Kinetics and distribution of [Fe–I]transferrin injected into the ventricular system of the rat

We examined the kinetics and distribution of [⁵⁹Fe–¹²⁵I] rat Tf and unlabelled human Tf injected into a lateral cerebral ventricle (i.c.v. injection) in the rat. [⁵⁶Fe–¹³¹I]Tf injected intravenously served as a control of blood–brain barrier (BBB) integrity. In CSF of adult rats, ⁵⁹Fe and [¹²⁵I]Tf decreased to only 2.5% of the dose injected after 4 h. In brain parenchyma, [¹²⁵I]Tf had disappeared after 24 h, whereas approximately 18% of i.c.v.-injected ⁵⁹Fe was retained even after 72 h. The elimination pattern of [¹²⁵I]Tf from the CSF corresponded to that of [¹³¹I]albumin injected i.c.v., suggesting a nonselective washout of CSF proteins. [¹³¹I]Tf was hardly detectable in the brain, reflecting an unimpaired BBB during the experiments. Morphologically, ⁵⁹Fe and i.c.v. injected human Tf were confined to the ventricular surface and meningeal areas, whereas grey matter regions at distances more than 2–3 mm from the ventricles and the subarachnoid space were unlabelled. However, accumulation of ⁵⁹Fe was observed in the anterior thalamic and the medial habenular nuclei, and in brain regions with synaptic communications to these areas. In the newborn rats aged 7 days (P7) injected i.c.v. with [⁵⁹Fe–¹²⁵I]Tf and examined after 24 h, the amounts of [¹²⁵I]Tf in CSF were approximately 3.5 times higher than in adult rats collected after the same time interval, whereas the amounts of ⁵⁹Fe in CSF were at the same level in P7 and adult rats. In the brain tissue of the i.c.v. injected P7 rats, both [¹²⁵I]Tf and ⁵⁹Fe were retained to a significantly higher degree compared to that seen in adult brains. The rapid washout and lack of capability for i.c.v. injected [¹²⁵I]Tf to penetrate deeply into the brain parenchyma of the adult brain question the importance of Tf of the CSF, and choroid plexus-derived Tf, for Fe neutralization and delivery of Fe–Tf to TfR-containing neurons and other cells in the CNS. However, it may serve these functions in young animals due to a lower rate of turnover of CSF.     

Global reduction in cerebral blood flow and metabolism elicited from intrinsic neurons of fastigial nucleus

We sought to determine whether the global increase in regional cerebral blood flow (rCBF) produced by electrical stimulation of the rostral cerebellar fastigial nucleus (FN) is a consequence of excitation of intrinsic neurons of the FN or of axons of fibers passing through or projecting into it. Studies were conducted in rats anesthetized with chloralose, paralyzed and ventilated. rCBF was measured with [¹⁴C]iodoantipyrine as tracer and regional cerebral glucose utilization (rCGU) by [¹⁴C]2-deoxyglucose in homogenates of 11 brain regions. Neuronal perikarya in FN were excited chemically by local microinjection of the glutamate analogue kainic acid (KA) (5 nmol in 100 nl). KA elicited a transient and significant fall of arterial pressure and heart rate, the fastigial depressor response (FDR). Associated was a significant and symmetrical reduction in rCBF, to 44% of control in all regions except medulla. The response was site- and agent-specific and unrelated to the hypotension. KA also significantly and proportionally reduced, to 52% of control, rCGU in the same 10 areas of brain. In all regions the magnitudes of the reductions in rCBF and rCGU elicited by KA were linearly related. Intrinsic neurons of FN were chronically destroyed by local microinjection of the excitotoxin ibotenic acid (IBO) (10 μg/μl in 0.4 μl). Destruction of intrinsic FN neurons had no effect on resting rCBF nor on the global cerebrovascular vasodilation elicited by electrical stimulation of the FN. We conclude that: (a) excitation of intrinsic neurons of FN elicits a widespread reduction of cerebral metabolism and, secondarily, blood flow; (b) FN neurons do not exert a long-term tonic influence on brain blood flow nor metabolism; (c) the global increase in rCBF elicited by electrical stimulation of the FN is a consequence of excitation of axons projecting into or through the nucleus.     

Amino acid transport in cerebral microvessels during plasmodium yoelii infection in mice

Plasmodium yoelii infected cerebral microvessels of mice had an enhanced time-dependent, temperature-sensitive, and saturable uptake of [¹⁴C]-amino acid. viz. leucine, valine and glycine. Metabolic inhibitors caused a noticeable inhibition of amino acid uptake in normal microvessels as compared to infected cerebral microvessels indicating that the uptake of [¹⁴C]-L-leucine, [¹⁴C]-L-valine and [¹⁴C]-glycine is an energy dependent process.     

The differentiation of synthesis from incorporation of basic protein in quaking mutant mouse myelin

The incorporation of radioactive label into the myelin basic protein isolated from whole brain and from purified myelin of Quaking mice and normal littermates was compared. Four Quaking mice (32 days) and 4 littermate controls were injected intracranially with 150 μCi [2-²H]glycine and 25 μCi of [2-¹⁴C]glycine, respectively. One hour later, the 8 mice were sacrificed and their brains combined for common homogenization. The ³H/¹⁴C ratios of the small and large basic proteins in whole brain were 3.44 and 2.48 respectively, while the ³H/¹⁴C ratios for these proteins in myelin were 0.79 and 1.00, respectively. In the same experiment, the microsomal fraction had a ³H/¹⁴C ratio of 2.98 which is the expected ratio for normal incorporation. The results indicate that the synthesis of basic protein in whole brain of Quaking mouse proceeds at a normal rate, but specifically, the incorporation of basic protein into myelin is depressed suggesting a defect at the step of assembly of myelin components into a final membrane product.     

2,4-Dichlorophenoxyacetic acid intoxication increases its accumulation within the brain

Exposure to the phenoxyacetic acid herbicides has been shown to produce neurotoxicity. Therefore, adult mice (pregnant) and rabbits were used to examine the accumulation and regional distribution of 2,4-dichlorophenoxyacetic acid (2,4-D) within the brain following intraperitoneal injection of a low dose (0.2–0.4 mg/kg) of [¹⁴C]2,4-D. Controls, i.e. animals not previously exposed to 2,4-D, were compared to animals acutely pretreated with higher doses (40–160 mg/kg) of unlabeled 2,4-D. Both autoradiography and direct tissue analysis showed that in control animals brain levels were much lower than plasma in both adult (4%) and fetus (8%). In both species, small variations were seen between the brain regions, with brainstem and cerebellum somewhat higher than other regions. Pretreatment with unlabeled 2,4-D caused a 5- to 10-fold increase in accumulation of [¹⁴C]2,4-D in both mice and rabbits. On the other hand, 2-deoxyglucose entry into the brain was not altered by 2,4-D pretreatment. Thus, there was no generalized increase in blood-brain barrier permeability. Instead, increased 2,4-D accumulation appeared to be caused by its decreased elimination from the brain. Pretreatment with 40 mg/kg led to a CSF 2,4-D concentration of 10 μM, a concentration sufficient to inhibit choroid plexus transport of [¹⁴C]2,4-D by nearly 50% in vitro. These results suggest that exposure to organic anions like 2,4-D may lead to the retention of potentially toxic anions within the CNS via competitive inhibition of the organic anion transport system which normally reduces their brain and CSF concentrations to very low levels.     

Evidence for defective incorporation of proteins in myelin of the quaking mutant mouse

The defect in myelinogenesis present in the Quaking mutant mouse was investigated using a double radioisotope technique for comparing the incorporation of amino acid into myelin proteins of normal and mutant mice. Quaking mice and littermate controls recieved intracranial injections of 150 μCi [³H]glycine and 25 μCi of [¹⁴C]glycine respectively. After 2 h their brains were combined and jointly processed to obtain subcellular fractions. The ³H/¹⁴C ratio for the myelin subfraction was 1.88 as compared to a ³H/¹⁴C ratio of 3.0 for the other subfractions, indicating a 40% decrease in glycine incorporation into myelin of Quaking mice. Myelin proteins were separated by discontinuous gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and the ³H/¹⁴C ratios determined in each gel slice. In contrast to the microsomal subfractions which gave a ³H/¹⁴C ratio of 2.6 across the gel, the ³H/¹⁴C ratio of myelin showed large variations with values ranging from 0.54 for proteolipid protein to 2.0 for some of the high molecular weight proteins. During development, the Quaking mutant exhibited a preferential depression in glycine incorporation into proteolipid protein in 18-day-old mice, while in older animals (32–54 days) the fast migrating basic protein, as well as the proteolipid protein, was labeled to a significantly lesser extent.     

Transport, uptake, and metabolism of blood-borne vasopressin by the blood-brain barrier

Transport, binding, and metabolism of [phenylalanyl-3,4,5-³H(N)]arginine vasopressin (AVP) by the blood-brain barrier (BBB) was studied in adult guinea-pigs by means of a novel vascular brain perfusion (VBP)/capillary depletion technique and HPLC. A time-dependent, progressive brain uptake of ³H-radioactivity was measured over the 10 min period of VBP both in brain homogenates and in brain tissue depleted of cerebral microvessels. The unidirectional blood-to-brain transport constant, K_IN, estimated by multiple-time tissue uptake analysis of the homogenate and postcapillary supernatant, indicated that the BBB transfer rat ffor [³H]AVP (K_IN = 2.37±0.25 μl min⁻¹ per gram brain homogenate) was almost 10 times higher than for simultaneously perfused [¹⁴C]sucrose, a cerebrosvascular space marker. In contrast to homogenate and postcapillary supernatant, the [³H]radioactivity determined in the vascular pellet after dextran density centrifugation of the brain homogenate was very low and only somewhat higher than for [¹⁴C]sucrose. HPLC analysis of the perfused brain tissue revealed time-dependent degradation of the blood-borne neuropeptide. The percentage of intact [³H]AVP as determined in the postcapillary supernatant progressively declined during brain perfusion, from 49% at 1 min to 11.9% at 10 min. The major detectable labeled metabolite was [³H]phenylalanine, the labeled amino acid residue of [³H]AVP. The aminopeptidase inhibitor bestatin (0.5 mM), perfused simultaneously with [³H]AVP by the VBP technique, did not alter tissue uptake of [³H]AVP, indicating that there was no significant hydrolysis of peptide by the luminal BBB surface. The results suggest that rapid in vivo metabolism of AVP occurs after BBB transport in the brain parenchyma with no evidence of significant capillary sequestration, or degradation of AVP by the BBB.     

Diminished effect of inhibition of nitric oxide synthase on regional cerebral vascular resistance in conscious and in isoflurane anesthetized rats during hemorrhage

Effects of a nitric oxide (NO) synthase inhibitor on regional cerebral vascular resistance (rCVR) and regional cerebral blood flow (rCBF) were studied during a severe hemorrhage in conscious and in isoflurane anesthetized groups of rats. Half of each group was infused with N^G-nitro-l-arginine-methyl ester (l-NAME), a NO synthase inhibitor, at a rate of 2 mg·kg⁻¹·min⁻¹ for 30 min. Half of the lNAME infused and half of the normal saline infused rats were bled to reduce the mean arterial blood pressure (MAP) to 44–49 mmHg. rCBF was measured using [¹⁴C]iodoantipyrine. rCVR was calculated as the ratio of MAP to rCBF. In the conscious non-hemorrhagic rats, l-NAME markedly increased rCVR in all the brain regions that we studied. In the conscious rats without l-NAME treatment, hemorrhage decreased rCVR in most of the brain regions. With l-NAME treatment in this group, hemorrhage increased rCVR only in the rostral part of the brain. Isoflurane decreased rCVR in most of the brain regions except the cortical area. l-NAME markedly increased rCVR in all the brain regions that we studied in the isoflurane anesthetized rats. In the isoflurane anesthetized rats, hemorrhage did not reduce rCVR in any of the brain regions. In the isoflurane anesthetized hemorrhagic rats, l-NAME did not significantly affect rCVR in any of the brain regions that we studied. We found that l-NAME increased rCVR to a greater extent in the non-hemorrhagic rats than in the hemorrhagic rats in both the conscious and in the isoflurane anesthetized rats. The effect of l-NAME appeared to be similar both under conscious and under isoflurane anesthetized conditions.     

Glucose metabolism in cholinoceptive cortical ratbrain regions after basal forebrain cholinergic lesion

To address the question whether the changes in cortical glucose metabolism observedin patients with Alzheimers disease are interrelated with, or consequences of, basal forebraincholinergic cell loss, an experimental approach was employed to produce cortical cholinergicdysfunction in rat brain by administration of the cholinergic immunotoxin 192IgG-saporin. [¹⁴C] -glucose utilization in brain homogenates, -glucose-displaceable [³H]cytochalasin B binding to glucose transporters (GLUT),Northern and Western analyses, as well as in vivo [¹⁴]2-deoxyglucoseautoradiography were used to quantify the regional glucose metabolism.

Basal forebrain cholinergic lesion resulted in transient increases in glucose transporterbinding in cortical regions displaying reduced acetylcholinesterase activity, already detectableseven days after lesion with peak values around 30 days post lesion. Western analysis revealedthat the changes in total glucose transporter binding are mainly due to changes in the GLUT3subtype only, while the levels of GLUT1 and GLUT3 mRNA (Northern analysis) were notaffected by cholinergic lesion. Both immunocytochemistry and in situ hybridizationdemonstrated preferential localizations of GLUT1 on brain capillaries and GLUT3 on neurons,respectively. A lesion-induced transient decrease in [¹⁴C] -glucoseutilization seven days post lesion was detected in the lesion site, whereas cholinoceptive corticalregions were not affected. In vivo [¹⁴C]deoxyglucose uptake was transientlyincreased in cholinoceptive cortical regions and in the lesion site being highest between three toseven days after lesion.

The cholinergic lesion-induced transient up-regulation of cortical glucose transporters anddeoxyglucose uptake reflects an increased glucose demand in regions depleted by acetylcholinesuggesting functional links between cortical cholinergic activity and glucose metabolism incholinoceptive target regions.     

Imaging of [C]-labelled RO 15-1788 binding to benzodiazepine receptors in the human brain by positron emission tomography

The benzodiazepine antagonist Ro 15-1788 was labelled with [¹¹C] and examined for possible use as ligand for PET scan studies on benzodiazepine receptors in the brain of cynomolgus monkeys and human subjects. [¹¹C] Ro 15-1788 allowed the in vivo visualization of benzodiazepine receptor binding in cerebral and cerebellar cortical areas as well as in basal brain nuclei in PET scan images. [¹¹C] Ro 15-1788 exhibited a high ratio of specific benzodiazepine receptor binding (cerebral cortex) to non-specific binding (pons) and the kinetics of binding should be satisfactory for quantitative clinical PET scan studies using [¹¹C]. The in vivo binding of [¹¹C] Ro 15-1788 in the cerebral cortex of cynomolgus monkeys and healthy human subjects was reduced by approximately 90% within 10 min after the intravenous injection of a high dose of unlabelled Ro 15-1788 (0.5 mg/kg i.v.). Different areas of the healthy human brain showed an approximately 10-fold variation in maximal [¹¹C] Ro 15-1788 binding that corresponded to the previously known distribution of benzodiazepine receptors in these regions. The highest degree of binding was obtained in the medial occipital cerebral cortex followed by frontal cortex, cerebellum, thalamus, striatum and pons. Two psychiatric patients with anxiety syndromes who had been treated for a long time with high doses of benzodiazepines had roughly the same degree of maximal [¹¹C] Ro 15-1788 binding in brain regions as the healthy subjects but the rate of decline of [¹¹C] Ro 15-1788 in the brain was higher. This indicates that there is measurable competition between [¹¹C] Ro 15-1788 binding and clinical benzodiazepine concentrations in the body fluids of psychiatric patients. The results demonstrate that [¹¹C] Ro 15-1788 should be a valuable tool for quantitative analyses of benzodiazepine receptor characteristics and receptor occupancy in the brain of patients with neuropsychiatric disorders.     

Effects of choline and glucose on atropine-induced alterations of acetylcholine synthesis and content in the caudate nuclei of rats

Atropine-induced decrease in the concentration of acetylcholine (ACh) in the brain can be diminished by pretreating the animals with a large dose of choline^72,73. The injected choline might act by improving the supply of substrate for the synthesis of ACh, or by competing with atropine for presynaptic muscarinic receptors and thus decreasing the release of ACh. Since, under in vitro conditions, changes in the release of ACh are reflected by changes in its synthesis, experiments have been performed on slices of rat caudate nuclei to check whether an increase in the extracellular concentration of choline causes a decrease in the atropine-induced stimulation of ACh synthesis. The rate of [¹⁴C]ACh synthesis from [¹⁴C]glucose was faster in the presence of 530 μM than 30 μM choline, and 5 μM atropine stimulated the synthesis of [¹⁴C]ACh more at 530 μM than at 30 μM choline in the medium. These observations substantiate the view that the administration of choline in vivo acts by supporting the synthesis of ACh, rather than by inhibiting its release.

In subsequent experiments on rats in vivo, an attempt has been made to influence the ACh-depleting action of atropine by pretreatment with a large dose of glucose as a distant precursor of acetyl-CoA, the second substrate for the synthesis of ACh. The administration of atropine (25 mg/kg) diminished the content of ACh in the caudate nuclei by 48%; if the injection of atropine followed after an injection of glucose (20.2 mmol/kg), the content of ACh was diminished by only 25%. It appears likely that glucose acted by improving the availability of acetyl-CoA for the synthesis of ACh and that the supply of either precursor of ACh (i.e. choline and acetyl-CoA) may become rate-limiting when the demands on the synthesis of ACh in the brain are increased.     

Protein kinase C in focal ischemic rat brain: dual autoradiographic analysis of [C]iodoantipyrine (IAP) and [H]phorbol-12,13-dibutyrate (PDBu)

Protein kinase C (PKC) activity was measured in rat brain with 2 h of middle cerebral artery (MCA) and common carotid artery (CCA) occlusion, using dual autoradiography of [¹⁴C]iodoantipyrine (IAP) and [³H]phorbol-12,13-dibutyrate (PDBu). In the ischemic brain, it required more than 120 min of incubation to obtain a plateau in PDBu binding. In contrast, the binding of PDBu in non-ischemic brain reached a plateau with incubation for 60 min. This delay of PDBu binding in the ischemic brain suggests that the affinity of this ligand is reduced due to a change in structure of the cell membrane caused by ischemia. PDBu binding in the ischemic brain increased significantly compared to the non-ischemic brain. This finding provides further evidence that excessive activation of PKC in the ischemic brain may play an important role in ischemic neuronal damage. ©1997 Elsevier Science B.V. All rights reserved.     

Widespread reductions in cerebral blood flow and metabolism elicited by electrical stimulation of the parabrachial nucleus in rat

We have studied the effect of electrical stimulation of the parabrachial nucleus (PBN) and adjacent areas of dorsal pons on regional cerebral blood flow (rCBF) and glucose utilization (rCGU) in anesthetized (chloralose), paralyzed (tubocurarine) rats. rCBF and rCGU were measured in dissected tissue samples of 9 brain regions by the [¹⁴C]iodoantipyrine and [¹⁴C]2-deoxyglucose method, respectively. Electrical stimulation restricted to the medial parabrachial nucleus (PBNm, n=5) elicited significant (P < 0.05) reductions in rCBF in 7 out of 9 brain regions. Reductions were greatest in cerebral cortex (up to 35% in occipital cortex) and least in the white matter of the corpus callosum (23%). The effect on rCBF persisted after transection of the cervical sympathetic trunk (n=5). In contrast, stimulation of the lateral portion of PBN (n=5), periventricular gray (n=5) and interestingly, the nucleus locus coeruleus (n=5) failed to elicit similar changes in rCBF. PBNm stimulation also elicited decreases in rCGU (n=4) in 5 out of 9 brain areas, most notably regions of cerebral cortex. The decreases in rCGU (ΔrCGU) were linearly related to the decreases in rCBF (ΔrCBF) according to the equationΔrCBF=2.37 ΔrCGU+2.1 (r=0.72; P<0.001). We conclude that excitation of neural pathways originating in, or passing through, PBNm elicits a widespread reduction in cerebral metabolism and secondarily in blood flow (secondary vasoconstriction). Since projections of the PBNm do not involve the entire cortex, it seems likely that the effect is mediated via inhibition of diffuse cortical projections through a subcortical site.     

Loss of muscarinic M4 receptors in hippocampus of Alzheimer patients

We assessed muscarinic M₁, M₂ and M₄ receptor subtypes in the hippocampus of Alzheimer’s and control brains by receptor autoradiography using ligands such as [¹²⁵I]muscarinic toxin-1 ([¹²⁵I]MT-1, M₁ selective), [³H]AFDX-384 (M₂ partially selective) and [¹²⁵I]muscarinic toxin 4 ([¹²⁵I]M₄ toxin-1, M₄ selective). Our results revealed a significant decrease in muscarinic M₄ receptor binding in the dentate gyrus and CA4 regions of brain sections from Alzheimer’s patients compared to controls. No changes in the density of M₁ or M₂ receptor binding were observed. Our findings suggest that, relative to other muscarinic receptor subtypes, the M₄ receptor could be the subtype which is selectively compromised in Alzeheimer’s disease (AD).     

Glucose utilization increases in choroid plexus during slow wave sleep. A[C] deoxyglucose study in the cat

Glucose utilization (GU) was measured during spontaneous waking and slow-wave sleep (SWS) by adaptation of the [¹⁴C]deoxyglucose method to the unrestrained cat. In sleeping animals a greater autoradiographic signal between choroid plexus (CP) and the rest of brain was noticed. Quantification provided an index of the metabolic rate of CP and confirmed that mean values for GU were significantly higher in ‘sleeping’ than in ‘awake’ cats.     

Photoperiodic effects on puberty and specific 2-[I]iodomelatonin binding sites in Siberian hamsters

When juvenile male Siberian hamsters are transferred from a long photoperiod to a short photoperiod, sexual maturation is greatly delayed by a pineal-dependent process. We hypothesized that the eventual onset of puberty during short photoperiod exposure may be caused by a loss of receptors for the pineal hormone, melatonin. This study quantitated specific 2-[¹²⁵I]iodomelatonin binding sites in the suprachiasmatic nuclei and pars tuberalis of Siberian hamsters exposed to short photoperiod (10 h light per day) for either 12 or 30 weeks and in hamsters exposed to long photoperiod (16 h light per day) for the same time intervals. Photoperiodic exposure significantly affected testes weight. The hamsters exposed to long photoperiod for either 12 or 30 weeks had mean testes weights > 700 mg, in contrast to hamsters in short photoperiod for 12 weeks (mean testes weights < 30 mg) or 30 weeks (mean testes weights approximately 350 mg). The affinity of specific 2-[¹²⁵I]iodomelatonin binding sites in both regions was significantly lower in hamsters exposed to short photoperiod as compared to hamster exposed to long photoperiod, at either 12 or 30 weeks. In contrast, there were no effects of photoperiod or duration of exposure on the density of specific 2-[¹²⁵I]iodomelatonin binding sites in either the suprachiasmatic nuclei or the pars tuberalis. Furthermore, a change in the affinity of the specific 2-[¹²⁵I]iodomelatonin binding sites in the suprachiasmatic nuclei was observed between the hamsters housed in short photoperiod for 12 weeks (sexually immature) and the hamsters housed in short photoperiod for 30 weeks (undergoing puberty). These results demonstrate that although the onset of puberty after long-term exposure to short photopoeriod does not involve a loss of specific 2-[¹²⁵I]iodomelatonin binding sites in the suprachiasmatic nuclei or pars tuberalis, it is associated with a decrease in the affinity of specific 2-[¹²⁵I]iodomelatonin binding sites in these regions.     

Homologous desensitization of human corticotropin-releasing factor, receptor in stable transfected mouse fibroblast cells

Previous radioligand binding and second messenger studies have shown that corticotropin-releasing factor (CRF) modulates its receptor following both in vivo and in vitro treatment. In the present study, we determined the sequence of events leading to CRF-induced downregulation and desensitization of cloned CRF receptors in murine fibroblast cells (Ltk⁻) stably transfected with CRF, DNA (from human pituitary). Treatment of cells with rat/human CRF produced a dose- and time-dependent decrease in [¹²⁵I]Tyr^o-ovine CRF ([¹²⁵I]oCRF) binding and a concomitant decrease in CRF-stimulated adenylate cyclase activity. Significant decreases in [¹²⁵I]oCRF binding and agonist-stimulated cAMP production were evident minutes after CRF treatment with maximal (60–80%) reductions seen following 1 h of CRF treatment. Scatchard analysis revealed that the decrease in [¹²⁵I]oCRF binding was due to the downregulation of the receptor with no significant alteration seen in the affinity of the ligand. Since the transfected cell line is engineered using an artificial promoter, we did not detect any significant changes in CRF₁ receptor mRNA levels following CRF treatment for up to 24 h.     

The opiate receptor: a single 110 kDa recognition molecule appears to be conserved in Tetrahymena, leech, and rat

We compared the molecular nature of the rat brain opiate receptor with that of the invertebrate leech, Haemopis marmorata, and the protozoan, Tetrahymena, in order to examine the issue of apparent receptor heterogeneity with respect to biochemical structure. A binding study with rat brain membrane verified that [¹²⁵I]β-endorphin ([¹²⁵I]βE), a broad specificity ligand, is displaced by the antagonist (-)-naloxone, but not the inactive stereoisomer (+)-naloxone; agonists considered prototypes for μ, δ, and κ opiate receptors all displayed stereospecific binding displacement. For SDS-PAGE analysis of the opiate receptor [¹²⁵I]β-endorphin was covalently affixed to its recognition molecule with the cross-linking reagent DSS. Primary reaction products occur at 110, 58/55, and 29 kDa. Cross-linking products of all 3 molecular weights are effectively reversed by opiate ligands, regardless of their μ, δ, or κ specificities. Peptide mapping studies in SDS gels, using limited proteolysis, showed that the 110 kDa band can be digested into 58 and 29 kDa fragments and the 58 kDa band into a 29 kDa fragment. Additional smaller molecular weight fragments were generated from the 110, 58/55, and 29 kDa bands which shared their molecular weights. Two possible explanations for the extensive homology between the three major cross-linking products are: (1) the 110 kDa species is the opiate receptor, and the 58 and 29 kDa species are proteolytic fragments; and (2) one of the lower molecular weight species is the opiate receptor, and adjacent receptors are aggregated into the 110 kDa complex through cross-linking. An evolutionary conservation of the opiate receptor is suggested by the presence of the same 3 major cross-linking products in Tetrahymena, leech, and rat.     

The effect of an acute nicotine infusion on the local cerebral glucose utilization of the awake rat

The effect of acute infusion of nicotine on local cerebral glucose utilization (LCGU) was studied in discrete regions of the central nervous system of the rat by means of the quantitative autoradiographic 2-deoxy-d-[1-¹⁴C]glucose method described by Sokoloff et al.²⁶. Nicotine was administered in 3 dosages: 0.5 μg/kg/min and 5 μg/kg/min. The resulting plasma concentrations of nicotine were 10/39/114 ng/ml plasma. During the experiment, blood pressure, heart rate, body temperature, hematocrit, acid-base status and plasma glucose concentration showed no — or minor — changes. Nicotine significantly increased LCGU in a dose-dependent manner in the following 9 of 45 examined structures: substantia nigra (compact part), superior colliculus (superficial grey layer), interpeduncular nucleus and cingulate cortex (P < 0.05); lateral geniculate body, optic chiasm, anteroventral and anteromedial nucleus of thalamus and mamillary body (P < 0.05). For most of these structures with increased LCGU, other groups have reported a high regional receptor binding of nicotine (exception: mamillary body and optic chiasm). It is concluded that nicotine has distinct effects on the functional activity of localized brain areas.     

Vasopressin binds to microvessels from rat hippocampus

Recent evidence suggests that vasopressin may influence the permeability of the endothelium of brain capillaries. We measured the binding of [¹²⁵I]arginine-8-vasopressin ([¹²⁵I]AVP) to microvessels isolated from different regions of the rat brain. The study revealed saturable and specific binding of [¹²⁵I]AVP to microvessels isolated from hippocampus. Scatchard analysis confirmed a single class of high affinity sites with an equilibrium dissociation constant,K_d, of 3.2 nM and an apparent maximal binding capacity of 205 fmol/mg protein. No binding was observed to microvessels from neocortex and striatum.