Central Nervous Tissue Research Articles

Elevated Levels of Tau Protein in Cerebrospinal Fluid of Patients With Probable Creutzfeldt-Jakob Disease

IntroductionA definitive diagnosis of Creutzfeldt–Jakob disease (CJD) can only be made by neuropathologic examination and demonstration of typical pathologic changes and the pathologic prion protein in central nervous tissues. This study investigated the diagnostic sensitivity and specificity of the microtubule-association protein tau in cerebrospinal fluid (CSF) from Chinese patients with sporadic CJD. MethodsTwo hundred two CSF samples from clinically suspected patients with sporadic CJD were analyzed for tau protein by enzyme-linked immunosorbent assay and for the signal transduction regulatory protein 14-3-3 protein by immunoblot. ResultsRemarkably increased levels of tau protein and increased incidence of 14-3-3 positivity were observed in probable CJD, when compared with possible CJD and others. With a threshold of 1400 pg/mL, tau determination showed a sensitivity of 90% and a specificity of 94% for the diagnosis of probable CJD. The combination of raised tau and positive 14-3-3 increased the specificity but slightly reduced the sensitivity. Statistical analysis indicated that the raised level of tau positively correlated with the presence of 14-3-3 in CSF but not with other main clinical features, eg, age, gender, clinical manifestations and sampling time. ConclusionsThese data suggest that Chinese patients with probable CJD have similar increased levels of tau in the CSF as in Caucasian patients. Measurement of CSF tau will be another potential technique for antemortem CJD diagnosis.

1p36.32 rearrangements and the role of PI-PLC η2 in nervous tumours

Deletions in the distal region of the short arm of chromosome 1 (1p36) are widely diffuse, both in congenital 1p36 Deletion Syndrome and as somatic abnormalities in tumours. Rearrangements in 1p36 have been described in a broad spectrum of human neoplasias in addition to other chromosomal abnormalities. In neuroblastomas, wide hemizygous deletions in 1p36.23-1p36.32 have been described suggesting that the 1p36 region contains a tumour-suppressor gene involved in malignancy. A role for phosphoinositide (PI)-specific phospholipase C (PLC) η2, whose gene maps on 1p36.32, was suggested. PI-PLC η2 belongs to a family of enzymes related to the phosphoinositide signalling pathway, which provide an important intracellular signalling system involved in a variety of cell functions such as hormone secretion, neurotransmitter signal transduction, cell growth, membrane trafficking, ion channel activity, regulation of the cytoskeleton, cell cycle control and apoptosis. Expression of PI-PLC η2 occurs after birth and continues throughout the life. Synapse formation occurs during a short period of postnatal development. Thus, it is likely that PI-PLC η2 acts in formation and maintenance of the neuronal network in the brain. The fact that PI-PLC η2, a highly neuron-specific isozyme, is abundantly expressed in the postnatal brain suggests the importance of PI-PLC η2 in formation and maintenance of the neuronal network in the postnatal brain. Further studies are required to verify the possible involvement of PI-PLC η2 mutation/deletion in central nervous tumour tissues presenting abnormalities of the 1p36 chromosomal band.

Tissue Binding Patterns and In Vitro Effects of Campylobacter jejuni DNA-Binding Protein from Starved Cells

Campylobacter jejuni (C. jejuni) is frequently associated with axonal Guillain-Barré syndrome (GBS). We reported that C. jejuni DNA-binding protein from starved cells (C-Dps) binds to and damages myelinated nerves in vivo. We studied the binding patterns of C-Dps to nervous tissues and its in vitro effects on neural cells. Immunohistochemically, C-Dps labeled the nodes of Ranvier, the outermost parts of internodal myelin and the basement membrane in the peripheral nerves, and neurons and myelin in the central nervous tissues. Its binding was blocked by sulfatide. C-Dps bound to the cell surfaces of nerve growth factor (NGF)-treated PC12 cells leading to dose-dependent LDH release, which was inhibited by either heat-denaturation of C-Dps or coincubation with an anti-C-Dps mAb. However, its binding to the surfaces of cultured NSC34 cells, S16 cells, or dorsal root ganglion cells, did not induce cytotoxicity. These findings suggest a possible involvement of C-Dps in C. jejuni-related GBS.

GC/MS detection of central nervous tissues as specified BSE risk material in meat products: validation by an externally controlled blind trial

The addition of central nervous tissues (CNT), such as brain and spinal cord, in the manufacturing of meat products is either forbidden--if the material falls under the legal definition of specified risk material (SRM)--or must be labelled on the packed product. To foster official food control, several CNT detection methods were developed, but only fatty acid patterns as detected by gas chromatography/mass spectrometry (GC/MS) allow the further characterization of the detected CNT as to both the animal species and--surprisingly--the age of the animal from which the CNT was derived in accordance with the legal definition. Complementing a previous report in this journal by Lücker et al. 2010 (doi: 10.1007/s00216-010-3956-5) on CNT quantification by GC/MS, we now report results of the validation of this new analytical approach by an externally controlled blind trial elucidating its potential to identify the species and age of the CNT detected. The 72 samples (24 standards of emulsion-type sausage, each heated in three different batches: 75°C, 30 min; 115°C, 25 min; 133°C, 40 min) containing porcine, ovine or bovine muscle tissue and differing amounts of CNT (bovine or ovine brain: 0, 0.1, 0.5, 1.0, 3.0% m/m) were produced externally and provided blind for analyses to our laboratory. In accordance with the previous study, heating had no detectable effect on the GC/MS analysis. Judged by the present sensitivity of this method (cut-off 0.2% CNT), all of the samples containing 0.5% or more CNT (n = 57, 100%) were identified correctly as CNT-positive. The CNT species was identified correctly in 54 samples (94.7%), with three samples of one specific standard (0.5% ovine CNT) falsely classified as bovine CNT. However, the CNT age of these samples was correctly classified (more than 12 months). Overall, 57 samples (100%) were correctly classified as SRM-positive and 15 samples (100%) as SRM-negative. To the best of our knowledge, this is the first time that a legal demand for the (1) detection of traces of a specific tissue in a food matrix, (2) the identification of its taxonomic origin and (3) the classification of its age has been shown to be analytically possible in totally blind samples. The very positive validation results of this externally controlled blind trial recommend the present GC/MS approach for the detection of CNT in meat products as a reference method. However, our results also demonstrate the need for further studies, in particular to increase sensitivity and to conduct ring trials including more than one laboratory.

Analysis of single nucleotide polymorphisms of PRNP gene in twenty-four ethnic groups of India

Human prion protein (PRNP), a copper binding sialoglycoprotein, is the causative agent for transmissible spongiform encephalopathies (TSEs), a group of neurodegenerative diseases that are generally associated with aggregation of amyloid plaques within the central nervous system disrupting the normal tissue structure (Liberski et al. 2001; Aguzzi et al. 2008). While more than 20 PRNP pathogenic mutations have been reported in patients, polymorphisms in the gene have been suggested to influence the incidence of the diseases with a Met129Val polymorphism (rs1799990) being the most important among them (Soldevila et al. 2006). This particular variant has been found to be associated with different prion related diseases as well as cognitive behaviour and long-term memory. Here, as a part of the effort from Indian Genome Variation Consortium (IGVC), we attempt to establish the baseline allele and genotype frequency of four PRNP SNPs including rs1799990 among 24 ethnic groups representing the Indian population. The allele and genotype frequency of rs1799990 is found to be different among different ethnic groups. This pilot study would serve as a platform for future epidemiologicalworks with respect to PRNP SNPs in India.

GC/MS detection of central nervous tissue as specified BSE risk material in meat products and meat and bone meals: thermal stability of markers in comparison with immunochemistry and RT-PCR

Methods for the detection of central nervous tissue (CNT) are urgently needed in food control as a means for controlling strict adherence to both food labeling and banning of specified BSE risk material. Here, we report data on heat stability of the CNT markers neuron-specific enolase (NSE) in western blotting, glial fibrillary acidic protein (GFAP) in an enzyme linked immunoassay, mRNA(GFAP) in a real-time PCR assay, and several fatty acids (C22:6, C24:0-OH, C24:1ω9/ω7, C24:1ω9-OH/ω7-OH, and C24:0) in gas chromatography mass spectrometry (GC/MS). The sample matrix, a standard material of emulsion-type sausage with varied contents of CNT (brain), was heat-treated in three studies: (1) routine meat technological heat treatment with low (85 °C, 30 min), medium (115 °C, 30 min), and high (133 °C, 30 min, 3 bar) heating of 72 anonymous samples from a blind trial; (2) heat treatment under experimental conditions (100, 110, …, 200 °C, 45 min); and (3) fractionized heating of central nervous system (up to three times) under moderate routine technological conditions (85, 100, and 115 °C, 30 min). The markers of the immunochemical methods showed a low GFAP or very low NSE temperature stability at medium and high temperature conditions. The real-time PCR assay gave inconsistent, non-quantitative results, which indicated an uncontrollable matrix effect. The relevant GC/MS markers (C24:0-OH, C24:1ω9/ω7, and C24:1ω9-OH/ω7-OH) proved to be extremely stable. Neither meat and bone meal conditions (133 °C) nor experimental heating (up to and above 140 °C) showed any reduction of GC/MS CNT quantification. On the contrary, a slight but significant increase was noted over a certain temperature range (120-140 °C) for most fatty acids, possibly due to an improved extractability of the fatty acids. We conclude that a quantitative approach is highly unreliable when using immunochemical methods; moreover, these methods might be basically prone to false-negative results depending on heat treatment and matrix composition. Therefore, antibodies with higher affinity to heat-treated CNT marker epitopes are needed. Relevant amounts of CNT (≥0.5%) in low- and medium-heated products would still be reliably detectable by the GFAP ELISA, which justifies its use as a screening method in official food control. The results obtained by the real-time PCR assay were contradictory to recently published data, indicating a need for further protocol optimization and collaborative trials. Up to date, the analytical approach using GC/MS is the only valid procedure as pertaining to heat stability and quantitative analysis; consequently, it should be recommended as the reference procedure in official food control for CNT detection in heat-treated meat products.

Genotyping of Porcine Teschovirus from Nervous Tissue of Pigs with and without Polioencephalomyelitis in Indiana

Porcine teschovirus (PTV) was isolated in cell culture and/or demonstrated by polymerase chain reaction in samples of brain and/or spinal cord in pigs in Indiana during the 2002-2007 period. Testing was initiated on pigs originating from populations exhibiting nervous clinical disease and/or pigs with microscopic lesions in central nervous tissues, indicating viral encephalitis and/or myelitis. Virus was demonstrated in pigs with and without lesions as well as with and without nervous clinical disease. Nucleotide sequence analysis of the 5'-nontranslated region of the viral genome revealed that these isolates had low-level genetic heterogeneity but were homologous to porcine PTV serotype 1 (PTV-1). These findings indicate that low-to-moderate virulence strains of PTV with some homology to PTV-1 are endemic in many swineherds of Indiana and are associated with subclinical and clinical nervous disease in weaned pigs.

Extramedullary hematopoietic tumor mimicking a thoracic nerve root schwannoma

Extramedullary hematopoiesis secondary to chronic anemia is well reported throughout the literature. A rare presentation of this condition is in the central nervous tissue reported most frequently as an epidural mass causing spinal cord compression. The authors report the case of a 51-year-old man with beta-thalassemia and chronic anemia who was found to have a 4-cm paravertebral mass suggestive of a schwannoma. The patient underwent transthoracic resection of the mass. Histological examination confirmed an extramedullary hematopoietic tumor. In this article the authors propose a method to distinguish extramedullary hematopoietic tumors from schwannomas. To the authors' knowledge, this is the first reported case in the neurosurgical literature of this phenomenon.

Antinociceptive Effects of AS1892802, a Novel Rho Kinase Inhibitor, in Rat Models of Inflammatory and Noninflammatory Arthritis

Rho kinase (ROCK) is involved in various physiological functions, including cell motility, vasoconstriction, and neurite extension. Although a functional role of ROCK in nociception in the central nervous tissue has been reported in neuropathy, the peripheral function of this protein in hyperalgesia is not known. In this study, antinociceptive effects of AS1892802 [1-[(1S)-2-hydroxy-1-phenylethyl]-3-[4-(pyridin-4-yl)phenyl]urea], a novel and highly selective ROCK inhibitor, were investigated in two rat models of arthritis. Orally administered AS1892802 exhibited potent antinociceptive effect in both an adjuvant-induced arthritis (AIA) model (inflammatory arthritis model) and a monoiodoacetate-induced arthritis (MIA) model (noninflammatory arthritis model), with an ED(50) of 0.15 mg/kg (MIA model). Fasudil, a ROCK inhibitor, and tramadol were also effective in both models; however, diclofenac was effective only in the AIA model. The onset of antinociceptive effect of AS1892802 was as fast as those of tramadol and diclofenac. AS1892802 did not induce gastric irritation or abnormal behavior. Because AS1892802 rarely penetrates the central nervous tissue and is also effective by intra-articular administration, it seemed to function peripherally. These results suggest that AS1892802 has an attractive analgesic profile for the treatment of severe osteoarthritis pain.

Molecular cloning and temporal–spatial expression of I element in gregarious and solitary locusts

It has been reported that many genes and small RNAs are associated with density-dependent polyphenism in locusts. However, the regulatory mechanism underlying gene transcription is still unknown. Here, by analysis of transcriptome database of the migratory locust, we identified abundant transcripts of transposable elements, which are mediators of genetic variation and gene transcriptional regulation, mainly including CR1, I, L2 and RTE-BovB. We cloned one I element, which represents the most abundant transcripts in all transposable elements, and investigated its developmental and tissue-specific expression in gregarious and solitary locusts. Although there are no significant differences of I element expression in whole bodies between gregarious and solitary locusts at various developmental stages, this I element exhibits high expression level and differential expression pattern between gregarious and solitary locusts in central and peripheral nervous tissues, such as brain, antenna and labial palps. These results suggest that I element is potentially involved in the response of neural systems to social environmental changes in locusts.

Neurocan contributes to the molecular heterogeneity of the perinodal ECM

Neurocan is a central nervous tissue-specific chondroitin sulfate proteoglycan of the lectican family. Mainly expressed during modeling and remodeling stages of this tissue, it is thought to play an important role via binding to various extracellular matrix and cellular components. In adults, neurocan expression is associated with the perineuronal net structures. This study shows the neurocan immunolocalization at the node of Ranvier in mouse central nervous tissues. The N-terminal fragment of neurocan (Ncan130) was the predominant form detected in the optic nerve. The expression of neurocan in the white matter of brain tissue and nerve tracts revealed differential expression profiles compared with those of versican V2 and brevican, other perinodal extracellular matrix molecules. Double immunolabeling for neurocan and a nodal marker, Bral1, or a paranodal marker, caspr, demonstrated that neurocan was localized at the node of Ranvier. Neurocan expression was found at many--not all--nodal regions, and neurocan-positive nodes outnumbered brevican-positive nodes. The nodal localization of neurocan was diminished in Bral1-deficient mice. Taken together, these findings indicate that neurocan contributes to the molecular heterogeneity of the perinodal matrix, and its nodal expression is dependent on Bral1.

Lutein and docosahexaenoic acid prevent cortex lipid peroxidation in streptozotocin-induced diabetic rat cerebral cortex

The mechanisms underlying diabetic encephalopathy, are largely unknown. Here, we examined whether docosahexaenoic acid (DHA) and lutein could attenuate the oxidative changes of the diabetic cerebral cortex. The levels of malondialdehyde (MDA) were significantly increased and glutathione (GSH) and glutathione peroxidase activity (GPx) were decreased in diabetic rats. The number of 4-hydroxynonenal (4-HNE) positive cells was increased. Treatment with insulin, lutein or DHA and the combination of each antioxidant with insulin, significantly restored all markers concentrations mentioned above, and the increase in 4-HNE inmunofluorescence. We combined 4-HNE immunofluorescence with NeuN (Neuronal Nuclei) staining. The latter demonstrated extensive overlap with the 4-HNE staining in the cortex from diabetic rats. Our findings demonstrate a clear participation of glucose-induced oxidative stress in the diabetic encephalopathy, and that the cells suffering oxidative stress are neurons. Lowering oxidative stress through the administration of different antioxidants may be beneficial for the central nervous tissue in diabetes.

Blueberry supplementation attenuates microglial activation in hippocampal intraocular grafts to aged hosts.

Transplantation of central nervous tissue has been proposed as a therapeutic intervention for age-related neurodegenerative diseases and stroke. However, survival of embryonic neuronal cells is hampered by detrimental factors in the aged host brain such as circulating inflammatory cytokines and oxidative stress. We have previously found that supplementation with 2% blueberry in the diet increases graft growth and neuronal survival in intraocular hippocampal grafts to aged hosts. In the present study we explored possible biochemical mechanisms for this increased survival, and we here report decreased microglial activation and astrogliosis in intraocular hippocampal grafts to middle-aged hosts fed a 2% blueberry diet. Markers for astrocytes and for activated microglial cells were both decreased long-term after grafting to blueberry-treated hosts compared with age-matched rats on a control diet. Similar findings were obtained in the host brain, with a reduction in OX-6 immunoreactive microglial cells in the hippocampus of those recipients treated with blueberry. In addition, immunoreactivity for the pro-inflammatory cytokine IL-6 was found to be significantly attenuated in intraocular grafts by the 2% blueberry diet. These studies demonstrate direct effects of blueberry upon microglial activation both during isolated conditions and in the aged host brain and suggest that this nutraceutical can attenuate age-induced inflammation.

Intercomparison of induced fields in Japanese male model for ELF magnetic field exposures: effect of different computational methods and codes

The present study provides an intercomparison of the induced quantities in a human model for uniform magnetic field exposures at extremely low frequency. A total of six research groups have cooperated in this joint intercomparison study. The computational conditions and numeric human phantom including the conductivity of tissue were set identically to focus on the uncertainty in computed fields. Differences in the maximal and 99th percentile value of the in situ electric field were less than 30 and 10 % except for the results of one group. Differences in the current density averaged over 1 cm(2) of the central nerve tissue are 10 % or less except for the results of one group. This comparison suggests that the computational uncertainty of the in situ electric field/current density due to different methods and coding is smaller than that caused by different human phantoms and the conductivitys of tissue, which was reported in a previous study.

Magnetoencephalography May Allow the Electromagnetic 'Biopsy' of Central Nervous System Neoplastic Tissue

AbstractDeveloping a non-invasive method for definitively diagnosing intracranial neoplasms would reduce the risks involved with diagnosis and improve the treatment of such tumors. We used magnetoencephalography to measure the power spectral densities of malignant brain tumor cells in vitro and found that meningioma and medulloblastoma cells produce unique electromagnetic signatures. Our results suggest that non-invasive identification of neoplastic tissue is possible through the use of magnetoencephalography.

Microtubule-associated Protein-2 is a Sensitive Marker of Primary and Metastatic Neuroblastoma

Microtubule-associated protein-2 (MAP-2) is a protein expressed in high levels in cells derived from the neural crest. To the best of our knowledge, MAP-2 expression has not been thoroughly evaluated in tissues outside of the central nervous tissue. We examined the diagnostic utility of MAP-2 as a marker of neuroblastoma and attempted to characterize the expression of this protein in other tumors in the morphologic differential diagnosis of neuroblastoma. MAP-2 showed significant cytoplasmic reactivity in 95% of primary and 100% of metastatic neuroblastomas. Included within this set of tumors were 3 undifferentiated neuroblastomas, all of which showed strong staining. MAP-2 did not show significant staining in the majority of other small round blue cell tumors within the morphologic differential. Additionally, MAP-2 showed comparable sensitivity in staining primary neuroblastomas as compared with synaptophysin, chromogranin, CD56, and beta-catenin. In contrast to other markers of neuroblastoma, MAP-2 did not show significant cross reactivity to native bone marrow precursors, thus eliminating a potential source of confusion. In normal tissues, MAP-2 staining was essentially restricted to organs derived from the neural crest (adrenal medulla, endocrine organs). Variant patterns of staining were seen in exocrine organs, monocyte/macrophages and solitary fibrous tumor/hemangiopericytoma family of tumors. Rarely, high-grade adult sarcomas exhibiting strong cytoplasmic MAP-2 staining were seen. MAP-2 is a sensitive and specific marker of neuroblastoma, both in the primary tumor and bone marrow biopsy settings. We think that MAP-2, in conjunction with synaptophysin, is a very powerful immunohistochemical marker in differentiating neuroblastoma from its morphologic mimics.

Molecular Genetic and Immunophenotypical Analysis of Pax6 Transcription Factor and Neural Differentiation Markers in Human Fetal Neocortex and Retina In Vivo and In Vitro

Neurotransplantation of various cells, including heterotransplantation of fetal cerebral stem/progenitor cells into the eye is used in experimental studies of central nervous tissue repair during neurodegeneration. For evaluation of this approach, human fetal (weeks 9-20) stem/progenitor cells of the neocortex and retina were studied in vivo and in vitro by quantitative PCR and immunohistochemical staining. Native tissues and cultures were characterized by expression of Pax6 transcription factor (critical for the development of the retina and neocortex) and differentiation markers (nestin, betaIII-tubulin, glial fibrillary acidic protein, recoverin, NeuN, neurofilaments, Ki-67). The expression of Pax6 gene in the retina during active neurogenesis was stable and much higher than in the neocortex. In primary cultures, the pattern of Pax6 gene expression is retained and repeats that in native tissues. Immunohistochemical analysis revealed similarity of nestin and betaIII-tubulin expression in the neocortex and retina during the early (9-10 weeks) and later (20 weeks) periods and differences in cell phenotypes and their distribution. Culture studies showed that neocortical and retinal stem/progenitor cells are determined and exhibit specific differentiation characteristic of the corresponding native tissues. It can be hypothesized that heterotransplantation of the cerebral progenitor cells into the retina of experimental animals can lead to realization of their neurotrophic effect, but not to their functional integration.

Quality of life considerations for patients with chronic hepatitis C

Chronic infection with the hepatitis C virus (HCV) has a profound effect on health-related quality of life (HRQoL) - with fatigue, depression and neurocognitive deficits among the most common complaints. Neuropsychiatric symptoms have prompted research to determine whether the HCV acts within the central nervous system. Replicating virus has been found in central nervous tissues, and changes in neurotransmitter levels in the frontal white matter of patients with chronic hepatitis C are correlated with impaired attention and concentration. Other symptoms of chronic hepatitis C that decrease HRQoL include associated sexual dysfunction and depression. Treatment of chronic HCV infection may temporarily worsen HRQoL, and common adverse effects of currently available agents include fatigue, muscle aches, depression and cognitive deficits. The relationship between sustained viral response and improvement in HRQoL is nonetheless well accepted. Although treatment-related adverse effects may dissuade people from starting therapy and reduce compliance with associated reductions in sustained viral response, for the majority of patients viral clearance produces improvements in both HRQoL and long-term prognosis. Novel agents, with improved adverse effect profiles, may afford more patients the opportunity to achieve a sustained viral response.

Is Toxoplasma gondii a Causal Agent in Migraine?

BackgroundMany different tissues may be parasitized by Toxoplasma gondii, particularly, lung, heart, lymphoid organs, and the central nervous tissues. Tissue cysts of this parasite in the brain may spontaneously rupture, releasing parasites that cause antibody titers to rise. In immunocompetent subjects with acquired toxoplasmosis, the most frequent symptoms were lymphadenopathy and headache. In the neurogenic inflammation theory of the pathogenesis of migraine, the cause of initial release of ions and inflammatory agents has not been established. In this study, we aimed to investigate if T. gondii infection is a possible cause of neurogenic inflammation of migraines. MethodsThe anti–T. gondii antibody status of 104 patients with migraine were studied and compared with those of control groups, 50 healthy subjects and 50 subjects with headache due to rhinosinusitis, by using a micro–enzyme-linked immunosorbent assay technique. ResultsForty-six (44.2%) patients with migraine, 13 (26.0%) healthy control subjects, and 12 (24%) control subjects with rhinosinusitis were positive for anti–T. gondii IgG antibody. The rate of positivity in the migraine patient group was statistically different from those of the control groups (P < 0.05). ConclusionsThe results show the presence of chronic Toxoplasma infection in patients with migraine. Toxoplasma infection may contribute to neurogenic inflammation as the pathogenesis of migraine, as many studies in the literature have reported that Toxoplasma infection causes biochemical and immunologic changes.

Ins(1,4,5)P3 receptor type 1 associates with AKAP9 (AKAP450 variant) and protein kinase A type IIβ in the Golgi apparatus in cerebellar granule cells

Interconnections between the Ca2+ and cAMP signalling pathways can determine the specificity and diversity of the cellular effects mediated by these second messengers. Most cAMP effects are mediated by PKA (protein kinase A), which is anchored close to its membranous substrates by AKAPs (A kinase-anchoring proteins). In many cell types, the activation of InsP3R (inositol 1,4,5-trisphosphate receptor), an endoplasmic reticulum Ca2+ channel, is a key event of Ca2+ signalling. The phosphorylation of InsP3R1 by PKA stimulates Ca2+ mobilization. This control is thought to be tight, involving the association of PKA with InsP3R1. The InsP3R1 isoform predominates in central nervous tissue and its concentration is highest in the cerebellar microsomes. We investigated the complex formed by InsP3R1 and PKA in this fraction, vith a view to identifying its components and determining its distribution in the cerebellar cortex. Immunoprecipitation experiments showed that InsP3R1 associated with PKA type IIbeta and AKAP450, the longer variant of AKAP9, in sheep cerebellar microsomes. The co-purification of AKAP450 with InsP3R1 on heparin-agarose provided further evidence of the association of these proteins. Immunohistofluorescence experiments on slices of cerebellar cortex showed that AKAP450 was colocalized with InsP3R1 and RIIbeta (regulatory subunit of PKA IIbeta) in granule cells, but not in Purkinje cells. AKAP450 was localized in the Golgi apparatus of these two cell types whereas InsP3R1 was detected in this organelle only in granule cells. Taken together these results suggest that InsP3R1 forms a complex with AKAP450 and PKAIIbeta, localized in the Golgi apparatus of cerebellar granule cells. In contrast, the association of InsP3R1 with PKA in Purkinje cells would require a different macromolecular complex.