Quantitative receptor autoradiography: tissue defatting eliminates differential self-absorption of tritium radiation in gray and white matter of brain

A four-fold greater absorption (quenching) of tritium emissions by white matter relative to gray matter produces a false ‘contrast’ effect in autoradiographs of³H-ligand binding to brain sections. The differential absorption is eliminated by tissue defatting prior to autoradiographic exposure.     

High affinity serotonin binding sites in human brain: a comparison of cerebral cortex and basal ganglia

Summary The high-affinity binding of³H-serotonin and³H-DP-AT was studied in membrane preparations and tissue sections of cerebral cortex and basal ganglia of human brain. In tissue sections,³H-serotonin bound to sites present at high density in the cerebral cortex, hippocampus and basal ganglia.³HDPAT bound predominantly to the outer layers of the cerebral cortex and the hippocampus, no significant binding was observed in the basal ganglia. In cortical membranes³H-serotonin bound to a heterogeneous population of sites, one of which was similar to a binding site labelled by³H-DPAT (the putative 5HT-1A receptor).³H-serotonin binding to membranes prepared from the putamen displayed low affinity for DPAT, mesulergine and spiperone, RU 24969 was considerably less potent than serotonin itself. This³H-serotonin binding site does not have the properties of those sites described in rodent brain (5 HT-1 A, 5 HT-1 B, and 5 HT-1 C), and may represent a novel serotoninergic binding site.     

Autoradiographic distribution of [3H]YM-09151-2, a high-affinity and selective antagonist ligand for the dopamine D2 receptor group,in the rat brain and spinal cord

We determined the regional distribution of the dopamine D₂ receptor group in the rat central nervous system by quantitative receptor autoradiography with a high-affinity and selective antagonist, [³H]YM-09151-2. Saturation and competition experiments demonstrated that the binding of [³H]YM-09151-2 to striatal sections was saturable (Bmax=37.3 fmol/section), of high affinity (Kd=0.315 nM), and was inhibited selectively by prototypic D₂ ligands. The anatomical localization of binding sites was determined by comparison of autoradiograms and the original ³H-ligand-exposed sections stained with cresyl violet. Very high levels of [³H] YM-09151-2 binding were found in the caudate-putamen, nucleus accumbens, tuberculum olfactorium and the insula of Calleja, to each of which midbrain dopaminergic neurons project densely. High levels of binding were also observed in other regiions rich in dopaminergic neurons and fibers including the glomerular layer of the olfactory bulb, the intermediate lobe of the pituitary, lateral septum, substantia nigra pars compacta, interfascicular nucleus, dorsal raphe nucleus, locus coeruleus, and nucleus of the solitary tract. Some regions poor in dopaminergi innervation, however, had high levels of [³H]YM-09151-2 binding including the molecular layer of gyrus dentatus, all layers of CA1 and the nonpyramidal layer of CA4 of hippocampus, and the deeper layer of medial entorhinal cortex. Motor neurons present in brainstem motor nuclei and spinal ventral horn were also strongly labeled. Neocortical, cerebellar, and thalamic regions had low levels of binding, except lobules 9–10 of the cerebellum, the olivary pretectal nucleus, zona incerta and lateral mammillary nucleus, in which moderate to high levels of binding were detected. Our findings concerning the widespread but region-specific localization of [³]YM-09151-2 binding sites in the brain and spinal cord may prove useful for analyzing varoius dopaminergic functions in the central nervous system. © 1994 Wiley-Liss, Inc.     

Opioid receptors in midbrain dopaminergic regions of the rat II. Kappa and delta receptor autoradiography

Summary Opiates and opioid peptides are known to influence the dopaminergic (DA) neurons in the midbrain. The purpose of this study was to map and quantify the density of kappa and delta opioid receptor subtypes in the retrorubral field, substantia nigra, and ventral tegmental area and related nuclei, which contain DA nuclei A8, A9, and A10, respectively. Sections through the rostral-caudal extent of the rat midbrain were stained with an antibody against tyrosine hydroxylase, as a DA cell marker, and comparable sections were processed for in vitro receptor autoradiography using the kappa-selective ligand, U-69593, and the delta-selective ligand, D-Pen², D-Pen⁵-enkephalin. In general, both kappa and delta ligands exhibited low levels of specific binding in regions occupied by the midbrain DA neurons.Kappa binding (4–8 fmol/mg tissue) was high throughout the rostral-caudal extent of the substantia nigra, in rostral portions of the ventral tegmental area, and in the nucleus paranigralis; low binding occurred in the retrorubral field and central linear nucleus raphe.Delta binding (6–18 fmol/mg tissue) was high in the caudal portion of the substantia nigra pars reticulata, and in the medial terminal nucleus of the accessory optic system (a region previously shown to contain DA dendrites). The kappa and delta receptor binding is heterogeneously distributed in regions occupied by midbrain dopaminergic neurons, and several fold lower than the binding of mu opioid receptors in the same brain regions.     

Laminar distribution of MK-801, kainate,AMPA, and muscimol binding sites in cat visual cortex: A developmental study

We used quantitative autoradiography to determine whether the development of glutamate receptors correlates with the sensitive period for monocular deprivation in the visual cortex. To study glutamate receptors, we incubated sections of cat visual cortex with tritiated (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10imine-maleate (MK-801), tritiated kainate, and tritiated amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA). [³H]MK-801 is a noncompetitive ligand for the N-methyl-D-aspartate (NMDA) receptor. [³H]kainate and [³H]AMPA are competitive ligands for non-NMDA receptors. We used [³H]muscimol, which binds to GABA_A receptors, so that we would have one control ligand that binds to a nonglutamate receptor. When all layers were combined, the results confirmed our previous studies with homogenate binding. [³H]MK-801 and [³H]kainate binding were significantly greater at 42 days than at earlier or later times. [³H]AMPA and [³H]muscimol binding did not show such a peak. This suggests that MK-801 and kainate binding sites are more likely to be involved in plasticity than are AMPA and muscimol binding sites. In layers 2/3, MK-801 had the greatest age-dependent changes; in layers 5 and 6, kainate binding changed most with age. This suggests that the mechanisms of plasticity may vary with cortical layer. © 1996 Wiley-Liss, Inc.     

Substance P receptors: Localization by light microscopic autoradiography in rat brain using [H]SP as the radioligand

Substance P (SP) is a putative neurotransmitter in both the peripheral and central nervous systems. In the present report we have used a modification of the Young and Kuhar technique to investigate some of the SP receptors binding properties and the distribution of SP receptors in rat brain. Tritiated SP ([³H]SP) adsorbed extensively to glass but this adsorption was greatly reduced by preincubating the slide-mounted tissue sections in a solution containing the cationic polymer polyethylenimine. [³H]SP was found to bind to rat tissue in a saturable fashion with a B_max of 14.7 fmol/mg tissue wet weight and a K_d of 1.1 nM. The rank order of potencies for displacing [³H]SP binding from rat tissue sections was SP > SP sulphoxide > DiMeC₇ > Eledoisin >0. SP(5–11) > SP(COOH) > SP(1–9) amide.Using autoradiography coupled with LKB tritium-sensitive Ultrofilm or the dry emulsion-coated coverslip technique the distribution of [³H]SP binding sites was found to be very dense within the olfactory bulb, amygdalo-hippocampal area and the nucleus of the solitary tract. Heavy concentrations of receptors were observed in the septum, diagonal band of Broca, striatum, subiculum, hypothalamus, locus coeruleus, parabrachial nucleus and lobule 9 and 10 of the cerebellum. Moderate to low concentrations of receptors were observed in the cerebral cortex, globus pallidus, raphe nuclei and the trigeminal nucleus. Very low densities were observed in most aspects of the dorsal thalamus, substantia nigra and cerebellum (other than lobule 9 and 10).Comparisons of the present data with SP peptide levels indicate that in some areas of the brain there is a rough correlation between peptide and receptor levels. However, in other brain areas (olfactory bulb, globus pallidus and substantia nigra) there is little obvious correlation between the two.     

Quantitative autoradiography of muscarinic cholinergic receptors in the rat brain

Using the in vitro autoradiographic technique with tritium-sensitive LKB sheet film and the liquid scintillation counting method, the distribution and the binding parameters of the muscarinic cholinergic receptors (MChR) were determined in various discrete regions of the rat brain. The results obtained in the present study were as follows:

(1) Specific binding of [³H]QNB to the slide-mounted tissue sections increased slowly when incubated at room temperature; saturation occurred 2 h after incubation. Only 23% of [³H]QNB bound to the tissue section was dissociated 5 h after the addition of 20 μM atropine to the medium. These findings were very different from those obtained in the study using the tissue homogenates.

(2) The regional distribution of MChR was determined using both autoradiographic and liquid scintillation counting methods. The distribution of MChR was heterogeneous, with highest densities in the striatum and nucleus accumbens and lowest in the globus pallidus, nucleus interpenduncularis and nucleus septi. Moreover, MChR were unevenly distributed within the subfields of each region.

(3) In saturation binding studies using the slide-mounted tissue sections of 20 μm thickness the (K_d)_app-values were similar but not exactly identical in 5 discrete regions, i.e. the striatum, somatosensory cortex, hippocampus (the subiculum + CA1 field), nucleus accumbens and gyrus dentatus, determined in the present study. The (K_d)_app-value of each region was about 700 pM which was about 20 times higher than that obtained in the study using the tissue homogenates. However (K_d)_app-values obtained with 5 and 10 μm tissue sections were approximately 3-fold lower.

Subchronic treatment with phencyclidine in adolescence leads to impaired exploratory behavior in adult rats without altering social interaction or N‐methyl‐D‐aspartate receptor binding levels

Although both the onset of schizophrenia and human phencyclidine (PCP) abuse typically present within the interval from adolescence to early adulthood, the majority of preclinical research employing the PCP model of schizophrenia has been conducted on neonatal or adult animals. The present study was designed to evaluate the behavioral and neurochemical sequelae of subchronic exposure to PCP in adolescence. Male 35–42‐day‐old Sprague Dawley rats were subcutaneously administered either saline (10 ml · kg^?1) or PCP hydrochloride (10 mg · kg^?1) once daily for a period of 14 days (n = 6/group). The animals were allowed to withdraw from treatment for 2 weeks, and their social and exploratory behaviors were subsequently assessed in adulthood by using the social interaction test. To examine the effects of adolescent PCP administration on the regulation of N‐methyl‐D‐aspartate receptors (NMDARs), quantitative autoradiography was performed on brain sections of adult, control and PCP‐withdrawn rats by using 20 nM ³H‐MK‐801. Prior subchronic exposure to PCP in adolescence had no enduring effects on the reciprocal contact and noncontact social behavior of adult rats. Spontaneous rearing in response to the novel testing arena and time spent investigating its walls and floor were reduced in PCP‐withdrawn animals compared with control. The long‐term behavioral effects of PCP occurred in the absence of persistent deficits in spontaneous locomotion or self‐grooming activity and were not mediated by altered NMDAR density. Our results document differential effects of adolescent PCP administration on the social and exploratory behaviors of adult rats, suggesting that distinct neurobiological mechanisms are involved in mediating these behaviors. © 2014 Wiley Periodicals, Inc.     

A new method for receptor autoradiography: [H]Opioid receptors in rat brain

Opioid receptors can be labeled with [³H]ligands in lightly fixed tissue sections mounted on microscope slides. The preparation of these sections does not seem to alter any of the known characteristics of opioid receptors. The light microscopic autoradiographic distribution of these binding sites can be observed if one attaches emulsion-coated coverslips to these slides to obtain autoradiograms. The distribution of [³H]diprenorphine binding sites determined by this in vitro method is identical to the distribution found in earlier studies utilizing in vivo labeling of opioid receptors. In addition, [³H]opioid peptide binding sites and [³H]dihydromorphine binding sites may be similar, perhaps identical, to those for [³H]diprenorphine, an opiate antagonist.

This method has several important advantages over earlier methods for determining the autoradiographic localization of receptors. It is possible, by washing, to reduce nonspecific binding to low levels. Since receptor labeling is performed with single tissue sections mounted on slides, one can examine different receptors in different but adjacent sections. One can use ligands that are not entirely suitable for in vivo labeling (For example, one can use [³H]peptides which do not normally cross the blood-brain barrier). One can perform autoradiographic studies after laveling tissues under a wide variety of conditions (For example, one can examine the effects of various ions and nucleotides on ligand binding distributions). This technique seems to be free of various artifacts, such as edge artifacts, which were common by techniques used in our laboratory earlier. Since one does not have to load an entire animal with radioactive ligand as one must with in vivo labeling, this approach is economically advantageous. One can use postmortem tissue, including that from humans to study receptor distribution with a high anatomical resolution.     

Laminar distribution of NMDA receptors in cat and monkey visual cortex visualized by [3H]-MK-801 binding

Glutamate is the major excitatory neurotransmitter of the mammalian central nervous system. Two major classes of glutamate receptors have been reported. The actions of glutamate on its N-methyl-D-aspartate (NMDA)-type receptor may underlie developmental and adult plasticity as well as neurotoxicity. The NMDA-type of glutamate receptor in cat and monkey visual cortex was visualized by means of in vitro receptor autoradiography with the noncompetitive NMDA-receptor antagonist [³H]-MK-801. The kinetics, performed on tissue sections, revealed an apparently single, saturable site with an approximate dissociation constant (KD) of 18.5 nM in cat and 15.9 nM in monkey visual cortex. Autoradiography, performed on frontal sections of cat and monkey visual cortex, revealed a heterogeneous laminar distribution of NMDA receptors. Cat areas 17,18,19, and the lateral suprasylvian areas exhibited a similar NMDA-receptor distribution. In these areas, NMDA receptors were most prominent in layer II and the upper part of layer III. In monkey striate cortex, NMDA receptors were primarily concentrated in layers II, upper III, IVc, V, and VI. In monkey secondary visual cortex, [³H]-MK-801 labeling was most prominent in layers II, V, and VI; whereas in the temporal visual areas included in this study layer II displayed the heaviest receptor labeling. In neither cat nor monkey could we observe significant differences in NMDA-receptor distribution between different retinotopic subdivisions within a single visual area. Neither did we detect any periodic changes in NMDA-receptor distribution that would correspond to the compartments defined by cytochrome-oxidase in monkey V1 and V2. © 1993 Wiley-Liss, Inc.     

Kappa opioid receptor agonists inhibit sigma-1 (σ1) receptor binding in guinea-pig brain, liver and spleen: autoradiographical evidence

The present study examined whether the kappa-opioid agonists U50,488H (trans-(±)-3,4-dichloro-N-methyl-N[-2-(1-pyrrolidinyl)cyclohexyl]-benzeacetamide methane sulphonate), bremazocine, spiradoline and ICI 197067 bind to a sites in guinea-pig tissues using in vitro, semi-quantitative receptor autoradiography and receptor binding, and compared the binding profile so obtained with those for several selective σ ligands. Guinea-pigs were killed and their brains, livers and spleens were removed, tissue sections cut and processed for σ binding site autoradiography using (+)-[³H]-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-[³H]-3-PPP), or tissue was wiped and determined by liquid scintillation. Serial slide-mounted sections were incubated with 9–10 concentrations (1 nM-10 μM) of kappa opioids and their potency to inhibit (+)-[³H]-3-PPP binding compared with that of the σ ligands haloperidol, DTG (1,3 di(o)-tolylguanidine), (+)-3-PPP, (+) and (−)pentazocine, SR 31742A and rimcazole (n = 3, duplicate determinations). Binding of (+)-[³H]-3-PPP to untreated, matched serial tissue sections was used as control. K_d values were estimated in brain, liver and spleen using quantitative, saturation binding analysis, IC₅₀ values were determined from the binding data obtained by slide wiping experiments for each drug, and K_i values were calculated using the Cheng-Prussoff equation. All four kappa opioids inhibited (+)-[³H]-3-PPP binding to σ₁-receptors with order of potency: brain: U50,488H = spiradoline > bremazocine > ICI 197067; liver: spiradoline > U50,488H > ICI 197067 > bremazocine; spleen: U50,488H > spiradoline > ICI 197067 > bremazocine. By comparison, the σ ligands inhibited (+)-[³H]-3-PPP binding to matched, serial slide-mounted brain tissue sections (similar results in liver and spleen) with order of potency: SR 31742A > haloperidol > (+)pentazocine . (+)-3-PPP > DTG > (−)pentazocine > rimcazole. (+)-[³H]-3-PPP autoradiography confirmed these binding data. It is concluded that the kappa opioids tested moderately inhibit (+)-[³H]-3-PPP binding to σ₁-receptors in guinea-pig brain, liver and spleen tissue with K_i values comparable to some selective σ ligands and therefore are not opioid selective.     

Kappa opioid receptor agonists inhibit sigma-1 (σ1) receptor binding in guinea-pig brain, liver and spleen: autoradiographical evidence

The present study examined whether the kappa-opioid agonists U50,488H (trans-(±)-3,4-dichloro-N-methyl-N[-2-(1-pyrrolidinyl)cyclohexyl]-benzeacetamide methane sulphonate), bremazocine, spiradoline and ICI 197067 bind to a sites in guinea-pig tissues using in vitro, semi-quantitative receptor autoradiography and receptor binding, and compared the binding profile so obtained with those for several selective σ ligands. Guinea-pigs were killed and their brains, livers and spleens were removed, tissue sections cut and processed for σ binding site autoradiography using (+)-[³H]-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-[³H]-3-PPP), or tissue was wiped and determined by liquid scintillation. Serial slide-mounted sections were incubated with 9–10 concentrations (1 nM-10 μM) of kappa opioids and their potency to inhibit (+)-[³H]-3-PPP binding compared with that of the σ ligands haloperidol, DTG (1,3 di(o)-tolylguanidine), (+)-3-PPP, (+) and (−)pentazocine, SR 31742A and rimcazole (n = 3, duplicate determinations). Binding of (+)-[³H]-3-PPP to untreated, matched serial tissue sections was used as control. K_d values were estimated in brain, liver and spleen using quantitative, saturation binding analysis, IC₅₀ values were determined from the binding data obtained by slide wiping experiments for each drug, and K_i values were calculated using the Cheng-Prussoff equation. All four kappa opioids inhibited (+)-[³H]-3-PPP binding to σ₁-receptors with order of potency: brain: U50,488H = spiradoline > bremazocine > ICI 197067; liver: spiradoline > U50,488H > ICI 197067 > bremazocine; spleen: U50,488H > spiradoline > ICI 197067 > bremazocine. By comparison, the σ ligands inhibited (+)-[³H]-3-PPP binding to matched, serial slide-mounted brain tissue sections (similar results in liver and spleen) with order of potency: SR 31742A > haloperidol > (+)pentazocine . (+)-3-PPP > DTG > (−)pentazocine > rimcazole. (+)-[³H]-3-PPP autoradiography confirmed these binding data. It is concluded that the kappa opioids tested moderately inhibit (+)-[³H]-3-PPP binding to σ₁-receptors in guinea-pig brain, liver and spleen tissue with K_i values comparable to some selective σ ligands and therefore are not opioid selective.     

Development of MK-801, kainate,AMPA, and muscimol binding sites and the effect of dark rearing in rat visual cortex

We used quantitative autoradiography to determine whether the development of glutamate receptors correlates with the plastic period for monocular deprivation in rat visual cortex. To study glutamate receptors, we incubated sections of rat visual cortex with tritiated (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10imine maleate (MK-801), tritiated kainate, and tritiated amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA). [³H]MK-801 is a noncompetitive ligand for the N-methyl-D-aspartate (NMDA) receptor. [³H]kainate and [³H]AMPA are competitive ligands for non-NMDA receptors. To compare glutamate binding sites with a nonglutamate binding site, we studied [³H]muscimol, which binds to γ-aminobutyric acid (GABA)_A receptors. [³H]MK-801 binding was maximal at postnatal day 26 (P26) and decreased in adulthood. [³H]AMPA binding was maximal at P18. [³H]kainate binding and [³H]muscimol binding were not age dependent. Dark rearing partially prevented the age-dependent decrease in [³H]MK-801 binding but had no effect on [³H]kainate or [³H]AMPA binding. Dark rearing decreased muscimol binding in adult animals. These results suggest that NMDA receptors, but not other glutamate receptors or GABA_A receptors, are likely to be critical for developmental plasticity in rat visual cortex. J. Comp. Neurol. 383:73–81, 1997. © 1997 Wiley-Liss, Inc.     

Neuropeptide FF receptors in rat brain: A quantitative light-microscopic autoradiographic study using [125I][D.Tyr1, (NMe)Phe3]NPFF

The anatomical localization of neuropeptide FF receptors was examined by in vitro autoradiography techniques in rat brain sections by using [¹²⁵I][D.Tyr¹, (NMe)Phe³]NPFF. The specific binding of [¹²⁵I][D.Tyr¹, (NMe)Phe³]NPFF reached 90% of the total binding at 0.05 nM in rat spinal cord sections. Up to 40% of the specific binding of [¹²⁵I][D.Tyr¹, (NMe)Phe³]NPFF to rat spinal cord sections was still detectable following fixation with glutaraldehyde. Afterwards, the distribution of NPFF receptors was studied by light microscopy and their densities by microdensitometry with an image analysis system. In the light microscope, [¹²⁵I][D.Tyr¹, (NMe)Phe³]NPFF labelling appeared more or less uniformly distributed over nerve-cell bodies and surrounding neuropil. High concentrations of binding sites were detected in the presubiculum, parafascicular thalamic nucleus, gracile nucleus, spinal trigeminal tract nucleus, and a number of brainstem nuclei, with virtually no labelling in the cerebellum. In several areas a rostrocaudal gradient of sites concentration was observed. Neuropeptide FF receptors are well-placed to control incoming sensory and autonomic information processing. In contrast, the more recently developed areas of the forebrain possessed low density of sites. The distribution of [¹²⁵I][D.Tyr¹, (NMe)Phe³]NPFF binding sites should suggest anatomical substrates for the actions of neuropeptide FF. © 1996 Wiley-Liss, Inc.     

[H]Spiperone binding sites in brain: autoradiographic localization of multiple receptors

[³H]Spiperone ([³H]SP) binding sites were localized by light microscopic autoradiography, after in vitro labeling. The kinetic and pharmacological characteristics of these binding sites were studied in slide-mounted sections of rat forebrain, and optimal labeling conditions were defined. Autoradiograms were obtained by apposing emulsion-coated coverslips to labeled sections. Diffetrential drug sensitivity allowed the selective displacement of [³H]SP from dopamine receptors by ADTN, from serotonin receptors by cinanserin, from both by haloperidol and from unique spiperone sites by unlabeled spiperone. The various sites presented a differential anatomical localization. For example, only dopaminergic sites were found in the glomerular laver of the olfactory bulb; only serotonergic sites were found in lamina IV of the neocortex, and a high concentration of unique spiperone sites was found in parts of the hippocampus.     

Quantitative analysis of [H]spiroperidol binding to rat forebrain sections: Plasticity of neostriatal dopamine receptors after nigrostriatal injury

The binding of [³H]spiroperidol to rat coronal sections in vitro was investigated using two procedures: swabbing studies, in which the tissue sections are wiped from the microscope slides after incubation in the presence of [³H]spiroperidol, and autoradiographic studies, in which the autoradiographic negatives are analyzed using computer-assisted densitometry. In the swabbing studies, the pharmacological and kinetic properties of butaclamol-displaceable binding were investigated, and the following results suggest that [^3H]spiroperidol binds specifically to only a single site within the basal forebrain of tissue sections and that the site is the dopamine D-2 receptor. (1) The pseudo-first order and first order plots for the rate of association to and dissociation from tissue sections appeared to be linear. (2) Dopamine antagonists, such asd haloperidol and butaclamol, were much more effective than dopamine agonists or the serotonin S-2 ligand, ketanserin, in inhibiting [³H]spiroperidol binding. (3) The ability of dopamine agonists to inhibit [³H]speroperidol binding was markedly reduced by the guanine nucleotide, Gpp(NH)p. (4) Saturation analysis of specific [³H]speroperidol binding revealed aK_d and B_max of 0.93 nM and 447 fmol/mg protein, and a Hill coefficient of 1.05. The findings are also compatible with the possibility that [³H]spiroperidol binds to several sites that have identical affinities for this ligand.Densitometric studies were used to assess the effect of lesions on [³H]spiroperidol binding in the neostriatum. Intrastriatal injection of kainic acid substantially reduced 1 μM (+)—butaclamol-displaceable binding, indicating that the receptors are in large part on intrinsic striatal neurons. Neostriatal [³H]spiroperidol binding was investigated 7 days after destruction of the mesotelencephalic dopamine system by the ventral tegmental injection of 6-hydroxydopamine. As determined by saturation analysis, the average values forK_d and B_max were 0.66 nM and 1212 fmol/mg protein in the intact striatum, and 0.82 nM and 1504 fmol/mg in the denervated striatum. The finding of a significant 23.8% increase in receptor density by the end of the first postoperative week, a period during which behavioral supersensitivity to apomorphine increases rapidly, supports the hypothesis that a proliferation of D-2 receptors underlies the behavioral manifestations of denervation supersensitivity.     

Quantitative measurement of local cerebral metabolic rate for glucose utilizing tritiated 2-deoxyglucose

The [¹⁴C]2-deoxyglucose (2-DG) technique has been widely utilized for quantitative measurement of local cerebral metabolic rate for glucose (1CMRG) in animals. The technique as presently used is limited by the energy of¹⁴C beta-particles, which can travel relatively great distances in tissue. This results in limited audiographic resolution and in computed¹⁴C concentrations which are a function of tissue section thickness. [³H]2-DG has less energetic beta-particles; hence, autoradiographs have better resolution and optical densities are independent of tissue thickness for sections greater than 5 μm.We have developed a method for quantitation of 1CMRG in rats using [³H]2-DG and a newly developed ultrasensitive X-ray film. Autoradiographic tissue standards were prepared by injecting rats with [³H]2-DG and assaying micro-samples of brain for³H concentration.Ten rats were used in this study. Five rats received [³H]2-DG (300 μCi/100 g) and 5 rats received [¹⁴C]2-DG (7.5 μCi/100g).The mean 1CMRG values for selected areas of the central nervous system demonstrated no significant difference (P > 0.05) between the [¹⁴C]2-DG and the [³H]2-DG groups. Values for 1CMRG from the [³H]2-DG group showed no variation attributable to inadequate microtome precision. The improved resolution obtained by utilizing [³H]2-DG is especially evident where gray matter (high 1CMRG) is immediately adjacent to white matter (low 1CMRG).     

Enkephalin-like immunoreactivity in rat area postrema: ultrastructural localization and coexistence with serotonin

The ultrastructure of enkephalin-containing neurons and their capacity to take-up [³H]serotonin were examined in the area postrema. Untreated adult rats and rats with intraventricular infusions of 10⁻⁴ M tritiated serotonin, 5-hydroxytryptamine ([³H]5-HT) were perfused with 4% paraformaldehyde and 0.2–0.5% glutaraldehyde. Coronal Vibratome sections through the area postrema from both groups of animals were immunocytochemically labeled with an antiserum to leucine Leu⁵-enkephalin. The sections from the animals infused with the isotope subsequently were processed for autoradiography. Enkephalin-like immunoreactivity (ELI) was detected in perikarya, dendrites, axons and axon terminals most frequently located along the ventricular and ventrolateral portions of the area postrema. The labeled perikarya were few in number and were characterized by a thin rim of cytoplasm containing peroxidase immunoreactivity. Dendrites and terminals containing ELI formed synapses primarily with unlabeled axon terminals and dendrites, respectively. However, terminals containing ELI also formed synaptic junctions with other unlabeled axon terminals. Appositions between enkephalin-containing processes and modified glia were occasionally seen near the ventricular surface. In sections processed for both immunocytochemistry and autoradiography, approximately 5% of the terminals containing ELI showed uptake of [³H]5-HT. We conclude that neurons containing ELI are primarily, but not exclusively, associated with other intrinsic neurons or afferents in the rat area postrema and that some of the enkephalin-labeled terminals have the capacity to take-up serotonin. Specificity of uptake of [³H]5-HT in neurons containing endogenous serotonin and factors which may contribute to the low probability of detecting both peroxidase and autoradiographic markers in single sections are discussed.     

Muscarinic receptors in the central nervous system of the rat. I. Technique for autoradiographic localization of the binding of [3H]propylbenzilylcholine mustard and its distribution in the forebrain

[³H]Propylbenzilylcholine mustard ([³H]PrBCM) is a synthetic, potent muscarinic antagonist, which binds specifically and irreversibly by means of a covalent linkage to muscarinic receptors.Ten μm coronal cryostat sections taken through unfixed rat brain at the level of the maximum extent of the caudate nucleus were mounted on glass slides and incubated with 2.4 nM [³H]PrBCM at 30°C for 25 min. They showed a total binding of 3250 pmol/g protein, of which 2130 pmol/g protein was sensitive to pretreatment with 10^?6 M atropine. The specific (atropine-sensitive) binding was saturable. Saturation was reached at 15 min, with a rate constant of 1.3 × 10⁶M^?1 sec^?1. Binding was unaffected by drugs acting at nicotinic receptors (d-tubocurarine, hexamethonium), or by physostigmine, but was inhibited by muscarinic drugs (pilocarpine, oxotremorineT 3-quinuclidinylbenzilate).Postfixation for 15 min in Carnoy's fixative reduced the specific binding by 10 % and the non-specific by 50 %. Prefixation (i.e. before incubation with [⁸H]PrBCM) with any fixatives containing formaldehyde largely prevented specific binding, but a range of concentrations of glutaraldehyde (2 % to 0.05 %) caused only small reductions in specific binding (e.g. 0.1 % glutaraldehyde caused only a 6% reduction). Clear, regionally specific patterns of localization of specific label in light microscope autoradiographs could be obtained from cryostat sections prefixed with 0.1 % glutaraldehyde, incubated with 2.4 nM [³H]PrBCM for 15 min at 30°C, and postfixed for 15 min in Carnoy's solution. Of the 105 forebrain areas studied 12 had grain counts between 6 and 9 times the non-specific level and a further 30 had counts 4 to 6 times non-specific.The higher grain counts were in the external plexiform layer of the olfactory bulb, anterior olfactory nucleus, olfactory tubercle, pyriform cortex, stratum radiatum of the hippocampus, stratum moleculare of the dentate gyrus, lateral amygdaloid nucleus, cortico-amygdaloid transition zone, anteroventral thalamic nucleus, hypothalamic supraoptic nucleus, caudate-putamen, nucleus accumbens, and in laminae 3 and 6 of the neocortex (parietal region). There were high grain densities over the choroid plexus of the lateral but not the third or fourth ventricles.     

Quantitative autoradiography of [H]corticosterone receptors in rat brain

We have quantified corticosterone receptors in rat brain by optical density measurements of tritium-film autoradiograms. Rats were injected i.v. with 500 μCi [³H]corticosterone to label brain receptors. Frozen sections of brain were cut with a cryostat and exposed for 2 months against tritium-sensitive sheet film (LKB Ultrofilm). Tritium standards were used to convert optical density readings into molar concentrations of receptor. High levels of corticosterone receptors were present throughout the pyramidal and granule cell layers of the hippocampus. Moderate levels of receptors were found in the neuropil of the hippocampus, the lateral septum, the cortical nucleus of the amygdala and the entorhinal cortex. All other brain regions had low levels of receptors. These results extend previous non-quantitative autoradiographic studies of corticosterone receptors and provide a general procedure for the quantitative autoradiography of steroid hormone receptors in brain tissue.