Evaluation of [3H]paroxetine as an in vivo ligand for serotonin uptake sites: A quantitative autoradiographic study in the rat brain

Paroxetine, a selective inhibitor of serotonin uptake and an antidepressant, was used in conjunction with quantitative ex vivo autoradiography to study the feasibility of imaging serotonin terminals in the living brain. Tritiated paroxetine was injected in the rat tail vein, and the brain was processed for quantitative autoradiography 3 hours later. Animals received either [³H]paroxetine alone (100 μCi/animal) or a mixture of labeled paroxetine (100 μCi) and an excess of unlabeled drug (0.5 or 2 mg/kg intravenously [i.v.]). Computerized image analysis of the resulting autoradiograms revealed high densities of radioactivity in brain regions known to contain high densities of serotonergic terminals and high specific binding of [³H]paroxetine in vitro, such as the raphe nuclei, interpeduncular nucleus, basolateral amygdala, substantia nigra, and some hypothalamic nuclei. Radioactivity uptake in these brain regions was effectively blocked (50-72%) by coadministration of excess unlabeled paroxetine. However, cortical and hippocampal binding of paroxetine in vivo was moderately high, in contrast to the relatively sparse serotonergic innervation in these regions. Only a relatively small proportion of cortical and hippocampal binding (20-40%) could be blocked by excess unlabeled paroxetine, indicating that most of the radioactivity in these regions is not associated with serotonin terminals or uptake sites. The usefulness of [³H]paroxetine as an in vivo ligand for imaging serotonin terminals in the human brain is limited by these nonserotonergic binding sites. © 1993 Wiley-Liss, Inc.     

Entorhinal cortex lesion or intrahippocampal colchicine injection increases peripheral type benzodiazepine binding sites in rat hippocampus

The peripheral type benzodiazepine binding site (PTBBS) has been proposed to be a good marker for reactive glial cells following brain insults. In the present study, homogenate binding of 3H-Ro5-4864 and quantitative autoradiography of 3H-PK-11195 binding (two ligands for the PTBBS) were used to assess the distribution, time-course and extent of reactive gliosis in the hippocampus following deafferentation by unilateral entorhinal cortex lesion or neuronal death produced by intrahippocampal colchicine injection. Intrahippocampal colchicine injections produced a 3-fold increase in 3H-Ro5-4864 binding in the dentate gyrus within 2 days. This effect was doubled in animals pretreated with the lysosomal inhibitor chloroquine. Quantitative autoradiography of 3H-PK-11195 binding 1 or 2 weeks after colchicine injection indicated that the increase in binding was restricted to the dorsal hippocampus both rostrally and caudally and was present in the dentate gyrus and CA1. Following a unilateral electrolytic lesion of the entorhinal cortex, the binding of 3H-Ro5-4864 to homogenates of the dentate gyrus was doubled 18 h after the lesion, reached a maximum at 4 days post-lesion, and returned to control values by 2 months after the lesion. A transient increase in binding was also observed 2 and 4 days post-lesion in the dentate gyrus contralateral to the lesion side. Autoradiography of 3H-PK-11195 binding indicated that the increase in PTBBS following entorhinal cortex lesion was restricted to the molecular layer of the dentate gyrus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoradiographic analysis of the distribution of vasoactive intestinal peptide binding sites in the vertebrate central nervous system: a phylogenetic study

The distribution of vasoactive intestinal peptide (VIP) binding sites in the brain of several vertebrate species was examined by in vitro autoradiography on slide-mounted sections. This study included fish, frog, snake, pigeon, rat, mouse, guinea pig, cat and monkey brain. A fully characterized, monoiodinated form of vasoactive intestinal peptide (M-¹²⁵I-VIP), which maintains the biological activity of the native peptide in the central nervous system (CNS), was used throughout the study. Among the lower vertebrate species, no significant specific binding was found in the fish brain, whereas in the frog and snake brain, specific VIP binding sites were observed, mainly in the telencephalon. In the pigeon brain, high densities of VIP binding sites were localized in the hyperstriatum, neostriatum, archistriatum, hippocampal area, dorsolateral cortical area and in the optic tectum. Ectostriatum and paleostriatum augmentatum displayed lower densities of specific binding. In mammals, the highest concentrations of VIP binding sites were observed in the rodent brain. In the rat, mouse and guinea pig brain, high densities were detected in the olfactory bulb, external layers of the cerebral cortex, dentate gyrus, midline thalamic nuclei, geniculate nuclei, some hypothalamic nuclei, superior colliculus coeruleus. Intermediate densities were found in amygdala, caudate-putamen, septum and nucleus accumbens, CA1–CA3 fields of the hippocampus and central gray. The cerebellum of these species presented high densities of VIP binding sites, with species to species differences in their localization. The non-specific binding was, however, increased in the rodent cerebellum. Lower densities of VIP binding sites were observed in the cat and monkey CNS. In these two species, the non-specific binding was considerably higher than in the lower mammals brain. In the cat and monkey brain, as in the lower mammals, the highest densities were revealed in the neocortex, dentate gyrus, thalamic nuclei and some midbrain structures including substantia nigra and locus coeruleus. In all the species studied, the white matter was never labeled with M-¹²⁵I-VIP. This study suggests that VIP binding sites appear relatively early in the evolution of the vertebrate CNS. The most important densities of specific VIP binding sites are observed in the pigeon and rodent brain, whereas the cat and monkey present a marked increase in non-specific binding. It is interesting to note that the distribution of VIP binding sites as revealed by autoradiography is quite conservative in terms of evolution and indicates an association, although non-exclusive, of VIP receptors with brain regions involved in the processing of specific sensory inputs.     

Long experimental durations are required for double label [C]- and [H]2-deoxyglucose autoradiographic methods

Double-label 2-deoxyglucose (2-DG) studies using sequential [¹⁴C]- and [³H]2-DG injections demonstrate increased [¹⁴C]2-DG uptake durating the first and second stimulation periods. To understand why this occurs, the rat mystacial vibrissae were stimulated at various times following [¹⁴C]2-DG injection. Local cerebral glucose utilization (LCGU) increased when whisker stimulation was performed at 0–90 min following [¹⁴C]2-DG injection. LCGU did not increase when whisker stimulation was performed at 90–150 min following [¹⁴C]2-DG injection. To minimize contamination of the two tracers in double label 2-DG mapping studies, the time between [¹⁴C]- and [³H]2-DG administration should be increased to 90 min.     

Autoradiographic discrimination of melanocortin receptors indicates that the MC3 subtype dominates in the medial rat brain

In the present study, we developed an autoradiographic method to visualize the distribution of melanocortin (MC) receptors 3 and 4 in sagittal sections of the rat brain. The method takes advantage of the MC3 and MC4 receptor selective compounds, γ1-MSH and HS014. First, we characterized the binding of γ1-MSH, HS014 and the radioligand [ ]NDP–MSH to the rat MC3 and MC4 receptors expressed in COS cells. [ ]NDP–MSH was found to be non-selective, whereas γ1-MSH showed a 40-fold preference for the rat MC3 receptor, and HS014 an over 300-fold preference to the rat MC4 receptor. Second, to discriminate between the MC3 and MC4 receptors in rat brain sections, the sections were incubated with [ ]NDP–MSH in the presence of graded concentrations of the MC3 selective ligand, γ1-MSH, or the MC4 selective ligand, HS014. From the autoradiograms thus made, competition curves of γ1-MSH and HS014 could be constructed for different regions of the rat brain. Our results indicate that in the nucleus accumbens shell, the medial preoptic area, and the ventromedial nucleus of the hypothalamus, there is a clear dominance of the MC3 receptor, whereas in the lateral septum and the olfactory tubercle, there seem to be present both MC3 and MC4 receptors, although the MC3 receptor may still be the dominating subtype. In the optic layer of the superior colliculus, our data indicate a more abundant expression of the MC4 receptor. In the ventral tegmental area, there might be an additional MSH-peptide binding site of unknown origin.     

Regulation of ionotropic glutamate receptors following subchronic and chronic treatment with typical and atypical antipsychotics

Quantitative in vitro receptor autoradiography was used to examine changes in three ionotropic glutamate receptor subtypes using ³H-MK801 (NMDA-R antagonist), ³H-CNQX (AMPA-R antagonist) and ³H-kainic acid (kainate-R agonist) following subchronic (28 days) and chronic (8 months) treatment of rats with a typical antipsychotic, haloperidol (1.5 mg/kg per day), atypical antipsychotic, clozapine (25 mg/kg per day), the dopamine D₂/D₃ receptor antagonist, raclopride (10 mg/kg per day), and the dopamine D1 (D₁/D₅) receptor antagonist SCH23390 (0.5 mg/kg per day). Subchronic and chronic drug treatments did not significantly alter ³H-CNQX or ³H-kainate binding in any of brain regions examined. Subchronic SCH23390 treatment elevated ³H-MK801 binding in the hippocampal formation with significant increases in the CA₁ and dentate gyrus, suggesting a specific role for dopamine D1 receptors in the regulation of hippocampal NMDA receptor function. Subchronic, but not chronic, haloperidol and clozapine treatment significantly reduced ³H-MK801 binding in the medial prefrontal cortex. This suggests that typical and atypical antipsychotics may exert some of their clinical effects by affecting NMDA receptors in the medial prefrontal cortex. Both subchronic and chronic clozapine treatment decreased ³H-MK801 binding in the caudate putamen. The minimal extrapyramidal side effects produced by clozapine may result, in part, from the reduction in NMDA receptor binding in the caudate putamen. Received: 29 February 1996 /Final version: 15 July 1996     

Autoradiographic distribution of sigma receptors in human neocortex, hippocampus, basal ganglia, cerebellum, pineal and pituitary glands

Autoradiographic distributions of [3H]1,3-di-O-tolylguanidine, [3H]DTG, binding to sigma (sigma) receptors were studied in key human brain regions. High densities occurred in the substantia nigra pars compacta and cerebellum. Pineal and pituitary levels were moderate. In neocortex, binding was high in laminae II-IVA and much lower in the midzone. Moderate binding occurred over hippocampal dentate granular cells and in the striatum. These results differ from those reported for the rodent.     

Spinal cord transection produces a long-term increase in GABAB binding in the rat substantia gelatinosa

Quantitative autoradiography was used to determine changes in GABA_B receptor binding in the substantia gelatinosa of the lumbar spinal cord at 4 days and at 6 weeks after a midthoracic spinal transection in rats. In the 4 day lesion animals, there was no significant change in either the density or the affinity of the GABA_B binding. At 6 weeks, however, there was a 35% increase in binding density, with no significant change in affinity. The results suggest that alterations in spinal synaptic mechanisms can slowly evolve following loss of descending input to the spinal cord. © 1993 Wiley-Liss, Inc.     

Regional distribution of substance P binding sites in the brainstem of the human newborn

The distribution of [³H]substance P ([³H]SP) binding sites in the brainstem of the human was investigated in eleven cases (aged 1 h to 6 months) by in vitro quantitative receptor autoradiography. The binding of [³H]SP to newborn brainstem tissue was found to be saturable (for the eight cases examined,K_d andB_max (M±S.E.M.) were 0.29±0.03 nM and 206±21 fmol/mg tissue, respectively). Competition studies showed unlabeled SP to be the most peptide for displacing [³H]SP binding from tissue sections. The desaturating effect of GTP on the specific binding of [³H]SP was also investigated, but was not found to be significant. Autoradiographic analysis showed that the neurokinin-1 (NK-1)/SP binding sites were widely but unevenly distributed, and that they varied with age. The highest densities of (NK-1)/SP binding sites were observed in the locus coeruleus, olivaris inferior nuclei, raphe magnus and obscurus nuclei, while low to moderate densities were observed in other brainstem structures. These findings support the idea that SP is involved in cardiovascular regulation, and that it may interact with the catecholaminergic and/or serotonergic system.     

Expression of non-angiotensin II [I]CGP 42112 binding sites on activated microglia after kainic acid, induced neurodegeneration

[¹²⁵I]CGP 42112, first developed to identify angiotensin II receptor subtype 2 (AT₂), was recently shown to bind to a novel non-angiotensin binding site in injured rat brain tissue. We addressed the question whether non-angiotensin [¹²⁵I]CGP 42112 binding appears after kainic acid induced hippocampal neurodegeneration, a process of neuronal cell death at a distance from the toxin injection site. After intraventricular kainic acid injection, we found non-angiotensin [¹²⁵I]CGP 42112 binding in the hippocampal areas CA3 (4 and 14 days after injection), CA1 and CA4 and the subiculum (14 days after injection). In addition, 14 days after kainic acid injection, [¹²⁵I]CGP 42112 binding was found in 50% of the animals, in the thalamus, amygdala and piriform cortex, areas receiving projections from the hippocampus and suffering kainic acid induced delayed neurodegenaration. The loss of neurons in these regions was accompanied by an accumulation of activated microglia as demonstrated by immunostaining with the specific antibodies OX-42 and ED1. The time course and regional pattern of OX-42/ED1 positive immunostaining was identical with the appearance and distribution of the non-angiotensin [¹²⁵I]CGP 42112 binding site. The non-angiotensin [¹²⁵I]CGP 42112 binding was not detected in brain regions unaffected by kainic acid injection. Our findings indicate the expression of a novel [¹²⁵I]CGP 42112 binding site on activated microglia. This site appears at a distance from the lesion and may be of importance in the process of neuronal death and brain tissue repair.