Transferrin is required for normal distribution of Fe and Mn in mouse brain

Hypotransferrinemia (hpx/hpx) is a genetic defect in mice resulting in <1% of normal plasma transferrin (Tf) concentrations; heterozygotes for this mutation (+/hpx) have low circulating Tf concentrations. These mice provide a unique opportunity to examine the role of Tf in Fe and Mn transport in the brain. Twenty weanling wild-type BALB/cJ mice, 15 +/hpx mice, and 12 hpx/hpx mice of both sexes were injected i.v. with either ⁵⁴MnCl₂ or ⁵⁹FeCl₃ either 1 h or 1 week before killing at 12 weeks of age. Total brain counts of ⁵⁴Mn and ⁵⁹Fe were measured, and regional brain distributions were assessed by autoradiography. Hypotransferrinemia did not affect total brain Mn uptake. However, 1 week after i.v. injection, hpx/hpx mice had less ⁵⁴Mn in forebrain structures including cerebral cortex, corpus callosum, striatum, and substantia nigra. The +/hpx mice had the highest total brain ⁵⁹Fe accumulation 1 h after i.v. injection. A striking effect of regional distribution of ⁵⁹Fe was noted 1 week after injection; in hpx/hpx mice, ⁵⁹Fe was located primarily in choroid plexus, whereas in +/+ and +/hpx mice ⁵⁹Fe was widely distributed, with relatively high amounts in cerebral cortex and cerebellum. We interpret these data to mean that Tf is necessary for the transport of Fe but not Mn across the blood–brain barrier, and that there is a Tf-independent uptake mechanism for iron in the choroid plexus. Additionally, these data suggest that endogenous synthesis of Tf is necessary for Fe transport from the choroid plexus.     

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.     

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).     

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.     

Inhibition of nitric oxide synthase attenuates blood-brain barrier disruption during experimental meningitis

Increased permeability of the blood-brain (B-B) barrier is observed during meningitis. Preventing B-B barrier alterations is important because adverse neurological outcomes are correlated with breeches in barrier integrity. It was hypothesized that pathological production of nitric oxide (NO) contributes to B-B barrier disruption during meningitis in the rat. Experimental meningitis was induced by intracisternal (i.c.) administration of lipopolysaccharides (LPS) or vehicle. Groups of rats were concomitantly infused intravenously (i.v.) with saline or the NO synthase inhibitor, aminoguanidine (AG). Eight h after i.c. dosing, B-B barrier alterations were quantitated pharmacokinetically using [¹⁴C]sucrose. Serum and regional brain tissues were obtained 0–30 min after tracer dosing and sucrose influx transfer coefficients ( K_{in (app)}) were calculated from the brain tissue data. Compared to the control groups (i.c. vehicle/i.v. saline), the K_{in (app)} of the i.c. LPS/i.v. saline group increased 1.6–2.1-fold in various brain regions, thus confirming previous observations of increased [¹⁴C]sucrose barrier penetration during meningeal inflammation. Remarkably, i.v. administration of AG to i.c. LPS-treated rats significantly inhibited meningeal NO synthesis and decreased K_{in (app)} permeability alterations in the B-B barrier, compared to i.c. LPS/i.v. saline-treated rats. Regional brain K_{in (app)} estimates in the i.c. LPS/i.v. AG group were similar to control groups (i.c. vehicle/i.v. AG and i.c. vehicle/i.v. saline). In conclusion, these data suggest the general concept that excessive NO production during neuroinflammatory diseases contributes to disruption of the blood-brain barrier.     

PHARMACOKINETICS, ABSORPTION, DISTRIBUTION AND DISPOSITION OF [I]-OLIGOTIDE FOLLOWING INTRAVENOUS OR ORAL ADMINISTRATION IN THE RAT

Oligotide (O) was labelled with ¹²⁵I. The radiolabelled compound ([¹²⁵I]-Oligotide ([¹²⁵I]-O)) retained the biological activity of parent O. Following single intravenous administration the half lives of radioactivity associated with O and/or O related components in plasma were 9–10 min and 9–10 h for and β phases respectively. Following single oral administration the half life of radioactivity associated with O and/or O related components in plasma was 11.45 – 12.76 h for β fase. Following multiple oral administration once daily for 7 days, the half life of radioactivity associated with O and/or O related components following the 7th dose was 10–12 h for β phase. The areas under plasma total radioactivity versus time curves were dose-dependent. Following single intravenous administration the major proportion of the administered dose was excreted via urine, while following single oral administration excretion via urine and faeces accounted for similar proportions of the administered dose. Following both single and oral administration the levels of radioactive components derived from [¹²⁵I]-O in organs examined were generally highest in highly perfused organs. © 1997 Elsevier Science Ltd     

Evidence for low molecular weight,non-transferrin-bound iron in rat brain and cerebrospinal fluid

Transferrin (Tf) donates iron (Fe) to the brain by means of receptor-mediated endocytosis of Tf at the brain barriers. As Tf transport through the brain barriers is restricted, Fe is probably released into the brain extracellular compartment as non-Tf-bound iron (NTBI). To evaluate NTBI in the brain and cerebrospinal fluid (CSF), different aged rats (P15, P20, P56) were injected intravenously with [⁵⁹Fe-¹²⁵I]Tf followed by sampling of CSF and brain tissue. Between 80 and 93% of ⁵⁹Fe in CSF was absorbed with anti-Tf and 1 and 5% with anti-ferritin antibodies. The fraction of ⁵⁹Fe from CSF passing through a 30,000 molecular weight (MW) cutoff filter was approximately 5% (P15), 10% (P20), and 15% (P56). Measurements of Fe and Tf concentrations in CSF of P20 rats revealed that the Fe-binding capacity of Tf was exceeded. In the supernatants of brain homogenates, between 94 and 99% of ⁵⁹Fe was absorbed with anti-Tf and anti-ferritin antibodies. The respective fractions of ⁵⁹Fe in the supernatants passing through the 30 kD cutoff filter were 4% (P15), 2% (P20), and 6% (P56). In brain homogenates mixed before filtering with desferroxamine (DFO) or nitrilotriacetic acid (NTA) which complex loosely protein-bound Fe and non-protein-bound Fe, these ⁵⁹Fe fractions were 2-fold higher. The results indicate that NTBI is present extracellularly in CSF and probably in brain interstitium. J. Neurosci. Res. 54:486–494, 1998. © 1998 Wiley-Liss, Inc.     

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.     

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.     

Calibration of I-polymer standards with I-brain paste standards for use in quantitative receptor autoradiography

¹²⁵I-Polymer standards were calibrated by interpolation of their optical densities in [¹²⁵I]-brain paste standard curves to obtain dpm/mg protein. There was a linear relationship between the calibrated polymer standards and the dpm/mg polymer, as provided by the manufacturer. One dpm/mg polymer was equivalent to 7.34 ± 0.22 dpm/mg protein. Receptor quantification in selected rat brain areas with comparison to either brain paste or calibrated polymer standards yielded similar results.     

Iron and transferrin uptake by brain and cerebrospinal fluid in the rat.

Iron and transferrin uptake into the brain, CSF and choroid plexus, and albumin uptake into the CSF and choroid plexus, were determined after the intravenous injection of [59Fe-125I]transferrin and [131I]albumin into control rats aged 15, 21 and 63 days and 21-day iron-deficient rats. Iron uptake by the brain was unidirectional, greatly exceeded that of transferrin and was equivalent to 39 and 36% of the plasma iron pool per day in the 15-day control and 21-day iron-deficient rats. The rate of transferrin catabolism in the rats was only about 20% of the plasma pool per day. Iron and transferrin uptake into the brain and CSF decreased with increasing age and was greater in the iron-deficient than in the control 21-day rats. The quantity of 125I-transferrin recovered in the CSF could account for only a small proportion of the iron taken up by the brain. Albumin transfer to the CSF also decreased with age but was lower than that of transferrin and was not affected by iron deficiency. Similarly, the plasma: CSF concentration ratios of transferrin and albumin, as determined immunologically, decreased with age and were greater for transferrin than albumin. It is concluded that iron uptake by the brain is dependent on iron release from transferrin at the cerebral capillary endothelial cells with recycling of transferrin to the plasma and transfer of the iron into the brain interstitium. Only a small fraction of the transferrin bound by brain capillaries is transcytosed into the brain and CSF, this being one source of CSF transferrin while other sources are local synthesis and transfer from the plasma by the choroid plexuses.     

Rat brain angiotensin II receptors: Effects of intracerebroventricular angiotensin II infusion

Angiotensin II (Ang II) was infused into a lateral cerebral ventricle of male Sprague-Dawley rats and its effects on blood pressure, water balance and specific [125I]Ang II binding to brain and adrenal tissues were studied. The infusion was maintained at a rate of 500 ng/microliter/h for 6 days using subcutaneously implanted osmotic minipumps. A control group was infused intracerebroventricularly (i.c.v.) with 0.9% saline at a rate of 1 microliter/h for 6 days. Angiotensin II treated rats showed a four-fold increase in water intake and urine volume and a moderate increase in blood pressure; these effects were not observed in rats given saline i.c.v. There was no significant difference in [125I]Ang II binding site density or binding affinity in either the hypothalamus-thalamus-septum-midbrain (HTSM) or the brainstem between Ang II-treated and saline-treated groups. In addition, [125I]Ang II binding sites in the adrenals were also unaffected by i.c.v. infusion of Ang II. The results suggest that brain Ang II receptors are unresponsive to increased Ang II levels in cerebrospinal fluid.     

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).     

Light-microscopic localization of somatostatin binding sites in the locus coeruleus of the rat

Somatostatin (SS14) binding sites within locus coeruleus (LC) were localized at the light microscope level by [¹²⁵I][Tyr⁰,d-Trp⁸]SS14 radioautography combined with an immunohistochemical/neurotoxic lesioning approach. In intact rats, the dense accumulation of SS14 binding sites of LC conspicuously overlapped with the cluster of tyrosine hydroxylase (TH) immunoreactive neurons; SS14 specific binding was directly proportional to the number of TH immunostained (TH+) cell bodies per mg of tissue throughout LC. Complete lesion of catecholaminergic nerve cell bodies of LC by intracerebroventricular injection of 6-hydroxydopamine (6-OHDA) resulted in the total abolition of SS14 specific binding in the structure. In addition, specifically bound [¹²⁵I][Tyr⁰,d-Trp⁸]SS14 and TH+ cell density were quantified serially in a set of rats bearing various partial neurotoxic lesions; a highly significant correlation was found between the two parameters at each of the 16 coronal levels of LC examined. The coefficient of proportionality was identical at all levels. These results strongly suggest that somatostatin binding sites are uniformly localized on all noradrenergic neurons of LC.     

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.     

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.     

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.     

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.