Comparison of the Mechanical Properties and Push-out Bond Strength of Self-adhesive and Conventional Resin Cements on Fiber Post Cementation.

OBJECTIVES: The purpose of this study was to compare the mechanical properties and push-out bond strength of self-adhesive resin cements (SACs) and a conventional resin cement (CRC). METHODS AND MATERIALS: Eighty bovine incisors were divided into four groups for cementation of a fiberglass post (Whitepost - FGM Dental Group, Coral Springs, FL) with different resin cements: three SACs (Maxcem Elite, MAX - Kerr; Calibra Universal, CAL - Dentsply; and RelyX Unicem 2, RUN - 3M Oral Care) and one CRC (RelyX Ultimate, RXU - 3M Oral Care). The groups were subdivided into two groups each (n=10) for evaluation of the push-out bond strength test (POBS) after 24 hours of water storage or after thermal aging (5000 cycles), following 24 hours of storage. The failure modes were evaluated using a stereomicroscope. Flexural strength (FS) and modulus of elasticity (EM) were determined using a three-point bending. Also, pH of the cements was measured over 48 hours and filler morphology was observed by scanning electron microscopy. Appropriate statistical analyses were performed by SPSS 21.0 (SPSS Inc., Chicago, IL, USA), with a significance level set at 5%. Results: RXU presented the highest POBS at both evaluation times. Among the SACs, RUN and CAL presented significantly lower POBS than MAX in cervical and middle-thirds at the 24-hour evaluation, and in all root regions after thermocycling. Adhesive failure between the cement and dentin were the most prevalent fractures at both times evaluated. MAX presented the lowest FS and RUN showed the highest EM. The pH reached the minimal point at the 30-minute evaluation for RXU and MAX. For RUN and CAL, the minimal pH was observed at the 60-minute evaluation. RXU and RUN presented spherical and regular filler particles, while MAX and CAL presented irregularly shaped and sized filler particles. CONCLUSIONS: The mechanical behavior of SACs is not superior to CRC; however, among all the SACs evaluated, MAX presented the highest POBS and stability after thermocycling evaluation. MAX also reached the closest neutral pH after 48 hours. Therefore, SACs with low initial pH and strong neutralization reactions are recommended, because these characteristics may lead to better mechanical properties and stability.

CNS germ cell tumor (CNSGCT) of childhood: presentation and delayed diagnosis.

OBJECTIVE: To describe the relationship between symptomatology and time to diagnosis of an institutional series of patients with CNS germ cell tumor (CNSGCT) over a 16-year period. METHODS: Thirty consecutive patients newly diagnosed with CNSGCT (mean age 10.9 years; range 6 to 17 years; 70% boys) were evaluated at our institution between 1990 and 2006. RESULTS: Duration of symptoms prior to diagnosis ranged from 5 days to 3 years (mean 8.4 months). Tumor location included pineal (14), suprasellar (8), pineal/suprasellar (3), pineal/thalamic (4), and basal ganglionic/thalamic (3). Five patients had disseminated disease at the time of diagnosis. Features including headache, nausea, vomiting, and visual changes led to earlier diagnosis. Symptoms including movement disorders, enuresis, anorexia, and psychiatric complaints delayed diagnosis in 9 of 30 patients, diagnosed 7 months to 3 years (mean 22.3 months) from symptom onset. In 7 of 9 patients with delayed diagnosis, enuresis was present. Seventeen of 30 patients had signs of endocrine dysfunction at presentation that included diabetes insipidus (4), hypothyroidism (8), and growth hormone deficiency (4). Ophthalmologic findings of decreased visual acuity, visual field deficits, or ocular abnormalities were present in 13 patients. Duration of symptoms did not correlate with tumor subtype or event-free survival. In three patients with basal ganglionic/temporal lobe, thalamic, or pineal/suprasellar signal abnormalities on MRI, neuroradiographic diagnosis was difficult. CONCLUSIONS: Diagnosis of CNS germ cell tumor is often delayed, and presentation may include movement disorders or mimic psychiatric disease. MRI interpretation can be challenging and may require serum/CSF markers and biopsy for diagnosis.

Periventricular heterotopia may result from radial glial fiber disruption.

Periventricular heterotopia (PVH) are collections of neurons and glia heterotopically located adjacent to the ventricles. The pathogenesis of periventricular heterotopia is believed to be a failure of cells to migrate from the ventricular zone. Mutations in filamin-1 (FLN1) have recently been identified as a genetic defect that results in an X-linked dominant form of PVH. In addition to this X-linked form, PVH may be found sporadically or occasionally as part of other syndromes. The pathogenesis(es) of PVH has not been entirely elucidated for patients with or without FLN1 mutation. In an attempt to better understand the pathogenesis of PVH, we examined 5 fetuses (gestational ages 21 to 34 wk), 3 females and 2 males, with PVH. Neuropathologic examination of these 5 fetuses revealed several to multiple periventricular nodules. No case showed the extensive periventricular heterotopia most commonly found in females with FLN1 mutations. By immunohistochemistry, neurofilament-positive cells were identified within the PVH in 3 of 5 cases and glial fibrillary acidic protein-positive cells surrounded the nodules in all 5 cases, but positive cells were only found within the nodules of 3 cases. Surprisingly, small collections of CD68-positive macrophages were found at the base of the nodules in 4 of the 5 cases. Moreover, in all cases, the radial glia highlighted with vimentin, showed disorganization specifically around the nodules. These data suggest that at least one pathogenesis for PVH is a disruption of the radial glial organization, resulting in a failure of cells to migrate from the ventricular zone.

N-acetylaspartylglutamate activates cyclic AMP-coupled metabotropic glutamate receptors in cerebellar astrocytes.

N-Acetylaspartylglutamate (NAAG) is the most prevalent peptide in the mammalian nervous system. NAAG meets the traditional criteria of a neurotransmitter, including localization in synaptic vesicles, depolarization-induced release, low potency activation of some N-methyl-D-aspartate receptors, and highly selective activation of a cAMP-coupled metabotropic glutamate receptor (mGluR) with potency approaching that of glutamate. The peptide is present in cultured cortical glia in high concentration and is hydrolyzed by cell surface peptidase activity. In the present work, we tested the hypothesis that NAAG selectively activates a subclass of metabotropic receptors on cultured rat cerebellar glia, primarily astrocytes. These glial cells express mRNA for mGluR subtypes 1, 3, 4, 5, 6, 7, and 8. We were not able to detect message for mGluR2 in these cells using the reverse transcriptase-polymerase chain reaction. Cerebellar glia responded to NAAG, glutamate, and trans-ACPD with a decrease in forskolin-stimulated cAMP formation. AP4, an agonist of the group III receptors mGluR4, mGluR6, mGluR7, and mGluR8, had little or no effect on stimulated cAMP levels. Treatment with low micromolar NAAG significantly increased uptake of radioactive thymidine by cultured astrocytes through activation of metabotropic glutamate receptors. Antagonists of group II mGluRs prevented the decrease in cAMP and the increase in uptake of thymidine by NAAG. Cultured cerebellar astrocytes expressed 20 pmol NAAG per mg protein, a value that is at least 30-fold lower than that expressed by mixed glial cultures prepared from mouse cortex. We conclude that cerebellar astrocytes respond to NAAG via the mGluR3 receptor and that the peptide may selectively activate this receptor subtype in astrocytes following release from neurons or glia.

Extradural origin of the posteroinferior cerebellar artery: an anatomic study with histological and radiographic correlation.

OBJECTIVE: The posteroinferior cerebellar artery (PICA) usually arises from the intradural segment of the vertebral artery (VA). The extradural origin of the PICA is infrequent. Its preoperatory identification is important in surgical strategy during the exposure of the VA. METHODS: During an anatomic prosection, the VA was exposed at the craniocervical junction in cadaveric adult specimens. The extradural origin of the PICA was encountered bilaterally in one specimen and on one side in a second specimen. An anatomic study with histological and radiographic correlation was performed. RESULTS: Perforating branches originate from the PICA. They supply the middle and inferior third of the olive and the lateral aspect of the medulla. The PICA has cortical branches that lead to the cerebellum. Injury to the PICA can produce an infarction of these neural structures that can be asymptomatic or cause major neurological deficits. Radiographic results obtained using a lateral projection provided the most reliable delineation of the extradural origin of the PICA. When this artery originates at, or posterior to, the posterior aspect of the occipital condyle, an extradural origin is likely. CONCLUSION: Bilateral selective vertebral angiography should be performed with special attention to the relationships of PICA origins before any surgical exposure of the VA at the craniocervical junction, unless magnetic resonance angiography provides this information without question. A thorough understanding of the relative dominance of the VAs and PICAs, the location of the PICA origin, and the collateral circulation of the posterior fossa are prerequisites to surgery in this region. The preoperative identification of an extradural PICA is important in planning surgical strategy and in avoiding complications during operations near the foramen magnum.

Analysis by polymerase chain reaction of alpha 1 and alpha 6 GABAA receptor subunit mRNAs in individual cerebellar neurons after whole-cell recordings.

With the use of the single-cell polymerase chain reaction (PCR), the GABAA receptor subunit mRNA content was analyzed in granule and Purkinje neurons from rat cerebellar slices. We used an experimental protocol to assess simultaneously the presence of two subunits in each cell while electrophysiological recordings were performed with the whole-cell patch-clamp technique. Based on a computer alignment of the nucleotide sequence corresponding to alpha 1 and alpha 6 GABAA receptor subunits, homologous regions were identified that allowed coamplification of both mRNAs using a single primer combination. The presence of selective restriction sites within the targeted templates allowed us to identify which receptor subunit mRNAs were coamplified by performing restriction enzyme-mediated cleavage of the amplification products. In all Purkinje neurons assayed, alpha 1 subunit mRNA but not alpha 6 mRNA was detected. In contrast, among individual granule neurons we found a heterogeneous distribution of the mRNA for the alpha 1 and alpha 6 GABAA receptor subunits. A comparison of the results of the PCR amplification and the analysis of GABA-mediated inhibitory synaptic currents does not allow us to identify kinetic characteristics of synaptic currents that clearly correlate with the presence or the absence of alpha 6 subunit mRNA.

Temporal and depolarization-induced changes in the absolute amounts of mRNAs encoding metabotropic glutamate receptors in cerebellar granule neurons in vitro.

Cerebellar granule neurons in primary culture express metabotropic glutamate receptors (mGluRs) coupled to the stimulation of phosphoinositide hydrolysis and to the inhibition of cyclic AMP (cAMP) formation. To evaluate which mGluR mRNAs are expressed in granule neurons under different depolarizing conditions, we measured the absolute amounts of selected receptor mRNAs in neurons cultured for 3-13 days in the presence of either 10 or 25 mM KCl. mGluR-specific primer pairs and internal standards, corresponding to unique regions of mGluR1a, mGluR2, mGluR3, mGluR4, and mGluR5, were constructed and used in a competitive PCR-derived assay to quantify the corresponding mRNA levels. For phosphoinositide-coupled receptors, the absolute content of mGluR1a mRNA was three to 10 times higher than the content of mGluR5 mRNA. The expression of mGluR5 mRNA increased up to 9 days in vitro and was much higher in 10 mM than in 25 mM KCl. For the cAMP-coupled receptors, there was a large amount of mGluR4 mRNA and a much smaller content of the mGluR3 and mGluR2 mRNAs. Maintaining the granule neurons in vitro in 10 mM KCl increased the absolute amount of mRNAs encoding mGluR2 and mGluR4 at 9 and 13 days in vitro. In contrast, the content of the mGluR3 mRNA was consistently higher in neurons cultured in 25 mM KCl. These data are consistent with the possibility that in primary cultures of cerebellar neurons, phosphoinositide responses may be predominantly mediated by mGluR1a, rather than mGluR5, and that cAMP inhibition involves preferentially mGluR4 and mGluR3.

Developmental expression of the alpha 6 GABAA receptor subunit mRNA occurs only after cerebellar granule cell migration.

Using a competitive polymerase chain reaction (PCR) and appropriate internal standards, we have analyzed absolute amounts of the alpha 6 GABAA receptor subunit mRNA in the postnatally developing cerebellum and neocortex. The PCR data have shown that absolute amounts of the alpha 6 receptor subunit mRNA in the cerebellum increase dramatically (nearly 100-fold) during the second postnatal week, reaching maximal levels by postnatal day 21 (1 fmol/microgram total RNA). The absolute amount of the alpha 6 GABAA receptor subunit mRNA in the cortex at postnatal day 1 was 2 amol/microgram total RNA and increased to 7 amol/micrograms total RNA by postnatal day 14. No further increase in alpha 6 mRNA expression in the adult cortex was observed. Microscopic analysis of emulsion coated and counterstained sections indicated that alpha 6 GABAA receptor subunit mRNA labeling was only detected in the internal granule cell layer and not in either the external granule cell layer or in migrating granule cells. The alpha 1 GABAA receptor subunit mRNA increased in the cerebellar cortex with a similar temporal profile, although its distribution extended to additional cell types (Purkinje cells, stellate/basket cells and possibly cerebellar astrocytes). The temporal expression of these two GABAA receptor subunit mRNAs is coincident with the formation of synaptic contacts in the granule cell dendrites suggesting that afferent pathways innervating these neurons following cell migration may play a critical role in increasing the expression of mRNAs encoding the alpha 1 and alpha 6 GABAA receptor subunits.

Expression patterns of gamma-aminobutyric acid type A receptor subunit mRNAs in primary cultures of granule neurons and astrocytes from neonatal rat cerebella.

Using a competitive polymerase chain reaction assay, we have quantitated the absolute amounts of mRNA encoding 14 distinct subunits of the gamma-aminobutyric acid type A (GABAA) receptor in primary cultures of rat cerebellar granule neurons and cerebellar astrocytes. We found that the total amount of GABAA receptor subunit mRNA in astrocytes was 2 orders of magnitude lower than in neuronal cells. Furthermore, granule cell cultures expressed all 14 different GABAA subunit mRNAs, while the astroglial cultures contained detectable amounts of all the subunits expressed by granule cells except the alpha 6 and the gamma 2L subunits. Of the alpha subunit family members, the alpha 1, alpha 5, and alpha 6 mRNAs were prominent in granule cells, while the alpha 1 and alpha 2 mRNAs were abundant in astrocytes. Of the beta receptor subunit mRNAs, the beta 1 and beta 3 mRNAs were abundantly expressed in both cultures. The gamma 2S and gamma 2L mRNAs constituted the great majority of gamma subunit mRNAs in neurons, while the gamma 1 subunit mRNA was the most abundant gamma subunit mRNA in astrocytes. When various allosteric modulators of GABAA receptors were tested electrophysiologically, methyl 6,7-dimethoxy-4-ethyl-beta-carboline- 3-carboxylate (DMCM) was the only one to modulate chloride currents elicited by GABA in a significantly different manner in granule cells (negative modulation) compared with astrocytes (positive modulation). The latter effect was previously observed in transiently expressed recombinant GABAA receptors containing a gamma 1 instead of a gamma 2 subunit. Our quantitative mRNA results suggest that an important molecular determinant responsible for the DMCM-positive modulatory effect on astroglial native GABAA receptors is the presence of the gamma 1 subunit in the receptor assembly.

Distinct developmental patterns of expression of rat alpha 1, alpha 5, gamma 2S, and gamma 2L gamma-aminobutyric acidA receptor subunit mRNAs in vivo and in vitro.

We have quantitated the alpha 1, alpha 5, gamma 2S, and gamma 2L gamma-aminobutyric acidA (GABAA) receptor subunit mRNAs in the maturing cerebellum in vivo and in cerebellar granule neurons differentiating in vitro. Absolute amounts of mRNA were measured by reverse transcription and competitive polymerase chain reaction (PCR) analysis with appropriate internal standards. The alpha 1 and gamma 2L mRNA content increased continuously during postnatal cerebellar maturation and their changes with time matched very closely those of the cerebellar granule cells differentiating in vitro. The gamma 2S subunit mRNA showed a relatively constant pattern of expression both in vivo and in vitro, with comparable absolute concentrations in both developmental paradigms. The alpha 5 mRNA was initially high in vivo and decreased (eight-fold) to adult levels as postnatal cerebellar development progressed. In vitro the amount of alpha 5 GABAA receptor subunit mRNA was higher than in vivo at 3 days, increased by more than twofold by 8 days, and declined to approximately the initial values at 23 and 28 days in vitro. Collectively, the results indicate that the alpha 1, alpha 5, gamma 2S, and gamma 2L GABAA receptor subunit mRNAs are regulated differentially in a temporal manner during in vivo and in vitro maturation. Moreover, a comparison of the ontogenetic profiles of the gamma 2S and gamma 2L mRNAs indicates that alternative splicing of the gamma 2 primary RNA transcript is regulated developmentally during postnatal maturation of the rat cerebellum.

Diazepam binding inhibitor peptide: cloning and gene expression.

Diazepam binding inhibitor (DBI) is a peptide, initially identified for its ability of displacing the binding of diazepam. The screening of lambda gt 10 cDNA libraries from rat brain with a 47merdeoxyoligonucleotide probe, complementary to a small portion of DBI coding region, allowed the isolation of cDNA clones encoding the entire aminoacid sequence of DBI. This sequence, when compared to that of mouse, human and bovine, revealed that DBI is a well conserved peptide, suggesting a similar function in different species. In order to characterize the function of DBI, studies on the regulation of DBI gene expression were undertaken. The expression of DBI mRNA occurs unevenly in the brain, as well as in peripheral tissues. Moreover, the biosynthesis of DBI is up-regulated in the cerebellum and cerebral cortex of rats made tolerant to diazepam, suggesting that changes in the biosynthesis of DBI might be one of the mechanisms eliciting tolerance to benzodiazepine. In peripheral tissues, the expression of DBI mRNA changes during development. In liver, the content of DBI mRNA was found maximal at postnatal day 1. In contrast, in kidney and heart a linear increase in levels of DBI mRNA was observed from postnatal day 1 to the adult stage, where it reached its maximum level. The tissue specific regulation of DBI mRNA expression, both pharmacologically or developmentally, leads to the hypothesis that DBI might have different functions in different tissues. This would be in line with recent findings that DBI might be also involved in the regulation of an important step of cell metabolism.

Neurosteroids act on recombinant human GABAA receptors.

The endogenous steroid metabolites 3 alpha,21dihydroxy-5 alpha-pregnan-20-one and 3 alpha-hydroxy-5 alpha-pregnan-20-one potentiate GABA-activated Cl- currents recorded from a human cell line transfected with the beta 1, alpha 1 beta 1, and alpha 1 beta 1 gamma 2 combinations of human GABAA receptor subunits. These steroids are active at nanomolar concentrations in potentiating GABA-activated Cl- currents and directly elicit bicuculline-sensitive Cl- currents when applied at micromolar concentrations. The potentiating and direct actions of both steroids were expressed with every combination of subunits tested. However, an examination of single-channel currents recorded from outside-out patches excised from these transfected cells suggests that despite the common minimal structural requirements for expressing steroid and barbiturate actions, the mechanism of GABAA receptor modulation by these pregnane steroids may differ from that of barbiturates.

Molecular cloning and expression of cDNA encoding a peripheral-type benzodiazepine receptor.

This report describes the cloning of a full length cDNA encoding PKBS, a protein of approximately 17 kDa associated with peripheral-type benzodiazepine binding sites. Cyanogen bromide digestion of purified PKBS yielded several peptide fragments which were subjected to gas-phase sequencing. Based on these partial amino acid sequences, oligonucleotide probes were used to screen a rat adrenal cDNA library. Several hybridizing clones were isolated which were found to contain overlapping sequences. The longest cDNA spanned 781 base pairs and specified an open reading frame of 169 amino acids which matched all of the peptide sequences. Northern analysis with this PKBS cDNA probe in different rat tissues revealed one RNA species of approximately 850 nucleotides exhibiting relative abundances qualitatively comparable with the densities of peripheral-type benzodiazepine binding sites in each tissue. The PKBS cDNA was cloned into an eukaryotic expression vector placing it under transcriptional control of the beta-globin promoter and SV40 enhancer. Transfection of the transformed human kidney 293 cell line with this recombinant vector resulted in stoichiometric increases of about 900 fmol/mg of protein in binding capacities for Ro5-4864 (4'-chlorodiazepam) and PK 11195, two peripheral-type benzodiazepine ligands. These increases were accounted for by the expression of binding sites with approximate dissociation constants of 5 nM for PK 11195 and 8 nM for Ro5-4864, thereby distinguishing the expressed binding sites as being characteristic of the receptor from rat origin rather than of the host human-derived cell line. The rank order of potency of different ligands to compete against [3H]Ro5-4864 binding in the transfected cells was PK 11195 greater than Ro5-4864 greater than diazepam greater than protoporphyrin IX greater than clonazepam, consistent with the specificity characteristic of rat peripheral-type benzodiazepine binding sites. These studies suggest that PKBS comprises binding domains for benzodiazepines and isoquinoline carboxamides and hence is apparently responsible for the manifestation of peripheral-type benzodiazepine recognition sites.

Differences in the negative allosteric modulation of gamma-aminobutyric acid receptors elicited by 4'-chlorodiazepam and by a beta-carboline-3-carboxylate ester: a study with natural and reconstituted receptors.

Cl- currents elicited by gamma-aminobutyric acid (GABA) application were recorded with the whole-cell tight-seal technique from voltage-clamped cortical neurons of neonatal rats in primary culture. The peripheral benzodiazepine recognition site ligand 4'-chlorodiazepam [Ro 5-4864; 7-chloro-1,3-dihydro-1-methyl-5-(4-chlorophenyl)-2H-[1,4]-benzodiazep in-2- one] inhibited the GABA-generated currents in a dose-dependent manner. Also, a beta-carboline (DMCM; 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate methyl ester), acting as a negative allosteric modulator of GABAA receptors, reduced the intensity of GABA-generated currents with similar efficacy but greater potency. Flumazenil (Ro 15-1788; 8-fluro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-[1,5-a] [1,4]-benzodiazepine-3-carboxylate ethyl ester) antagonized DMCM inhibition but not that elicited by 4'-chlorodiazepam. The isoquinoline carboxamide PK 11195, an antagonist of 4'-chlorodiazepam effects in other systems, failed to antagonize the action of 4'-chlorodiazepam. The transient expression of various molecular forms of GABAA receptors in the human embryonic kidney cell line 293 allowed a study of the minimal structural requirements for the inhibition of GABA-induced Cl- currents by bicuculline, picrotoxin, 4'-chlorodiazepam, and DMCM. GABA-elicited Cl- currents in cells coexpressing alpha 1 and beta 1 subunits of GABAA receptors were inhibited by bicuculline and picrotoxin, but not by DMCM or 4'-chlorodiazepam. Conversely, the GABA currents in cells coexpressing alpha 1 beta 1 and gamma 2 subunits were inhibited by bicuculline, picrotoxin, 4'-chlorodiazepam, and DMCM. Since the Cl- currents generated by GABA in some molecular forms of GABAA receptors are inhibited by bicuculline and picrotoxin only, 4'-chlorodiazepam cannot be acting isosterically with picrotoxin.

Heterogeneity of gamma-aminobutyric acid/benzodiazepine/beta-carboline receptor complex in rat spinal cord.

The properties of muscimol, beta-carboline (BC), and benzodiazepine (BZD) binding to crude synaptic membranes were studied in the spinal cord and cerebellum of rats. In cerebellar membranes, the density of high-affinity [3H]muscimol and [3H]6,7-dimethoxy-4-ethyl-beta-carboline ([3H]BCCM) binding sites is almost identical to that of [3H]flunitrazepam ([3H]FLU) or [3H]flumazenil (Ro 15-1788; ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a] [1-4]benzodiazepine-3-carboxylate). In contrast to the cerebellum, the number of muscimol and BC binding sites in rat spinal cord is approximately 20-25% of the number of FLU or flumazenil binding sites. Moreover, in spinal cord membranes, BC recognition site ligands displace [3H]-flumazenil bound to those sites, with low affinity and a Hill slope significantly less than 1; the potency of the different BCs in displacing [3H]flumazenil is 20-50-fold lower in the spinal cord than in the cerebellum. [3H]Flumazenil is not displaced from spinal cord membranes by the peripheral BZD ligand Ro 5-4864 (4'-chlorodiazepam), whereas it is displaced with low affinity and a Hill slope of less than 1 (nH = 0.4) by CL 218,872 (3-methyl-6-(3-trifluoromethylphenyl)-1,2,4-triazolol[4,3-b] pyridazine). These data suggest that a large number of BZD binding sites in spinal cord (approximately 80%) are of the central-type, BZD2 subclass, whereas the BZD binding sites in cerebellum are predominantly of the central-type, BZD1 subclass.(ABSTRACT TRUNCATED AT 250 WORDS)

Coexistence of GABA receptors and GABA-modulin in primary cultures of rat cerebellar granule cells.

GABA-modulin (GM), a basic polypeptide purified from rat brain synaptosomes, which is an allosteric inhibitor of GABA recognition sites, has been detected in primary cultures of cerebellar interneurons enriched in granule cells by immunohistochemistry, using a specific antibody raised in rabbit injected with GM purified from rat brain synaptosomes. In these cultures, GM is expressed by the granule cells, which are postsynaptic to GABAergic interneurons, but not by glial cells. In rat cerebellar sections anti-GM antiserum intensely strains the granular cell layer and Purkinje cell dendrites and cell bodies. GM has been purified from the cerebellar granule cell cultures and appears to be identical under biochemical, immunological, and functional criteria to authentic GM purified from rat brain synaptosomes. Granule cell cultures devoid of GABAergic neurons contain the GABA/BZ/Cl- receptor complex; in fact, intact cell monolayers, incubated in physiological buffer at 25 degrees C, express 3H-muscimol and 3H-flunitrazepam binding sites, which are comparable to the sites detected in cell membrane preparations and which modulate each other reciprocally. It is concluded that GM might participate in the supramolecular organization of the GABA receptor complex, perhaps functioning as a modulator of this receptor protein.

Modulation of gamma-aminobutyric acid-mediated inhibitory synaptic currents in dissociated cortical cell cultures.

Inhibitory gamma-aminobutyric acid-mediated synaptic currents were studied in dissociated primary cultures of neonatal rat cortex with the whole-cell patch-clamp technique. Immunocytochemical staining of the cultures showed the presence of a large number of glutamic acid decarboxylase-containing neurons, and electrical stimulation of randomly selected neurons produced in many cases chloride-mediated and bicuculline-sensitive inhibitory synaptic currents in postsynaptic cells. The amplitude and decay time of the inhibitory synaptic currents were increased by flunitrazepam and decreased by the beta-carboline derivative methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, two high-affinity ligands for the allosteric regulatory sites of gamma-aminobutyric acid receptors. The imidazobenzodiazepine Ro 15-1788, another high-affinity ligand of the gamma-aminobutyric acid receptor regulatory sites that has negligible intrinsic activity, blocked the action of flunitrazepam and beta-carboline. However, Ro 15-1788 also increased the decay rate of the inhibitory synaptic currents. This might suggest that an endogenous ligand for the benzodiazepine-beta-carboline binding site is operative in gamma-aminobutyric acid-mediated synaptic transmission.

Study of an octadecaneuropeptide derived from diazepam binding inhibitor (DBI): biological activity and presence in rat brain.

An endogenous brain neuropeptide with 104 amino acid residues that modulates gamma-aminobutyric acid receptor function was termed DBI because it displaces diazepam from its specific brain binding sites. Tryptic digestion of DBI generates an octadecaneuropeptide (ODN) that is more potent than the parent compound in the displacement of specifically bound beta-[3H]carboline-3-carboxylate methyl ester [( 3H]BCCM) and in proconflict action (Vogel test in thirsty rats). The proconflict action of ODN is antagonized by the imidobenzodiazepinone Ro 15-1788, which is a specific antagonist of beta-carboline and benzodiazepine recognition sites. The ODN amino acid sequence is Gln-Ala-Thr-Val-Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys. The pharmacological properties associated with this sequence were confirmed by comparing the activity of ODN generated from tryptic digestion of DBI with that of ODN obtained by synthesis. Amidation of the terminal lysine of ODN produces a peptide (ODN-NH2) devoid of pharmacological activity. Three peptides containing the COOH-terminal segment of ODN were synthesized. All these peptides [Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys (octapeptide), Pro-Gly-Leu-Leu-Asp-Leu-Lys (heptapeptide), and Gly-Leu-Leu-Asp-Leu-Lys (hexapeptide)] express the displacing and proconflict actions of ODN. In primary cultures of cerebellar granule cells of rat, DBI, ODN, octapeptide, heptapeptide, and hexapeptide preferentially displace [3H]BCCM over [3H]flunitrazepam; moreover, they displace bound [3H]BCCM completely but [3H]flunitrazepam only by 50%. These data suggest that ODN includes a specific ligand for the gamma-aminobutyric acid receptor regulatory site occupied by beta-carbolines. Using rabbit antibodies directed against the NH2-terminal portion of ODN, we detected ODN-like material in rat brain homogenates. However, whether this material is identical to the ODN generated by tryptic digestion of DBI remains to be established.