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.     

Distribution of GLP-1 Binding Sites in the Rat Brain: Evidence that Exendin-4 is a Ligand of Brain GLP-1 Binding Sites

The distribution and biochemical properties of glucagon-like peptide (GLP)-1(7–36)amide (GLP-1) binding sites in the rat brain were investigated. By receptor autoradiography of tissue sections, the highest densities of [¹²⁵I]GLP-1 binding sites were identified in the lateral septum, the subfornical organ (SFO), the thalamus, the hypothalamus, the interpenduncular nucleus, the posterodorsal tegmental nucleus, the area postrema (AP), the inferior olive and the nucleus of the solitary tract (NTS). Binding studies with [¹²⁵I][Tyr39]exendin-4, a GLP-1 receptor agonist, showed an identical distribution pattern of binding sites. Binding specificity and affinity was investigated using sections of the brainstem containing the NTS. Binding of [¹²⁵I]GLP-1 to the NTS was inhibited concentration-dependently by unlabelled GLP-1 and [Tyr39]exendin-4 with K ₁ values of 3.5 and 9.4 nM respectively. Cross-linking of hypothalamic membranes with [¹²⁵I]GLP-1 or [¹²⁵I][Tyr39]exendin-4 identified a single ligand-binding protein complex with a molecular mass of 63 000 Da. The fact that no GLP-1 binding sites were detected in the cortex but that they were detected in the phylogenetically oldest parts of the brain emphasizes that GLP-1 may be involved in the regulation of vital functions. In conclusion, the biochemical data support the idea that the central GLP-1 receptor resembles the peripheral GLP-1 receptor. Furthermore, the presence of GLP-1 binding sites in the circumventricular organs suggests that these may be receptors which act as the target for both peripheral blood-borne GLP-1 and GLP-1 in the nervous system.     

Rats bred for enhanced apomorphine susceptibility have elevated tyrosine hydroxylase mRNA and dopamine D2-receptor binding sites in nigrostriatal and tuberoinfundibular dopamine systems

From a Wistar population two rat lines were generated using as criterion the behavioral response to the dopamine agonist apomorphine. Rats of the apomorphine-susceptible (apo-sus) line revealed a vigorous gnawing response to apomorphine administration while the other rat line, the apomorphine-unsusceptible (apo-unsus) line, was selected for lack of response to the drug. In the present study using the 12th and 13th generation of these genetically selected lines, we have investigated whether this difference in apomorphine responsiveness was correlated with changes in dopamine neurochemistry. Therefore, we measured tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis, as well as dopamine D₁ and D₂ receptor mRNA levels in discrete brain regions by in situ hybridization. Dopamine (D₂/D₃) receptor binding was assessed with [¹²⁵I]iodosulpride in a membrane binding assay and by quantitative autoradiography on tissue sections. [³H]SCH 23390 was used to analyze D₁ receptor binding. Apo-sus rats displayed significantly higher TH mRNA levels in the A₉ cell group of the substantia nigra pars compacta and in the A₁₂ cell group of the arcuate nucleus. No difference was found in the A₁₀ cell group of the VTA and the A₆ cell group of the locus coeruleus. The density ofD_2/3 binding sites as well as D₁ receptor mRNA levels in the striatal projection area of the A₉ substantia nigra neurons, were significantly elevated in apo-sus rats. Dopamine D₂ receptor mRNA and D₁ receptor binding levels in caudate putamen and nucleus accumbens, however, were similar in rats of both lines. In conclusion, high apomorphine susceptibility is related to a potentially enhanced dopamine responsiveness selective for the nigrostriatal and tuberoinfundibular pathways.     

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.     

Localization and quantification of the dopamine transporter: comparison of [H]WIN 35,428 and [I]RTI-55

Transport into the presynaptic terminal by the dopamine transporter is the primary mechanism for removing dopamine from the synaptic cleft. This transporter is a specific marker for dopamine terminals and is a primary site for CNS actions of cocaine. Several radioligands have been developed for analysis of the dopamine transporter. The ligands vary in affinity and specificity, leading to differences in reported transporter density in brain regions. We compared two of the most commonly used ligands, [³H]WIN 35,428 and [¹²⁵I]RTI-55, analyzing the localization and density of sites in the rat brain using serial sections and quantitative autoradiography. Citalopram at 50 nmol/1 was used to block [¹²⁵I]RTI-55 binding to serotonin transport sites. Transporter density was highest in the striatum and both ligands labeled equivalent numbers of sites, with lateral to medial and anterior to posterior gradients. In most areas the density of sites measured with the two ligands was similar. However, [¹²⁵I]RTI-55 binding was significantly higher than [³H]WIN 35,428 binding in the substantia nigra zona compacta, ventral tegmental area, subthalamic nucleus and a number of other subcortical nuclear groups while [³H]WIN 35,428 binding was higher in lateral striatum and in olfactory tubercle. These differences could reflect different forms of the transporter, perhaps due to post-translational modifications, and they may provide a basis for differential pharmacological regulation of transporter function in discrete brain regions and disease states.     

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.     

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).     

Distinct binding sites for calcitonin gene-related peptide and salmon calcitonin in rat central nervous system

Calcitonin gene-related peptide (CGRP) binding sites have been identified in homogenates from the rat brain and spinal cord. Autoradiography with [¹²⁵I]rat CGRP (rCGRP) revealed high grain density over the lateral hypothalamus, vestibular nuclei, colliculi, medial geniculate body, corpus mamillare and the molecular layer of the cerebellum which lacked binding sites for [¹²⁵I]salmon calcitonin (sCT). In contrast, no rCGRP labeling was seen over the anterior and dorsomedial hypothalamus which showed high sCT binding. The different regional distribution of rCGRP and sCT binding sites indicates that the structurally related peptides interact with separate receptors. The overlap between the localization of CGRP binding sites and endogenous CGRP in many regions of the central nervous system suggests that CGRP exerts unique physiological functions in the central nervous system.     

Galanin Receptor Binding Sites in Adult Rat Spinal Cord Respond Differentially to Neonatal Capsaicin, Dorsal Rhizotomy and Peripheral Axotomy

The discrete distribution and possible changes in specific [¹²⁵I]galanin binding sites were evaluated in the rat spinal cord following neonatal capsaicin treatment, dorsal rhizotomy and sciatic nerve section. The highest density of [¹²⁵I]galanin binding sites in the normal rat spinal cord was particularly evident in the superficial layers of the dorsal horn whereas moderate to low amounts of labelling were associated with the deeper dorsal horn, areas around the central canal and the ventral horn. Capsaicin-treated rats, compared to littermate controls, showed a significant bilateral increase in [¹²⁵I]galanin binding in the superficial laminae of the dorsal horn. Similarly, unilateral dorsal rhizotomy evoked a significant increase in the density of [¹²⁵I]galanin binding sites in the superficial dorsal horn ipsilateral to surgery. Section of the sciatic nerve, on the other hand, induced a significant depletion in [¹²⁵I]galanin binding in laminae I and II of the ipsilateral dorsal horn. These results, in parallel to those reported for galanin immunoreactivity under similar conditions, suggest that [¹²⁵I]galanin binding sites are preferentially located postsynaptically to the primary afferent fibre terminals in the dorsal horn of the spinal cord. Thus it seems that galanin, at the level of the dorsal spinal cord, regulates the processing of nociceptive information by acting on its own class of specific receptors located postsynaptically to primary sensory terminals.     

Endocytosis of nerve growth factor by ‘differentiated’ PC12 cells studied by quantitative ultrastructural autoradiography

The endocytosis of [¹²⁵I]nerve growth factor (NGF) by rat pheochromocytoma cells (PC12 line), previously exposed to the growth factor (‘differentiated’ or ‘primed’ cells), was studied by ultrastructural quantitative autoradiography. Cells previously grown in the presence of NGF were incubated at 37 °C with [¹²⁵I]NGF for periods of up to 24 h. Under these culture conditions, PC12 cells have a rich network of neurites.

At the commencement of the experiment, after incubation of cells with [¹²⁵I]NGF for 1 min at room temperature, the plasma membranes of perikarya and processes showed similar levels of labeling by [¹²⁵I]NGF of0.186 ± 0.03grains/μm and0.152 ± 0.013grains/μm respectively.

The density of grains per micron of plasma membrane of perikarya reached a plateau between 15 min to 2 h of incubation of cells at 37 °C with [¹²⁵I]NGF (0.58 ± 0.15grains/μm and0.65 ± 0.18grains/μm, respectively).

The endocytosis of [¹²⁵I]NGF in perikarya of cells incubated for 6 h at 37 °C was studied by the ‘mask’ analysis method of Salpeter et al.²². At this time, the greatest amount of endocytosis was observed, corresponding to 28.4% of total grain counts. The following optimized computed source densities, or relative specific activities± standard errors of measurement (S.E.M.), were obtained: plasma membrane,16.52 ± 0.86; multivesicular bodies,9.58 ± 2.84; endosomes,5.00 ± 0.97; smooth vesicles and tubules,1.66 ± 0.38; lysosomes,1.13 ± 0.20; mitochondria,0.46 ± 0.10; nuclear membranes or envelopes,0.32 ± 0.14; nuclei,0.06 ± 0.01; the Golgi apparatus,0.08 ± 0.06; and other cytoplasmic elements0.07 ± 0.03.

Our findings indicate that smooth vesicles and tubules, endosomes, multivesicular bodies and lysosomes are part of the pathway(s) of endocytosis of NGF, while all other cytoplasmic and nuclear elements, including the nuclear membrane, are not.

The heavy plasma membrane labeling of NGF and the relatively low degree of its endocytosis are consistent with the hypothesis that the NGF action is mediated through plasma membrane activated second messenger(s).     

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.     

Evidence for Multiple Opioid Receptors in the Human Posterior Pituitary

The distribution and quantification of opioid receptor types in post-mortem human pituitary cryostat sections was determined by quantitative in vitro receptor autoradiography. Highly specific radioligands were used for each opioid receptor type, i.e. [¹²⁵I]-FK-33-824 for μ-opioid sites, [¹²⁵I][D.Ala²]-Deltorphin-I for δ -opioid sites and ³H-U69,593 for κ -opioid sites.
None of the five specimens tested exhibited any labelling in the anterior lobe of the pituitary for the three radioligands. As for the posterior pituitary, all 5 specimens contained both μ and κ -opioid binding sites whereas none of them showed δ -binding sites.
The presence of both μ- and κ -opioid binding sites in the human posterior pituitary contrasts with previous findings in the rat where only κ -sites have been found. The present study could contribute to understanding of the functional action of opioids in the human pituitary.     

Somatostatin Depletion by Cysteamine Increases Somatostatin Binding and Growth Hormone-Releasing Factor Messenger Ribonucleic Acid in the Arcuate Nucleus

We have previously described somatostatin (SRIF) pericellular binding sites in the vicinity of growth hormone-releasing factor (GRF)-containing cells in the ventrolateral part of the arcuate nucleus (ARC) of the male rat. To further assess the direct role of SRIF on GRF messenger ribonucleic acid (mRNA) levels in the mediobasal hypothalamus, we depleted endogenous SRIF by cysteamine (CS; 300 mg/kg body wt 6 h prior to sacrifice). In the ventrolateral part of the ARC, there was a 2-fold increase (P<0.05) in [¹²⁵I]SRIF specific binding and GRF mRNA-labelled cell numbers in the CS-treated group as compared to control animals. Furthermore, there was a positive correlation between [¹²⁵I]SRIF binding and the number of GRF mRNA-labelled cells (r = 0.89; P<0.01). In contrast, such effects were not observed along the base of the ventromedial nucleus where pericellular [¹²⁵I]SRIF binding was not associated with GRF mRNA-labelled cells. These results provide functional evidence for a direct SRIF inhibition, through specific receptors, of GRF mRNA levels in ARC neurons.     

Characterization of a Distinctive Motif of the Low Molecular Weight Neurotrophin Receptor that Modulates NGF-mediated Neurite Growth

The cytoplasmic region of the common neurotrophin receptor (p75^NGFR) (rat, human, chick) contains a putative membrane-associating domain implicated in intracellular signalling. A peptide (R3) identical to this domain (p75^NGFR 367–379) and various analogues of this peptide displayed circular dichroism spectra in aqueous and non-polar environments identical to the amphiphilic tetradecapeptide mastoparan (MP) and were internalized by PC12 rat pheochromocytoma cells. The R3 peptide enhanced neurite growth in PC12 cells, embryo chick primary sensory neurons and fetal rat primary sensory neurons in vitro in the presence of sub-saturating concentrations of NGF. Peptide analogues of R3 not faithful to the distance and angular relationships of ionic groups and the putative amphiphilic structure of p75^NGFR 367–379 displayed reduced potency to enhance NGF-mediated neurite growth. Exposure of NGF and the R3 peptide to a cell line displaying predominantly p75^NGFR (PC12^nnr5), had no influence on neurite growth. The R3 peptide had no effects on cell survival, cell binding or uptake of [¹²⁵I]NGF, affinity cross-linking of [¹²⁵I]NGF to p75^NGFR or trkA monomers and homodimers, or NGF-mediated trkA monomer tyrosine phosphorylation. The studies implicate a role for a highly conserved motif of p75^NGFR in the downstream modulation of NGF-mediated neurite growth.     

Differential alterations of ion channel binding sites in temporal and occipital regions of the cerebral cortex in Alzheimer''s disease

Three ion channel binding sites were examined by means of quantitative ligand binding autoradiography in temporal and occipital cortex from 9 patients with neuropathologically confirmed Alzheimer's disease (AD) and 7 matched control subjects. The following ligands were used: ¹²⁵I-apamin to label a population of Ca²⁺-sensitive K⁺ channels; [³H]PN200-110 to label L-type voltage-sensitive Ca²⁺ channels and [³H]glibenclamide to label ATP-sensitive K⁺ channels. Ion channel binding sites were compared to: choline acetyltransferase (ChAT) activity and plaque densities measured in the same tissue. In the temporal cortex in AD¹²⁵I-apamin binding was increased compared to controls (e.g. superficial layers: control= 0.71 ± 0.07;AD= 1.02 ± 0.07,mean±S.E.M. pmol/g tissue). In contrast, in adjacent sections [³H]glibenclamide binding was reduced in AD compared to controls (e.g. superficial layers: control= 25.3 ± 1.7;AD= 17.9 ± 1.4pmol/g tissue). [³H]PN200-110 binding in temporal cortex was not altered in AD compared to controls. In the occipital cortex¹²⁵I-apamin binding was increased in AD while both [³H]glibenclamide and [³H]PN-200-110 binding sites in this cortical area were not different from controls. Plaque density (per mm²) was higher in temporal (e.g. layers I–III, 43 ± 6) than in occipital cortex (layers I–III, 27 ± 4) in the AD patients while ChAT was reduced by 40% in temporal cortex and by 50% in occipital cortex compared to controls. The results suggests that the three ion channel binding sites are located on structural elements in the brain which are differentially affected by the pathophysiology of AD.     

Optic nerve transection decreases 2-[I]iodomelatonin binding in the chick optic tectum

The distribution of specific 2-[¹²⁵I]iodomelatonin binding sites in the various layers of the chick optic tectum was analyzed using quantitative receptor autoradiography. Following unilateral optic nerve transection, binding in the optic fiber layer and superficial retinorecipient layers of the contralateral tectum was significantly decreased at 7 and 14 days, but not at 1 day, following transection. The results are consistent with the presence of presynaptic melatonin receptors on axon terminals of retinotectal fibers.     

A radioisotopic method for the simultaneous quantitation of regional cerebral blood flow and glucose utilization in small dissected samples: Validation studies and values in the nitrous oxide-anesthetized rat

A method is described for the simultaneous determination of the rates of regional cerebral blood flow (rCBF) and regional cerebral glucose utilization (rCMRgl) in 6–7 mg brain samples dissected from multiple areas of interest. The method utilizes [¹³¹I]-iodoantipyrine ([¹³¹l]IAP) to measure rCBF by indicator fractionation, and [¹⁴C]2-deoxyglucose to measure rCMRgl. [¹³¹I]IAP was synthesized with specific activity exceeding 350 Ci/mmol and radiochemical purity greater than 99.5% by the radioiodination of antipyrine with Na¹³¹I. A triple-counting strategy was developed to quantitate¹⁴C activity of the dissected brain samples in the presence of¹³¹I. The factors contributing to the propagated error of the double-label separation strategy were defined and optimal assay parameters were determined. The separation strategy was validated by measuring rCBF simultaneously with both [¹³¹I]IAP (x) and [¹⁴C]IAP (y) in a series of rats. The equation of the regression line was y = 1.025 x −0.065 (correlation coefficient 0.985), denoting excellent agreement. In another series of 5 normocapnic rats anesthetized with nitrous oxide, rCBF and rCMRgl were measured simultaneously. In individual animals, the rates of rCBF within 14–16 brain areas were closely coupled to their respective rates of glucose metabolism. For the group data, the linear regression equation relating rCBF (y) to rCMRgl (x) was y = 1.76 x + 0.13 (correlation coefficient 0.93,P < 0.001). These studies provide direct evidence, based upon data obtained in the same brain, of a close coupling of regional metabolic rate and blood flow.     

Reduction of insulin binding in the arcuate nucleus of the rat hypothalamus after 6-hydroxydopamine treatment

Insulin receptors are present in the hypothalamus, but the cell types bearing them are unknown. In order to test the hypothesis that some insulin receptors in the hypothalamus are associated with catecholamine terminals, rats were injected with 50 μg or 75 μg doses (intracerebroventricular) of 6-hydroxydopamine (6-OHDA). Control rats received vehicle only. The animals were sacrificed 7 days after injection, and catecholamine and indolamine levels in the hypothalamus were measured by high performance liquid chromatography with electrochemical detection. Localization of specific binding sites for [¹²⁵I]-insulin in the arcuate (ARC), dorsomedial (DMN) and ventromedial (VMN) nuclei were determined by quantitative film autoradiography. Treatment with 6-OHDA resulted in a 70% reduction in hypothalamic norepinephrine content as compared to vehicle-treated controls (P < 0.01). A slight depletion of epinephrine, dopamine and indolamines was also detected. Computerized image analysis of the autoradiograms was used to determine radioactivity bound (DPM/mm²) in each nucleus. Highest binding was in the ARC and DMN, with much lower binding in the VMN. Insulin binding in the ARC of the 6-OHDA-treated group was decreased by 25% compared to controls (P < 0.01). No significant change in insulin binding was observed in the DMN or VMN. The 6-OHDA treatment had no significant effect on weight gain or on plasma insulin levels. The reduction of insulin binding in the ARC after 6-OHDA treatment supports the hypothesis that some insulin binding sites are located on catecholamine terminals in the arcuate nucleus.     

Binding of [125I]insulin to specific receptors and stimulation of nucleotide incorporation in cells cultured from rat brain

The occurrence of insuling receptors and biological responses to insulin has been investigated in trypsin-dissociated fetal rat brain cells maintained in culture for 8 days. Binding of [¹²⁵]insulin to brain cells in culture was time- and pH-dependent and 85–90% specific. Porcine insulin competed for [¹²⁵]insulin binding in a dose-dependent manner. Unrelated polypeptides, including angiotensin II, glucagon, bovine growth hormone, and bovine prolactin did not compete for [¹²⁵]insulin binding. The half-life of [¹²⁵]insulin dissociation from receptors at 24°C was 15 min and a plot of ln[B/Bo] vs time suggested two dissociation rate constants of2.7 × 10⁻⁴ sec⁻¹ and5.0 × 10⁻⁵ sec⁻¹. Scatchard analysis of the binding data gave a curvelinear plot which may indicate negative cooperativity or the occurrence of both high affinity(K_a = 2 × 10¹¹M⁻¹) and low affinity(K_a = 4 × 10¹⁰M⁻¹) sites. Of the estimated total of 4.9 × 10⁴ binding sites per cell, 28–30% appear to be high affinity sites.

Incubation of cultures with insuling caused a time- and dose-dependent stimulation of [³H]thymidine and [³H]uridine incorporation into TCA-precipitable material. Maximum stimulation of thymidine incorporation (2–5-fold) occured 11 h after incubation with 167 nM insulin. The same concentration of insulin caused a 2.2-fold increase in [³H]uridine incorporation in 2 h. These results indicate that cells cultured from rat brain contain specific insulin receptors capable of mediating effects of insulin on macromolecular synthesis in the central nervous system.     

Cross-reactivities of neuropeptide Y and peptide YY with pancreatic polypeptide antisera: evidence for the existence of pancreatic polypeptide in the brain

Highly purified neuropeptide Y (NPY) and peptide YY (PYY) did not cross-react in our human pancreatic polypeptide (hPP) radioimmunoassay, nor did ¹²⁵I-labelled NPY and PYY, even with anti-hPP serum at low dilution (1:1000). However, both [¹²⁵I]NPY and [¹²⁵I]PYY significantly cross-reacted with anti-bovine PP (bPP) serum at low dilution (1:1000, similar to that used in immunohistochemistry). These results suggest that radioassayable hPP-like peptide in the porcine or canine brain is probably pancreatic polypeptide itself, otherwise immunohistochemically detected bPP-like peptide may represent both NPY and PP.