from the brainstem to the cerebellum. At postnatal day (P)0, gp130 immunoreactivity first appears in the Purkinje cell layer, external granule cell layer, and cerebellar nuclei. As Purkinje cells differentiate, gp130 immunoreactivity progressively extends from the cell body along their developing dendritic arbor. All Purkinje cells show intense gp130 immunoreactivity in their cell bodies by P7. In contrast the gp130 immunoreactivity detected in fiber bundles at E15 and E17 is downregulated postnatally, and cannot be detected after P7. Granule cells show gp130 immunoreactivity at P0 in the external granule cell layer and subsequently in the internal granule cell layer. Astrocytes in the white matter express gp130 at P0, and show intense gp130 immunoreactivity between P7 and P14. As the cerebellum matures gp130 immunoreactivity in the white matter decreases. The present description of the differential spatial and temporal distribution of gp130 provides an initial step in defining specific cellular populations that are potential targets of IL-6 type cytokines during cerebellar ontogeny.

DNA repair protein Rad55 is a terminal substrate of the DNA damage checkpoints.

Checkpoints, which are integral to the cellular response to DNA damage, coordinate transient cell cycle arrest and the induced expression of DNA repair genes after genotoxic stress. DNA repair ensures cellular survival and genomic stability, utilizing a multipathway network. Here we report evidence that the two systems, DNA damage checkpoint control and DNA repair, are directly connected by demonstrating that the Rad55 double-strand break repair protein of the recombinational repair pathway is a terminal substrate of DNA damage and replication block checkpoints. Rad55p was specifically phosphorylated in response to DNA damage induced by the alkylating agent methyl methanesulfonate, dependent on an active DNA damage checkpoint. Rad55p modification was also observed after gamma ray and UV radiation. The rapid time course of phosphorylation and the recombination defects identified in checkpoint-deficient cells are consistent with a role of the DNA damage checkpoint in activating recombinational repair. Rad55p phosphorylation possibly affects the balance between different competing DNA repair pathways.

Age differences in dynamic measures of EEG.

Eighteen older adults and 18 younger adults were compared on two quantitative measures describing changes over time in the spatial distribution of running EEG. EEG was collected from 128 electrodes under resting eyes-open and eyes-closed conditions and during performance of a 13 minute sustained attention task. One EEG measure, the recrudescence rate, represented the number of changes in the location of the highest squared voltage per second. A second EEG measure consisted of the algorithmic complexity of changes in the location of the highest squared voltage over time. Regardless of the task condition, older adults had significantly higher scores than younger adults on both the recrudescence rate and the measure of algorithmic complexity. The implications of the results for neurologically-based theories of performance declines in older adults are discussed.

Developmental expression of corticotropin-releasing factor in the postnatal murine cerebellum.

Corticotropin-releasing factor (CRF) is present in climbing and mossy fibers and both have a distinct pattern of distribution in the adult cerebellar cortex. The intent of this developmental study is to determine when the lobular pattern of CRF distribution emerges, and to analyze the morphogenesis of CRF immunoreactive climbing and mossy fibers in individual cerebellar lobules. Between postnatal day (P)0 and P3, CRF-immunoreactive (IR) punctate elements are present throughout the cerebellum. By P3, there is a decrease in the density of staining in the white matter and punctate elements become concentrated within the developing cortex. Between P3 and P7 CRF-IR, varicosities circumscribe Purkinje cell bodies, and are present in the internal and external granule cell layers. Between P10 and P12, there is a major reduction in the density of CRF-IR puncta, especially in the internal and external granule cell layers. Varicosities remain around Purkinje cell bodies and some extend into the molecular layer. During this interval, CRF-IR profiles are first evident in axonal configurations characteristic of developing climbing fibers, although there are lobular differences in the degree of maturation of this afferent system. Axonal enlargements characteristic of immature mossy fibers can first be seen at P10 in lobules IX and X; they cannot be differentiated until P12-14 in more rostral or lateral lobules. CRF-IR fibers in lobules IX and X, the vestibulocerebellum, develop into mature climbing and mossy fibers before any other area of the cerebellum. In other lobules of the cerebellum the gradient of maturation for these axonal phenotypes is from medial to lateral and posterior to anterior. Between P10 and P12, CRF-IR climbing fibers are present in all lobules of the cerebellum. After P12, few climbing fibers are observed in the anterior lobe of the cerebellum at midvermal levels; those present are only faintly immunolabeled. Based on its early expression and uniform distribution between P0 and P10, CRF could have a role in cerebellar development. After this age, as climbing and mossy fiber terminal phenotypes mature, and the differential adult patterns of distribution emerge, CRF likely begins to function as a neuromodulator as has been shown in the adult cerebellum.

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.

Corticotropin releasing factor in the embryonic mouse cerebellum.

Corticotropin releasing factor (CRF) is a 41 amino acid peptide that has been localized throughout the mouse cerebellum on postnatal day (P0). The wide-spread distribution of CRF within this brain region at birth suggests that it likely is present during embryonic stages of development. Thus, the intent of this study was to use immunohistochemical techniques to determine when CRF is first present in the cerebellar anlage, to analyze its distribution within the developing cerebellum, and to correlate these findings with early events in cerebellar ontogeny. CRF can first be detected in the cerebellum on embryonic day (E) 10 in scattered puncta that appear to approximate cell bodies throughout the cerebellar plate. Between E11 and E14 the number of puncta increase in the intermediate zone and more dorsal aspect of the cerebellum and decrease in the ventricular zone. At E14, in addition to the puncta, lightly immunolabeled cell bodies are observed in the ventricular zone. Just prior to birth at E17, CRF-immunoreactive varicosities distribute along the multitiered Purkinje cell layer and the intermediate zone. The CRF-positive cell bodies increase in number and intensity of staining. The majority remain within the ventricular zone, although a few also are present in the intermediate zone; it is postulated that these may be glial cells or neurons that are transiently expressing CRF. In conclusion, CRF-positive punctate elements derived from an as yet unknown source are present in the embryonic cerebellum just prior to and during the birth of Purkinje cells and nuclear neurons. The presence of this peptide at this critical stage of cerebellar development and its continued expression throughout the postnatal period of ontogeny suggests that CRF may play an important developmental role.

Fine structural analysis of calcitonin gene-related peptide in the mouse inferior olivary complex.

Climbing fiber afferents to the cerebellum, from the inferior olivary complex, have a powerful excitatory effect on Purkinje cells. Changes in the responsiveness of olivary neurons to their afferent inputs, leading to changes in the firing rate or pattern of activation in climbing fibers, have a significant effect on the activation of cerebellar neurons and ultimately on cerebellar function. Several neuropeptides have been localized in both varicosities and cell bodies of the mouse inferior olivary complex, one of which, calcitonin gene related peptide (CGRP), has been shown to modulate the activity of olivary neurons. The purpose of the present study was to investigate the synaptic relationships of CGRP-containing components of the caudal medial accessory olive and the principal olive of adult mice, using immunohistochemistry and electron microscopy. The vast majority of immunoreactive profiles were dendrites and dendritic spines within and outside the glial boundaries of synaptic glomeruli (clusters). Both received synaptic inputs from non-CGRP labeled axon terminals. CGRP was also present within the somata of olivary neurons as well as in profiles that had cytological characteristics of axons, some of which were filled with synaptic vesicles. These swellings infrequently formed synaptic contacts. At the LM level, few, if any, CGRP-immunoreactive climbing fibers, were seen, suggesting that CGRP is compartmentalized within the somata and dendrites of olivary neurons and is not transported to their axon terminals. Thus, in addition to previously identified extrinsic sources of CGRP, the widespread distribution of CGRP within olivary somata and dendrites identifies an intrinsic source of the peptide suggesting the possibility of dendritic release and a subsequent autocrine or paracrine function for this peptide within olivary circuits.

Distribution of tyrosine hydroxylase-immunoreactive afferents to the cerebellum differs between species.

The indirect antibody peroxidase-antiperoxidase technique was used to determine the laminar and lobular distribution of catecholaminergic afferents in the adult mouse, opossum, and cat cerebellum. A monoclonal antibody to tyrosine hydroxylase (TH) revealed a plexus of thin varicose fibers that exhibited a different density and distribution pattern for each species. In the cat, TH-immunoreactive fibers were sparsely distributed to all laminae, lobules, and nuclei of the cat cerebellum except for an area of elevated density in the ventral folia of lobules V and VI. In the opossum, TH-positive fibers were uniformly and densely distributed in the granule and Purkinje cell layers; they were more abundant in vermal lobules V-VI than in more anterior and posterior lobules, particularly I and X. Numerous TH-immunoreactive fibers were found in all four cerebellar nuclei of the opossum. In the mouse, TH-positive fibers formed a dense plexus within all cerebellar lobules, laminae, and nuclei. The mouse also exhibited numerous TH-immunoreactive Purkinje cells that were localized predominantly within vermal lobules VI-X, the paraflocculus, and flocculus. In addition to the interspecies differences in the distribution of catecholaminergic fibers within the cerebellum, comparison of this plexus to that previously described for serotonin in these species reveals that the relative densities and distribution patterns of catecholaminergic and serotoninergic fibers also vary between species. It is thus hypothesized that in each species a given monoamine has a unique net effect on cerebellar output that is determined by its effects on different neuronal populations 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.