A quantitative histochemistry technique for measuring regional distribution of acetylcholinesterase in the brain using digital scanning densitometry.

Studies of brain acetylcholinesterase (AChE) are traditionally based on biochemical assays, immunoreactivity, and histochemistry. Conventional histochemistry yields rich morphological data from tissue sections but yields quantitative results only with great difficulty. Several histochemical methods developed in recent years, including microdensitometry, microphotometry, and video-based histochemistry, are effective in quantitative and detailed study of AChE in tissue sections. However, they are usually time-consuming. As we report here, we adapted digital scanning densitometry to quantitate AChE histochemical staining in brain sections. The AChE and butyrylcholinesterase (BuChE), as measured by the method, were heterogeneously distributed throughout the brain, results that are consistent with those obtained by biochemical methods. The staining intensity is dependent on section thickness, substrate concentration, and reaction time. The cholinesterase inhibitor methyl paraoxon significantly decreased AChE staining intensity. Furthermore, data acquired from densitometry are similar to those obtained by video-based microscopy or by spectrophotometry. The advantage of the densitometric measurements compared to other quantitative histochemical methods is that it is very rapid while collecting data that are equivalent in quality. Because the digital scanning densitometers provide high quality and sensitive imaging, wide dynamic ranges, and convenient image analysis software, they are very useful tools in quantitative histochemistry.

Sequence preference of mouse H1(0) and H1t.

Histone H1 proteins bind to DNA and are important in formation and maintenance of chromatin structure. Little is known about differences among variant H1 histones in their interactions with DNA. We examined the effects of histones H1(0) and H1t on thermal denaturation of several DNA species. One of the DNA molecules was a 214-base-pair fragment from the plasmid pBR322, which contains an AT-rich and a GC-rich region. Both H1(0) and H1t bound preferentially to one region of the DNA fragment, a region that is relatively GC-rich. This result indicates that histones H1(0) and H1t are not totally nonspecific but rather bind with some sequence preference to DNA. This conclusion was supported by studies of other DNA species, including two 92-base-pair fragments derived from the two regions of the 214-mer, and several synthetic homocopolymers of DNA. Data obtained with the homocopolymers suggested that the binding preference was not simple preference for GC base pairs. The binding of the two H1 variants was not identical: there appear to be differences in binding site sizes, affinities, and sequence selectivities between H1t and H1(0).

Autoradiographic study of [3H]flunitrazepam binding sites in the subnuclei of the thalamus of rats rendered tolerant to and dependent on pentobarbital.

We examined changes in benzodiazepine binding sites labeled by [3H]flunitrazepam in five nuclei of the thalamus, the central medial, central lateral, intermediodorsal, ventroposterior, and laterodorsal nuclei, in rats made tolerant to and dependent on pentobarbital. Animals were made tolerant by intracerebroventricular infusion with pentobarbital (300 microg (10 microl)(-1) h(-1) for six days) through pre-implanted cannulae. Pentobarbital dependence was assessed 24 h after abrupt withdrawal from pentobarbital. Pentobarbital-tolerant rats showed no significant change in [3H]flunitrazepam binding sites (Bmax and Kd) in any nucleus examined in the thalamus. In the rats made dependent on pentobarbital, significant increases in the Bmax of [3H]flunitrazepam binding without changes in Kd were noted in central medial and central lateral nuclei. GABAergic (gamma-aminobutyric acid) neurons in the ventrobasal nucleus and in nuclei in the midline group are important in seizure regulation and arousal. These findings suggest that alterations of benzodiazepine receptors in certain nuclei of thalami are involved in the physiological changes induced by pentobarbital dependence. There were no changes in the binding parameters for [3H]flunitrazepam in pentobarbital-tolerant rats.

Changes in [3H]forskolin binding to adenylate cyclase and [3H]phorbol dibutyrate binding to protein kinase C in pentobarbital tolerant/dependent rats.

These studies were designed to examine the effect of chronic administration of pentobarbital on activity of adenylate cyclase (AC) and protein kinase C (PKC) in the rat brain by autoradiography. Recently, it has been suggested that the phosphorylation of specific proteins may be involved in the development of physical dependence. An experimental model of barbiturate tolerance and dependence was developed using i.c.v. infusion of pentobarbital (300 microg/10 microl/hr for 7 days) by osmotic minipumps and abrupt withdrawal from pentobarbital. The levels of [3H]forskolin binding were elevated (28-67%) in cortex, thalamus, dentate gyrus, hippocampal CA3 and cerebellum of the pentobarbital withdrawal animals, while these changes were not observed in tolerant rats. The levels of [3H]phorbol dibutyrate binding were highly elevated (38-65%) in the region of cortex, caudate putamen, septum, thalamus, dentate gyrus, and cerebellum of rats withdrawal from pentobarbital. These results show that the levels of AC and PKC were significantly elevated in pentobarbital withdrawal rats, and suggest that the levels of AC and PKC are altered in a region-specific manner during pentobarbital withdrawal.

Purification of mouse H1 histones expressed in Escherichia coli.

We amplified the coding regions of the previously cloned H1 genes H1-1, H1 zero and H1t and inserted them into the expression vector pET-11d. The synthesis of the H1 histones can be induced in the appropriate strains of bacteria, and the H1 histones can be readily purified. We report detailed protocols for the purification of the expressed proteins using combinations of ion-exchange and reverse-phase HPLC. Sufficient amounts of each pure variant protein can be obtained for use in physical studies of H1-DNA interactions.

Carboxyl-terminal peptides from histone H1 variants: DNA binding characteristics and solution conformation.

The carboxyl-terminal domains of the histone H1 proteins bind to DNA and are important in condensation of DNA. Little is known about the details of the interactions between H1 histones and DNA, and in particular, there is little known about differences among variant H1 histones in their interactions with DNA. Questions concerning H1 histone-DNA affinity and H1 conformation were investigated using peptide fragments from the carboxyl terminal domains of four nonallelic histone H1 variant proteins (mouse H1-1, H1-4 and H1(0), and rat H1t). Three of the four peptides showed a slight preference for binding to a GC-rich region of a 214-base-pair DNA fragment, rather than to an AT-rich region. The fourth peptide, H1t, appeared to bind preferentially to the AT-rich region of the 214-base-pair fragment. The results show that these small peptides bind preferentially to a subset of DNA sequences: such sequence preference might be exhibited by the intact H1 histones themselves. CD spectra of the peptides, which are from regions of the proteins that are not compactly folded, showed that the alpha-helical content of the peptides was minimal if the peptides were in 10 mM phosphate buffer, but increased if the peptides were in 1M NaClO4 and 50% trifluoroethanol, conditions that are postulated to approximate certain aspects of binding to DNA. H1-4 peptide, which was predicted to be 70% alpha-helix, but was not alpha-helical in 10 mM phosphate buffer, appeared from difference CD spectra to be more alpha-helical when it was bound to DNA. The regions of the proteins from which these peptides are derived, which are extended in solution, may fold, forming alpha-helices, upon binding to DNA.

Chronic pentobarbital administration alters gamma-aminobutyric acidA receptor alpha 6-subunit mRNA levels and diazepam-insensitive [3H]Ro15-4513 binding.

In order to study the chronic effects of pentobarbital, a positive GABAA receptor modulator, on the inverse agonist binding of the benzodiazepine site, binding of [3H]Ro15-4513 and levels of GABAA receptor alpha 6-subunit mRNA were investigated in the brains of pentobarbital-tolerant/dependent animals, using receptor autoradiography and in situ hybridization histochemistry in consecutive brain sections. Pentobarbital was administered to rats either 60 mg/kg, i.p., once, for acute treatment, or 300 micrograms/10 microliters/h i.c.v. continuously for 6 days via osmotic minipumps to render rats tolerant to pentobarbital. Rats assigned to the dependent group were sacrificed 24 h after discontinuance of pentobarbital infusion, while those assigned to the tolerant group were sacrificed at the end of infusion. The alpha 6 subunit mRNA was increased in the tolerant group only. Diazepam-insensitive [3H]Ro15-4513 binding was increased in the cerebellar granule layer of pentobarbital-tolerant and -dependent rats. No alterations in these parameters were observed in acutely treated animals. These data suggest that chronic pentobarbital treatment induced expression of alpha 6-subunit mRNA. This was in contrast to alpha 1- and gamma 2-subunit mRNA, which in tolerant animals are unchanged, but for which withdrawal triggers a surge in levels. Because the alpha 6-subunit is a major component of the diazepam-insensitive [3H]Ro15-4513 binding site, the increased diazepam-insensitive [3H]Ro15-4513 binding implied de novo synthesis of the receptor subunit protein.

An autoradiographic study of [3H]flunitrazepam binding sites in the brain of rat made tolerant to and dependent on pentobarbital.

The effects of continuous administration of pentobarbital on the benzodiazepine receptor labeled by [3H]flunitrazepam were investigated. Animals were made tolerant to pentobarbital by intracerebroventricular (i.c.v.) infusion with pentobarbital (300 micrograms/10 microliters/h) for 6 days through pre-implanted canulae connected to osmotic mini-pumps. The dependent rats were assessed 24 h after cessation of pentobarbital infusion. Changes in [3H]flunitrazepam binding were examined in 37 brain regions at a concentration of [3H]flunitrazepam of 1 nM. In subsequent saturation studies, the binding parameters Bmax and KD were also investigated in 17 brain regions, most of which showed significant changes in [3H]flunitrazepam binding in experiments using a fixed concentration of radioligand. The pentobarbital-tolerant rats showed a significant increase in Bmax with an increase in KD for [3H]flunitrazepam in the ventroposterior nucleus of thalamus. In the dependent rats, a significant increase in Bmax for [3H]flunitrazepam binding, without a change in KD, was observed in all layers of the frontal cortex, the caudate-putamen, olfactory tubercle, and some nuclei in thalamus, compared to those in the control. Increased [3H]flunitrazepam binding in the molecular layer of the olfactory bulb, the ventral pallidum, and the cerebellum of the pentobarbital dependent rats at a fixed concentration of [3H]flunitrazepam was also observed. There was no significant change in [3H]flunitrazepam binding in the hippocampus and several nuclei of the brain stem. These findings suggest that benzodiazepine receptors are closely involved in the development of tolerance to and dependence on pentobarbital. Further studies on changes in gamma-aminobutyric acid (GABA)A receptor subunit mRNA or the effects of pentobarbital on GABAA receptor phosphorylation would be necessary for an explanation of the precise mechanisms underlying the development of tolerance to and dependence on pentobarbital.

Pharmacology of barbiturate tolerance/dependence: GABAA receptors and molecular aspects.

Barbiturates are central nervous system depressants that are used as sedatives, hypnotics, anesthetics and anticonvulsants. However, prolonged use of the drugs produces physical dependence, and the drugs have a high abuse liability. The gamma-aminobutyric acidA (GABAA) receptor is one of barbiturates' main sites of action, and therefore it is thought to play a pivotal role in the development of tolerance to and dependence on barbiturates. Recent advances in the study of the GABAA receptor/chloride channel complex allow us to examine possible mechanisms that underlie barbiturate tolerance/dependence in a new light. In this minireview, we mainly focus on molecular and cellular aspects of the action of barbiturates and the possible mechanisms that contribute to development of tolerance to and dependence on barbiturates.

A novel quantitative receptor autoradiography and in situ hybridization histochemistry technique using storage phosphor screen imaging.

A new technique of image acquisition for quantitative receptor autoradiography and in situ hybridization histochemistry was developed using storage phosphor screen imaging. This method was at least 4-5 times faster than conventional film densitometry. Two of the advantages of the phosphor screen method are high sensitivity and wide linear range of response. Other aspects of this method were compared with those of conventional densitometry. Use of storage phosphor screen imaging will allow greatly increased speed of pharmacological screening procedures that utilize quantitative autoradiography.

Spectroscopic studies of Manduca sexta and Sesamia nonagrioides chorion protein structure.

The secondary structure of Manduca sexta and Sesamia nonagrioides chorion proteins has been studied in intact chorions using laser-Raman and Fourier transform infra-red (FTIR) spectroscopy and in a solution containing extracted and reassembled chorion proteins using circular dichroism (CD) spectroscopy. Laser-Raman and IR spectra suggest the predominance of antiparallel beta-pleated sheet structure in intact chorion proteins of both Lepidoptera species. The bands at 1673, 1674 cm-1 (amide I) and 1234-1238 cm-1 (amide III) in the laser-Raman spectra can best be interpreted as resulting from abundant antiparallel beta-pleated sheet structure. Analysis of the amide I band suggests that chorion proteins consist of 60-70% antiparallel beta-pleated sheet and 30-40% beta-turns. Supporting evidence for the prevalence of antiparallel beta-pleated sheet in chorion proteins was supplied using FTIR spectroscopy by the observation of a very intense absorption band at 1635 cm-1 (amide I) and of a weak band at 1530, 1525 cm-1 (amide II) from chorions of both species. Surprisingly, analysis of the CD spectra of extracted and reassembled chorion proteins suggests that, in solution, they retain a regular secondary structure most probably dominated by beta-pleated sheet. We therefore suggest that the prominent regular beta-sheet structure of chorion proteins may exist in solution and dictate the aggregation and polymerization process in vivo.

Changes in [3H]flunitrazepam binding in the brain of rats made tolerant to and dependent upon pentobarbital.

Changes in benzodiazepine binding sites labeled by [3H]flunitrazepan (FNZ) in twenty discrete brain regions of rats made tolerant to and dependent upon pentobarbital were examined. Animals were rendered tolerant by intracerebroventricular (i.c.v.) infusion with pentobarbital (300 micrograms/ 10 microliters/ hr for six days) through pre-implanted cannulae connected to osmotic mini-pumps. The pentobarbital dependence was assessed 24 hr after abrupt withdrawal from pentobarbital. In the tolerant rats, a significant increase in [3H]FNZ binding sites was found in layer IV of frontal cortex and the molecular layer of olfactory bulb. [3H]FNZ binding sites in the pentobarbital dependent rats were significantly increased in layers I-III and V-VI of frontal cortex, caudate-putamen, olfactory tubercle, globus pallidus and ventral pallidum, in addition to those observed in the tolerant group. There was, however, no significant difference in the hippocampus and several regions in the hindbrain in either pentobarbital-treated group. Taken together with characteristics of subtypes of benzodiazepine receptors and changes in GABA-benzodiazepine receptor complexes elucidated in our previous studies, these findings suggest that both types of benzodiazepine receptors are involved in the development of pentobarbital intoxication mediated by GABAA receptors.

In situ hybridization evidence of differential modulation by pentobarbital of GABAA receptor alpha 1- and beta 3-subunit mRNAs.

Tolerance to and withdrawal from pentobarbital were induced in rats by continuous intracerebroventricular infusion via subcutaneously implanted osmotic minipumps. In situ hybridization of GABAA receptor alpha 1- and beta 3-subunit mRNA was conducted using synthetic 3'-end 35S-dATP-labeled oligodeoxynucleotide probes. Results were quantified by film densitometry. In animals that were tolerant to pentobarbital, levels of alpha 1-subunit mRNA were decreased in hippocampus, superior colliculus, and inferior colliculus, but levels of beta 3-subunit mRNA were not affected. Dramatically increased levels of GABAA receptor subunit mRNA were observed in animals 24 h after withdrawal from chronic pentobarbital treatment. These increases occurred in cerebral cortex and cerebellum for the alpha 1 subunit and in cerebral cortex only for the beta 3-subunit. These data provide further support to the structural and pharmacological GABAA receptor heterogeneity in discrete brain areas. The observed changes of subunit expression may underlie, at least in part, the receptor up- and down-regulation observed in receptor ligand binding studies.

Preferential binding of H1e histone to GC-rich DNA.

We have investigated the binding of a pure H1 histone, the mouse variant H1e, to a 214 bp fragment of DNA from pBR322. Binding was monitored by observing the effects of the protein on melting of the DNA and by a gel-mobility-shift assay. We found, using this highly purified system, that H1e protein binds preferentially and cooperatively to the GC-rich region of the DNA. A chemically synthesized peptide containing 25 residues, corresponding to a region of the carboxyl-terminal domain of H1e, shows the same sequence preference but does not exhibit cooperativity.

Differential effects on GABAA receptor gamma 2-subunit messenger RNA by tolerance to and withdrawal from pentobarbital--an in situ hybridization study.

The heterogeneity of the GABAA receptors has been confirmed structurally and functionally. The present study demonstrates the pharmacological heterogeneity of the GABAA receptors. Rats were rendered tolerant to pentobarbital by continuous intracerebroventricular infusion via osmotic minipumps and abruptly withdrawn from pentobarbital. In situ hybridization of mRNA coding for the GABAA receptor gamma 2-subunit showed decreases of mRNA levels in superior and inferior colliculus in pentobarbital tolerant rats compared to rats in withdrawal. In rats 24-hr after withdrawal from pentobarbital, increases of mRNA levels in neocortex, piriform cortex and in granular and Purkinje cell layers of the cerebellum were observed. These results indicate the fast adaptation of GABA synapses in response to abrupt withdrawal from chronic pentobarbital treatment. The differential responsiveness seen in different areas further confirms the pharmacological heterogeneity of the GABAA receptors. The observed increases and decreases of mRNA may underlie, at least in part, the previously reported changes in Bmax of GABAA receptor ligand binding sites.

Secondary structure of synthetic peptides derived from the repeating unit of a giant secretory protein from Chironomus tentans.

The secretory proteins of Chironomus tentans larvae, which are used to construct underwater feeding and pupation tubes, assemble into complexes in vitro. Members of a family of 1000 kDa proteins, the spIs, appear to form the fibrous backbone of the assembled complexes. The spIs consist of a core of tandemly repeating units of 60 to 90 amino acids that can be subdivided into two regions: the subrepeat region, made up of short internal repeats, and the constant region, which lacks simple subrepeats. We have synthesized peptides representative of the constant and subrepeat regions of one of the spIs, and have examined their secondary structure using Fourier transform IR and CD spectroscopy. The IR spectrum of the constant peptide indicates that this peptide has alpha-helical regions and beta-turns. The CD spectrum confirms this. The IR spectrum of the subrepeat peptide is similar to that of the poly(Gly)II helix, and also may indicate the presence of beta-turns. The CD spectrum is consistent with this helical structure. Extrapolation of these results to intact spIs is in agreement with secondary structure prediction and modeling studies. Our results indicate that the alpha-helices and poly(Gly)II-like helices are not arranged as coiled-coils, which are often found in fibrous proteins. We suggest that these structural elements may be in an unusual arrangement in the spIs, organized as alternating alpha-helices and poly(Gly)II or collagen-like helices, interspersed with beta-turns.

Disassembly and reassembly in vitro of complexes of secretory proteins from Chironomus tentans salivary glands.

The secretory proteins of Chironomus tentans larvae form insoluble fibers that are spun into threads used to construct underwater feeding and pupation tubes. We began in vitro studies of the mechanism of assembly into fibers, the structure of the assembled proteins, and the contribution of individual proteins to the assembled structure. From measurements of turbidity and electron micrographs, we observed that the secretory proteins were isolated as complexes. These complexes are most likely at initial stages of assembly; further assembly into insoluble fibers must occur in vivo. Denaturation and reduction disrupted the complexes, and removal of the denaturing and reducing agents resulted in reassembly of the complexes. The circular dichroic spectrum of the complexes indicated that the assembled proteins had the tertiary structure alpha + beta. The largest secretory proteins were purified and shown to have both similar morphology, using electron microscopy, and a similar dichroic spectrum to that of the native complexes. We concluded that the large secretory proteins form the fibrous backbone of the complexes that we observe.

Effects of anti-C23 (nucleolin) antibody on transcription of ribosomal DNA in Chironomus salivary gland cells.

Protein C23 (also called nucleolin or 100-kDa nucleolar protein) is a major nucleolar phosphoprotein involved in ribosome biogenesis. To determine the effects of protein C23 on preribosomal RNA (pre-rRNA) synthesis anti-C23 antiserum was microinjected into nuclei of Chironomus tentans salivary glands. Transcription was measured by incubation of the glands with 32P-labeled RNA precorsors followed by microdissection of nucleoli, RNA extraction, and electrophoretic analyses. Injection of the anti-C23 antibody caused a 2- to 3.5-fold stimulation of 32P incorporation into 38S pre-rRNA. No stimulation was observed in salivary glands injected with preimmune serum or antiserum preabsorbed with protein C23. The stimulatory effect was selective for pre-rRNA as indicated by the lack of stimulation of 32P incorporation into extranucleolar RNA. Injection of the antiserum produced little or no effect on pre-RNA processing as measured by the relative amounts of 32P-labeled intermediate cleavage products of pre-rRNA in stimulated versus control glands. When protein extracts of Chironomus tentans salivary gland nuclei were probed on Western blots with anti-C23 antibody the predominant cross-reacting species was a 110-kDa polypeptide which had an electrophoretic mobility similar to that of protein C23. These results suggest that protein C23 not only is involved in ribosome assembly but also plays a role in regulating the transcription of the preribosomal RNA.

Isolation and characterization of a mouse fully replication-dependent H1 gene within a genomic cluster of core histone genes.

We have used an oligonucleotide complementary to a sequence coding for the conserved central globular domain of H1s to screen a mouse genomic library for H1 genes. We then used a series of universal histone oligonucleotides to identify five different H1 genes which were linked to core histone genes. We characterized one of the H1 genes which was linked to an H2a, an H2b, an H3, and an H4 histone gene. This characterization involved: 1) sequencing of the coding region of the gene and several hundred base pairs of flanking region. 2) Comparison of this sequence to other H1 sequences from other organisms. This sequence analysis clearly showed that the gene coded for an H1 and identified H1 consensus sequences in the 5'- and 3'-flanking region. 3) Mapping of the 5'- and 3'-ends of the mRNA complementary to this gene by S1 nuclease analysis. 4) Identifying this gene and an adjacent H3 gene as being of the fully replication-dependent expression class, by measuring changes in the steady state levels of their mRNAs in the presence of hydroxyurea and during differentiation of murine erythroleukemia cells.