Georgia A. Bishop

Bardet–Biedl syndrome proteins are required for the localization of G protein-coupled receptors to primary cilia

Primary cilia are ubiquitous cellular appendages that provide important yet not well understood sensory and signaling functions. Ciliary dysfunction underlies numerous human genetic disorders. However, the precise defects in cilia function and the basis of disease pathophysiology remain unclear. Here, we report that the proteins disrupted in the human ciliary disorder Bardet–Biedl syndrome (BBS) are required for the localization of G protein-coupled receptors to primary cilia on central neurons. We demonstrate a lack of ciliary localization of somatostatin receptor type 3 (Sstr3) and melanin-concentrating hormone receptor 1 (Mchr1) in neurons from mice lacking the Bbs2 or Bbs4 gene. Because Mchr1 is involved in the regulation of feeding behavior and BBS is associated with hyperphagia-induced obesity, our results suggest that altered signaling caused by mislocalization of ciliary signaling proteins underlies the BBS phenotypes. Our results also provide a potential molecular mechanism to link cilia defects with obesity.

Type III adenylyl cyclase localizes to primary cilia throughout the adult mouse brain

Solitary primary cilia project from nearly every cell type in the human body. These organelles are considered to have important sensory and signaling functions. Although primary cilia have been detected throughout the mammalian brain, their functions are unknown. The study of primary cilia in the brain is constrained by the scarcity of specific markers for these organelles. We previously demonstrated that type III adenylyl cyclase (ACIII) is a marker for primary cilia on neonatal hippocampal neurons in vivo and in vitro. We further showed that ACIII localizes to cilia on cultured glial cells. Here, we report that ACIII is a marker for primary cilia throughout many regions of the adult mouse brain. Furthermore, we report that ACIII localizes to primary cilia on choroid plexus cells and some astrocytes in the brain, which to our knowledge is the first report of a marker for visualizing cilia on glia in vivo. Overall, our data indicate that ACIII is a prominent marker of primary cilia in the brain and will provide an important tool to facilitate further investigations into the functions of these organelles. J. Comp. Neurol. 505:562–571, 2007.

The distribution and origin of serotonin immunoreactivity in the rat cerebellum

The distribution of serotonin immunoreactivity in the rat cerebellum was studied using the indirect antibody peroxidase-antiperoxidase (PAP) technique of Sternberger. Furthermore, the origin of these chemically defined cerebellar afferents was studied using a procedure which combines the retrograde transport of horseradish peroxidase (HRP) with the PAP technique. Serotoninergic fibers and varicosities distribute throughout the cerebellar cortex. However, within the cortex there are density variations in the distribution of this indoleamine to the granule cell and molecular layers as well as differences in the spatial orientation of labeled elements, especially in the latter lamina. Serotonin-positive fibers are also present in the Purkinje cell layer. Some of the fibers pass from this layer into the overlying molecular layer while others form a plexus around the somata of Purkinje cells. Subsequent to injections of HRP into the vermis and immediately adjacent portions of the cerebellar cortex, several reticular and raphe nuclei (n.) were found to project to the cerebellum including the paramedian reticular nucleus, n. raphe pallidus, n. raphe obscurus, n. raphe magnus, n. reticularis gigantocellularis, n. reticularis paragigantocellularis, n. pontis oralis, n. reticularis tegmenti pontis and n. centralis superioris. Double-labeling experiments, however, reveal that the neurons giving rise to serotoninergic afferents to the cerebellum are located almost exclusively in the n. reticularis gigantocellularis, the n. reticularis paragigantocellularis and the n. pontis oralis. In conclusion, the findings of the present study further support the view that the cerebellar cortex is not uniform in its histological structure. Although serotoninergic elements are distributed throughout the cerebellar cortex, there are lobular variations in the laminar distribution of this indoleamine. These data suggest that serotonin may be exerting its physiological effect on different populations of cortical neurons in different lobules. Thus this putative neurotransmitter may play different roles in the circuitry of the cerebellum in disparate regions of the cortex. Further, the data obtained in the double-label experiments demonstrate a fairly restricted origin for serotoninergic afferents in the medullary and pontine reticular formation. Moreover, the majority are not located in the raphe nuclei.

Neuromodulatory effects of corticotropin releasing factor on cerebellar purkinje cells: An in vivo study in the cat

Corticotropin releasing factor, a 41 amino acid peptide, has been localized in climbing fibers and mossy fibers in the cats cerebellar cortex. In the present study, corticotropin releasing factor was iontophoretically applied to Purkinje cells, isolated extracellularly, to assess the effect of this peptide on the firing rate of the neuron. By itself corticotropin releasing factor had little or no effect on cellular activity. However, this peptide potentiated the excitatory effects of aspartate and glutamate, the putative neurotransmitters of the climbing fiber and mossy fiber-parallel fiber systems, respectively. In addition, corticotropin releasing factor blocked the suppressive effects induced by the iontophoretic application of GABA. Finally, it shortened or eliminated the period of suppression produced by activation of climbing fibers in the cerebellar cortex. These data suggest that corticotropin releasing factor functions as a neuromodulator rather than as a neurotransmitter in cerebellar circuitry.

Hippocampal neurons possess primary cilia in culture

Primary cilia are cellular appendages that provide important sensory functions and defects in primary ciliary signaling have been implicated in the pathophysiology of human diseases and developmental abnormalities. Almost all human cell types possess a primary cilium. Neurons throughout the brain possess primary cilia on which certain receptors localize, suggesting that neurons possess cilia‐mediated signaling. However, the functional significance of neuronal cilia is unknown. Although there is a great deal of interest in understanding the functions of neuronal cilia, their study is hampered by the lack of an in vitro model system. We report that the majority of hippocampal neurons cultured from postnatal mice possess primary cilia in vitro. Further, we describe cilia proteins that can be labeled to readily visualize neuronal primary cilia in culture. These findings are the first characterization of neuronal primary cilia in vitro and should greatly facilitate further investigations into the function of these organelles.

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.

Chapter 4 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.

Anatomical evidence for enkephalin immunoreactive climbing fibres in the cerebellar cortex of the opossum

SummaryEnkephalin immunoreactivity is present in the cerebellum of the adult opossum within axonal arbors that resemble mature climbing fibres. In the developing cerebellum, enkephalinergic axons form pericellular nests around the perikarya of Purkinje cells in a manner which resembles developing climbing fibres seen in Golgi impregnations. Serial electron micrographs of adult climbing fibres reveal elongate enkephalin immunoreactive profiles that contain synaptic vesicles and make contact with the thorns and shafts of Purkinje cell dendrites. These results suggest that a peptide, enkephalin or an enkephalin-like substance may mediate synaptic interactions between certain populations of climbing fibres and Purkinje cells in the cerebellum of the opossum. Enkephalin immunoreactive axonal arbors, present in the molecular layer, are localized in restricted areas of vermal lobules II–VIII and X. The intermediate cortex and hemispheres are devoid of enkephalinergic climbing fibres except in restricted areas of the paramedian lobule, paraflocculus and the flocculus. In an attempt to establish the origin of enkephalin axons in the cerebellum, a double labelling technique that combines retrograde labelling of cells with horseradish peroxidase and enkephalin immunohistochemistry has been employed. Enkephalin immunoreactive neurons within specific portions of the medial accessory olive are retrogradely labelled in this paradigm. The presence of enkephalin immunoreactivity in selected climbing fibres provides evidence for chemical heterogeneity within one of the major afferent systems to the cerebellum previously thought to be uniform in its transmitter content.

Peptides in cerebellar circuits

Abbreviations

The physiological effects of serotonin are mediated by the 5HT1A receptor in the cat's cerebellar cortex

Serotonin is present in a fine beaded plexus in the cerebellar cortex of several mammalian species. In the cat, serotoninergic afferents arise from neurons located within the lateral, paramedian and peri-olivary reticular nuclei (Kerr and Bishop, J. Comp. Neurol., 304 (1991) 502-515). In addition to serotoninergic afferents, these same nuclei also contain a separate population of neurons that give rise to mossy fibers to the cerebellar cortex. Physiological studies have shown that mossy fibers are excitatory to their target neurons. The intent of the present study was to determine the physiological effects of serotonin in the cats cerebellum in an in vivo preparation and to identify the receptor(s) that mediate the observed responses. Iontophoretic application of serotonin (5HT) onto Purkinje cells reduces the spontaneous firing rate of all cells tested (n = 12). Serotonin also blocks the excitatory effects elicited by the application of aspartate in 17 of 19 units tested and of glutamate (n = 62) in all cases. In addition, 5HT potentiated the inhibitory action of GABA (n = 12). Iontophoretic application of the 5HT1A agonists, 8-OH-DPAT and ipsapirone, mimic the suppressive action of serotonin in a dose-dependent manner. This response, as well as the 5HT mediated suppression are blocked by the application of spiperone, a 5HT1A antagonist. Compounds selective for the 5HT1C,2 and 3 receptors are physiologically ineffective. The present data are in partial agreement with previous studies in the rats cerebellar cortex.(ABSTRACT TRUNCATED AT 250 WORDS)