A comparative study on characterization and distribution of cholecystokinin binding sites among the rat, mouse and guinea pig brain

Using the in vitro quantitative receptor autoradiographical technique, [propionyl-3H]propionylated cholecystokinin octapeptide ([3H]pCCK-8) binding sites were investigated in tissue sections of rat, mouse and guinea pig brains. In all the tested animals, [3H]pCCK-8 bound very slowly to the tissue sections. Dissociation was also slow, and had a biphasic profile suggesting CCK-8 binding sites are heterogeneous. Dissociation rate constants were, however, unequal among these species. In the saturation binding studies, both Bmax and (Kd)app values varied among the animal species. The autoradiograms revealed marked species differences in [3H]pCCK-8 binding sites in the brain among 3 closely related species of rodents. [3H]pCCK-8 binding sites were undetectable in the nucleus accumbens/caudate-putamen and the amygdaloid complex of the mouse brain, and scarcely found in the ventromedialis of the hypothalamus of the mouse and guinea pig brain. Furthermore, moderate-to-high densities of [3H]pCCK-8 binding sites were observed in the cerebella of the mouse and guinea pig, whereas in the rat cerebellum the binding sites were undetected. The above-mentioned observations suggest the existence of species differences in the binding pattern of CCK-like peptides among closely related animal species. Furthermore, it would appear that CCK-like peptides in the brain may play different physiological roles among animal species.     

The effect of the beta-adrenergic receptor antagonist, propranolol, on the cerebral spread of a memory trace in mice

Bi-temporal injections of puromycin that primarily affect the hippocampal-entorhinal areas consistently induce amnesia of aversive maze-learning in mice for 3 days after training but are consistently ineffective if given 6 or more days after training. At these later times, additional puromycin sites covering widespread areas of the forebrain are necessary to induce amnesia. Consistent with other evidence, these observations are interpreted to indicate that the locus of the memory trace becomes more widespread during the 6-day period. A single subcutaneous injection of (-)-propranolol (50 micrograms/kg) given either before or 2 days after training suppressed engram spread for 60-90 days, at which time engram spread spontaneously occurred. This effect of propranolol was stereospecific. Suppression of engram spread persisted for a prolonged period in spite of the rapid recovery (about 4 hr), following treatment, of the normal level of specific binding of 3H-dihydroalprenolol in membrane preparations of the cerebral hemispheres and of 125I-pindolol in selected areas of the forebrain, diencephalon and brainstem.     

Labeling of bovine rod outer segment surface proteins with 125I

Osmotically intact purified bovine rod outer segments (ROS) were labeled with ¹²⁵I by lactoperoxidase-catalyzed iodination. Molecular weights of Coomassie-stained and ¹²⁵I-labeled polypeptides were estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Only four ¹²⁵I-labeled proteins were consistently detected, although 13 Coomassie-stained bands were resolved. Rhodopsin was labeled, in addition to proteins having average apparent molecular weights of 226 000 (226 K), 110 000 (110 K) and 66 000 (66 K). The high molecular weight labeled proteins (226 K and 110 K) did not comigrate with Coomassie-stained bands. The 226 K band was much more heavily labeled than the 110 K and 66 K proteins or rhodopsin. Mild trypsin digestion of intact labeled ROS resulted in degradation of the 226 K labeled band, but did not alter the electrophoretic mobility of any of the other labeled bands, or of the Coomassie-stained bands. None of the labeled proteins were extracted from pelletable ROS membranes by 10 mm Tris-Cl, 0·5 m-KCl, 8 m urea or digitonin at 0·5 mg/mg of ROS protein. Sucrose density gradient analysis showed that the labeled proteins were not associated with gradient fractions other than those containing ROS. These results suggest that the 226 K and 110 K proteins are components of the ROS plasma membrane, and support previous reports that rhodopsin is a component of this membrane.     

Local spinal cord glucose utilization in conscious and halo-thane-anaesthetized rats

The authors used the 2-[14C]deoxyglucose method to study local spinal and cerebral glucose utilization simultaneously during 1.2 per cent halothane anaesthesia in adult Sprague-Dawley rats. In conscious animals (n = 5) the rate of glucose utilization in lumbar spinal gray matter was about 50 per cent lower than that of cerebral cortex. Halothane anaesthesia (n = 6) reduced spinal cord and cerebral metabolic rate. Spinal glucose utilization was reduced 12-35 per cent, but this was less than the 45-70 per cent decrease halothane produced in 8 of 16 cerebral structures examined and was independent of the hypotension produced. These results indicate that halothane is a spinal metabolic depressant but that its effects on this tissue are substantially less than those it has on many cerebral structures.     

Localization and Characterization of Insulin-Like Growth Factor-I Receptors in Rat Brain and Pituitary Gland Using in vitro Autoradiography and Computerized Densitometry* A Distinct Distribution from Insulin Receptors

In order to identify likely sites of action of insulin-like growth factor-I (IGF-I) in rat brain and pituitary gland, we have used the technique of in vitro autoradiography and computerized densitometry to map, characterize and quantify its receptors in coronal and sagittal sections. A discrete and characteristic distribution of IGF-I receptor binding was demonstrated, with specific binding representing 85% of total binding. Displacement and specificity competition curves in the olfactory bulb were typical for authentic IGF-I receptors and computer analysis indicated a single class of binding site with a dissociation constant (K_d) of 13 nM for the choroid plexus and 5.1 nM for the olfactory cortex. IGF-I receptor density was very high in the choroid plexus in ail ventricles, but the binding in other circumventricular organs was variable, with high levels in the median eminence and the sub-fornical organ, and low levels in the organum vasculosum of the lamina terminalis. Highest binding was seen in the glomerular layer of the olfactory bulb and its associated regions the taenia tecta and anteromedial olfactory nucleus. The preoptic and septal regions showed moderate binding, while the hypothalamus, with the exception of the median eminence, showed low IGF-I binding. The pituitary gland showed very high binding density in both anterior and posterior lobes, similar to the median eminence. The thalamus had high IGF-I binding density, while it was low in basal ganglia. In the limbic system the hippocampal CA2, CAS, CA4 layers showed high binding, with little in CA1, while binding was high also in the adjacent amygdala. Binding was low in the mid and hindbrain, with the exception of the geniculate bodies, and the sensory nucleus of the trigeminal nerve. Binding was high in the primary olfactory and endopyriform cortex and in specific superficial layers. Cerebellar binding was also high in the molecular layer. Fibre layers showed no binding. Comparison with insulin receptors revealed common distribution in the choroid plexus, paraventricular nucleus, cerebellum, entorhinal cortex and amygdala, with receptor density three- to five-fold higher for IGF-I than for insulin. In contrast, in the hippocampus, insulin binding was high in the CA1 field, and low in CA2, CA3, CA4 while for IGF-I binding the converse was seen. The arcuate nucleus showed prominent insulin labelling and minimal IGF-I binding, while the median eminence showed low insulin and high IGF-I binding. The hypothalamus was more widely labelled with insulin, while in the thalamus the converse was true. Olfactory bulb laminae were labelled with differing intensity by insulin and IGF-I. In common with insulin receptor distribution was the high density of IGF-I receptors over areas of extensive dendritic arborizations which receive rich synaptic inputs, in the cerebellum, hippocampus and olfactory bulb. We conclude that IGF-I receptors are widespread throughout rat brain and pituitary gland, with concentration in regions concerned with olfaction, autonomie and sensory processing, as well as in regulation of growth hormone release, via feedback at the median eminence and pituitary gland. Many of these regions have in common high rates of metabolic and synthetic activity, which may be mediated by IGF-I and its receptors.     

Brain regional distribution of physostigmine and its relation to cerebral blood flow following intravenous administration in rats

3H-labeled physostigmine (50 micrograms.kg-1) was administered intravenously to rats, and its concentration in brain tissue and spinal cord was assessed by quantitative autoradiography. Regional cerebral blood flow (rCBF) was measured with iodo-14C-antipyrine autoradiography in control rats and in animals injected i.v. with a dose of physostigmine similar to that used for the distribution studies. Tissue concentration of 3H-physostigmine was correlated with rCBF for 37 brain regions. A high degree of correlation was found at 0.5 min after drug injection, r (correlation coefficient) = 0.87. This association decreased at later times (5 min r = 0.73, 12 min r = 0.24). Structures with high cholinesterase activity (caudate-putamen, amygdala, hippocampus) showed greater retention of physostigmine over time. The highest initial physostigmine concentrations were found in regions lacking a blood-brain barrier (pineal bland, median eminence, choroid plexus) (range = 10.4-23.8 nCi/mg) and the lowest in white matter (corpus callosum, internal capsule, hippocampus commisure, spinal cord dorsal column) (range = 1.2-2.6 nCi/mg). Initial concentrations of the drug in the areas in which physostigmine induced vasodilatation (motor, sensory, temporal and occipital cortex, claustrum, and superior collicullus) were not different from concentrations in areas of comparable basal rCBF in which no such effect was observed. Variations in drug access to brain regions, then, do not explain the topographical variations of the cerebrovascular action of physostigmine.     

Autoradiographic studies of [3H]-glycine, [3H]-GABA, and [3H]-muscimol uptake in the mudpuppy retina

Autoradiographic studies showed selective accumulation of [3H]-glycine, [3H]-GABA, and the GABA agonist [3H]-muscimol by neurons of the mudpuppy retina. [3H]-Glycine was taken up by bipolar cells, amacrine cells, and displaced amacrine or ganglion cells. Both [3H]-GABA and [3H]-muscimol were also accumulated by bipolar cells, amacrine cells and ganglion layer cells. However, the [3H]-GABA uptake pattern differed from that of [3H]-muscimol in showing labeling of horizontal cells, an increased percentage of cells in the ganglion cell layer, and a band in the most proximal portion of the inner plexiform layer. Variations in grain density suggested the presence of multiple subpopulations of [3H]-glycine- and [3H]-GABA-labeled amacrine cells. The labeled cells may play a role in inhibitory pathways in the inner retina.     

Expression and distribution of kinin B1 receptor in the rat brain and alterations induced by diabetes in the model of streptozotocin

A role for kinin B1 receptors was suggested in the spinal cord and peripheral organs of streptozotocin (STZ)-diabetic rats. The present study aims at determining whether B1 receptors are also induced and over-expressed in the brain of STZ-rats at 2, 7, and 21 days post-treatment. This was addressed by in situ hybridization using the [35S]-UTPalphaS-labeled riboprobe and by in vitro autoradiography with the radioligand [125I]-HPP-des-Arg10-Hoe 140. In control rats, B1 receptor mRNA was found widely distributed in many brain regions. Low mRNA levels were found in thalamus and hypothalamus (7-12 nCi/g) while high mRNA signals were detected in cortical regions and hippocampus (18-29 nCi/g). In diabetic rats, B1 receptor mRNA was markedly increased in hippocampus, temporal/parietal cortices and amygdala at 2 and 7 days (+88 to +150%). Low densities of B1 receptor binding sites were detected in all analyzed regions in control rats (0.18-0.37 fmol/mg tissue). In diabetic rats, B1 receptor binding sites were significantly increased in hippocampus, amygdala, temporal/parietal, and perhinal/piriform cortices (+ 55 to + 165 %) at 7 days only. Results highlight an early but transient and reversible up-regulation of B1 receptors in specific brain regions of STZ-diabetic rats. This may offer the advantage of reducing putative central side effects with B1 receptor antagonists if used for the treatment of diabetic complications in the periphery.