Slide-mounted Rat Brain Sections Research Articles

Preface

Preface

Preface

Preface

Immunocytochemical detection of regional protein changes in rat brain sections using computer-assisted image analysis.

We used several approaches to assess the reliability and sensitivity of computer-assisted densitometry to detect regional changes in tissue antigen content as a function of immunohistochemical staining density. We designed a model system to mimic variations in antigen concentration in postfixed, slide-mounted rat brain sections by varying the ratios of conjugated (biotinylated) to unconjugated secondary antibody. Antigen concentration was also varied in tissue discs made from mixing rat brain homogenate with increasing amounts of tissue embedding compound. The monoclonal antibody bd-17 to the beta2/3 subunit of the GABAA receptor was used as the primary antibody. Immunostaining density was visualized with diaminobenzidine (DAB). There was a significant, positive linear relationship (r = 0.97-0.99) between immunostaining intensity and antigen concentration. With this approach, changes in antigen content of less than 10%, as reflected in immunostaining intensity, were detectable in brain sections. The low degree of variability in measures of regional variation in immunostaining in sections from naive rats (n = 7) suggested that the method was suitable for quantitative analysis and indicated the reliability of the method. This systematic study of the utility of computer-assisted image analysis for semiquantitative immunohistochemical analysis found the method to be both reliable and sensitive.

Flunitrazepam increases the affinity of the GABA A receptor in cryostat-cut rat brain sections

We have recently observed autoradiographically the benzodiazepine facilitation of [ 3 H]GABA binding to cryostat-cut slide-mounted rat brain sections (Bristow and Martin, 1989). Since cryostat-cut sections are arguably less disrupted than homogenised membranes, we chose to use this technique to study the effect of flunitrazepam on the [ 3 H]GABA equilibrium binding parameters

125I]-Bolton-Hunter scyliorhinin II: A novel, selective radioligand for the tachykinin NK3 receptor in rat brain

The cyclic tachykinin scyliorhinin II (SCYII) has high affinity for the [neurokinin B (NKB)-preferring] NK3 receptor. SCYII was iodinated using [ 125I]-Bolton-Hunter reagent and the product BHSCYII purified using reverse phase HPLC. In rat brain membranes, binding of BHSCYII and of the relatively unselective radioligand [ 125I]-Bolton-Hunter eledoisin (BHELE) was saturable, reversible and to an NK3 site. In competition studies, the rank order of potency in inhibiting binding of BHSCYII and BHELE was: SCYII ≥ [MePhe 7]-NKB ≈ senktide > NKB ≥ kassinin ≥ eledoisin > [Pro 7]-NKB > neurokinin A > neuropeptide K ≥ substance P > [Sar 9,Met(O 2) 11]-substance P. In “cold” saturation experiments, binding of BHELE occurred to a single class of high affinity sites (K D, 18.6±0.91 nM). Binding of BHSCYII was of greater affinity than for BHELE and could be resolved into a high (K D, 1.33±0.98 nM; 27% of sites) and low affinity (K D, 9.84±2.75; 73% of sites) component. The total number of binding sites was similar for both radioligands (BHSCYII, 8.27±0.98; BHELE, 7.94±0.32 fmol/mg wet weight). In vitro autoradiography in slide-mounted sections of rat brain showed identical binding patterns for both radioligands (100 pM), with dense binding localized predominantly to the cortex, Ammon's horn field 1, premammillary nuclei and interpeduncular nucleus. In membranes from several peripheral tissues, specific binding of BHELE was much higher than for BHSCYII, suggesting few NK3 receptors in the periphery. In comparison with BHELE, BHSCYII exhibits lower nonspecific binding (15%), better selectivity and higher affinity for the tachykinin NK3 receptor in rat brain.

Demonstration of high- and low-affinity β-adrenergic receptors in slide-mounted sections of rat and human brain

Studies using isolated cell membranes have shown that β-adrenergic receptors exist in two interconvertible conformations, one with high affinity for agonists and the other with low affinity. Guanine nucleotides and sodium shift high-affinity receptor sites into low-affinity sites. We sought to demonstrate affinity states of the β-adrenergic receptor in slide-mounted sections of rat and human postmortem brain, and to determine using quantitative receptor autoradiography the regional distribution in the brain of high-affinity receptors relative to the total population of β-adrenergic receptors. The β-adrenergic agonist isoproterenol inhibited the binding of 125I-iodopindolol to slide-mounted sections of rat forebrain and human premotor cortex in a biphasic manner. Approximately 80% of the β-receptor sites in both rat and human brain showed high-affinity for isoproterenol (7–14 nM). Treatment with 0.1 mM non-hydrolyzable guanine nucleotide 5′-guanylylimidodiphosphate and 25 mM NaCl abolished high-affinity binding in rat brain sections and reduced it in human brain sections. These findings were confirmed by membrane studies using similar tissue samples. For autoradiographic studies, 17 nM isoproterenol displaced 71% of the high-affinity sites without affecting low-affinity β-adrenergic receptors. Digital subtraction was used to selectively visualize β-receptors in the high-affinity conformation. The proportion of β-adrenergic receptors in the high-affinity conformation differed significantly across rat brain regions and across human cortical layers. We conclude that β-adrenergic affinity states, presumably reflecting the interaction of the receptor with a G-protein, occur in slide-mounted sections of rat brain and persist in human postmortem material. Quantitative receptor autoradiographic analysis of receptor affinity states may provide a better index of receptor function than determination of binding site density alone. This work was previously presented in abstract form 3.

Localization and characterization of 35S-t- butylbicyclophosphorothionate binding in rat brain: an autoradiographic study

35S-t-butylbicyclophosphorothionate (TBPS) binding to slide-mounted rat brain sections was characterized for subsequent autoradiographic analysis. Cortical brain mash slices, preincubated with EDTA to remove endogenous GABA, were used for biochemical characterization. Steady state for 35S-TBPS binding was reached by 3 hr of incubation at 22 degrees C. The association rate constant (K1) and dissociation rate constant (K2) were 0.377 min-1 microM-1 and 0.011 min-1, respectively. Dissociation was monophasic and slow (t1/2 = 80 min). The kinetically derived KD was 29.4 nM. Scatchard analysis indicated a single population of binding sites with a KD of 21.0 +/- 2.2 nM and a Bmax of 1.59 +/- 0.13 pmol/mg protein. Both picrotoxin and muscimol inhibited 35S-TBPS binding completely with IC50s of 251 +/- 13 nM and 203 +/- 41 nM and nHs of 0.98 and 1.4, respectively. The distribution of 35S-TBPS binding sites in the rat brain resembles that of other ligands that bind to GABAA receptor complex with some regionally specific differences. Regions with a high degree of 35S-TBPS binding included the inferior colliculus, medial septal nucleus, central and paracentral nuclei of the thalamus, olfactory tubercle, zona incerta, dentate gyrus, and substantia nigra. 35S-TBPS preferentially bound to the molecular vs granular layer of the cerebellum. Omission of the preincubation markedly but variably decreased 35S-TBPS binding. The greatest regional decreases occurred in areas with a high degree of GABA synthesis. In addition, 35S-TBPS binding was inhibited to different degrees in the cell layers of the cerebellum. The addition of 1 microM GABA to the incubation medium of preincubated slices also produced variable decreases in 35S-TBPS binding to cerebellar layers. These findings support previous studies that demonstrate GABAA receptor heterogeneity. Our study confirms the suitability of 35S-TBPS for use as a ligand in autoradiography and demonstrates that the distribution of 35S-TBPS binding sites is significantly influenced by the preincubation-incubation conditions used.

Chronic administration of morphine and naltrexone up-regulate μ-opioid binding sites labeled by [ 3H][D-Ala 2, MePhe 4, Gly-ol 5]enkephalin: further evidence for two μ-binding sites

A variety of data support the hypothesis of an opiate receptor complex composed of distinct, yet interacting μ and δ binding sites (termed μ cx and δ cx to indicate binding sites ‘in the complex’), in addition to independent μ and δ binding sites, termed μ ncx and δ ncx, to indicate binding sites ‘not in the complex’. Ligand binding studies using membranes and slide-mounted sections of rat brain support the hypothesis that the irreversible μ-antagonist β-funaltrexamine (FNA) selectively alkylates the opiate receptor complex, altering the binding of μ agonists to the μ cx binding site and the binding of [ 3H][D-Ala 2, D-Leu 5]enkephalin to the δ cx site. Previous studies demonstrated that the chronic administration of morphine to rats selectively ‘upregulates’ the opiate receptor complex. In contrast, the chronic administration of naltrexone upregulates several types of opioid receptors, including κ, the δ ncx binding site, and multiple binding sites labeled by μ agonists. A prediction based upon these observations is that, using [ 3H][D-Ala 2, MePhe 4, Gly-ol 5]enkephalin to label μ binding sites, chronic morphine should upregulate only the μ cx binding site, whereas chronic naltrexone should additionally up-regulate the μ ncx binding site. In this study we test and confirm this hypothesis, using sensitivity to FNA to define the μ cx binding site. The implications of these data for models of the opioid receptors and the mechanism(s) of tolerance and dependence are discussed.

A comparative autoradiographic study of the distributions of substance P and eledoisin binding sites in rat brain

The relative potencies tachykinin peptide analogs competing for binding of [ 125I]Bolton Hunter-conjugated substance P ([ 125I]BH-SP) or [ 125I]Bolton Hunter-conjugated eledoisin ([ 125I]BH-ED) in slide-mounted rat brain sections are very different, indicating the presence of two distinct tachykinin binding sites. The structure-activity profiles resemble those described in peripheral bioassay studies in which two tachykinin receptors have been postulated. Autoradiography of the two iodinated ligands bound with selective and one-site in vitro incubation conditions shows two discrete and distinctly different distribution patterns in brain. Binding sites for [ 125I]BH-ED are densely distributed in the accessory olfactory bulb, intermediate layers of the cerebral neocortex, portions of the hippocampal CA fields, hypothalamic supraoptic and paraventricular nuclei, central portions of the interpenduncular nucleus, sphenoid nucleus, medial subdivision of the solitary tract complex, and the substantia gelatinosa of the spinal cord. Binding sites for [ 125I]BH-SP are present in many of these same structures, but the densities and distribution patterns are different. In addition, [ 125I]BH-SP binds in numerous structures not labeled by [ 125I]BH-ED. Neither pattern matches the locations of terminations of endogenous tachykinin pathways marked by immunohistochemistry. The results suggest that it would be inappropriate to name brain tachykinin receptors according to the endogenous ligand which binds with highest affinity.

Quantitative autoradiography of [3H]indalpine binding sites in the rat brain: I. Pharmacological characterization.

The binding of [3H]indalpine (4-[2-(3-indolyl)]ethyl piperidine) to slide-mounted sections of rat brain has been characterized. This 5-hydroxytryptamine (5-HT) uptake blocker binds to sections with high affinity (KD approximately 1 nM). The binding is saturable, and can be displaced by the addition of clomipramine (1 microM). Other drugs inhibiting the uptake of 5-HT also have the capacity to inhibit the binding of [3H]indalpine. A significant correlation (r = 0.86) was found between the capacity of these compounds to inhibit the uptake of 5-HT and their potencies as inhibitors of [3H]indalpine binding. Binding was Na+ - and Cl- -dependent and was inhibited competitively by 5-HT. Furthermore, electrolytic lesions of the dorsal raphe or medial forebrain bundle, which cause a degeneration of 5-HT cell bodies and fibers, respectively, resulted in a 30-40% reduction in the binding of [3H]indalpine. [3H]Indalpine binds to the 5-HT uptake recognition sites in a different manner from imipramine-like compounds.

Autoradiographic distribution of nicotine receptors in rat brain

The autoradiographic visualisation of 90%-specific tritiated nicotine binding to slide-mounted sections of rat brain is reported. Tritiated nicotine bound with high affinity (nanomolar K d ) and was selectively displaced by nicotinic agonists (e.g l-nicotine ∼ ACh > d-nicotine). The strikingly discrete distribution pattern obtained deviates from that of alpha-bungarotoxin, and suggests several possible roles for nicotinic transmission in the brain.

Autoradiographic localization of a binding protein(s) specific for prostaglandin D2 in rat brain.

The specific [3H]prostaglandin (PG) D2 binding was detected by using the slide-mounted sections of rat brain fixed by perfusion with 2% paraformaldehyde. The binding was reversible, saturable, high affinity, Na+ dependent, and highly specific for PGD2. These binding characteristics are essentially similar to those observed with the synaptic membrane of rat brain as previously reported. Using autoradiographic image analyses by computerized densitometry and color coding, we visualized the localization of [3H]PGD2 binding in rat brain. A high density of the binding sites was observed in the cerebral cortex, preoptic area, amygdala, hypothalamic nuclei (arcuate nucleus, ventromedial nucleus, and posterior hypothalamic nucleus), thalamic nuclei (reuniens nucleus and rhomboid nucleus), hippocampus, pineal body, and cerebellar cortex. The binding was not significantly observed in the striatum and also was negative in the white matter, arachnoid membranes, and vasculatures.

Characterization and visualization of cholecystokinin receptors in rat brain using [ 3H]pentagastrin

[ 3H]Pentagastrin binds specifically to an apparent single class of CCK receptors on slide-mounted sections of rat brain (K D=5.6 nM; B max=36.6 fmol/mg protein). This specific binding is temperature-dependant and regulated by ions and nucleotides. The relative potencies of C-terminal fragments of CCK-8(SO 3H), benzotript and proglumide in inhibiting specific [ 3H]pentagastrin binding to CCK brain receptors reinforce the concept of different brain and pancreas CCK receptors. CCK receptors were visualized by using tritium-sensitive LKB film analyzed by computerized densitometry. CCK receptors are highly concentrated in the cortex, dentate gyrus, granular and external plexiform layers of the olfactory bulb, anterior olfactory nuclei, olfactory tubercle, claustrum, accumbens nucleus, some nuclei of the amygdala, thalamus and hypothalamus.