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).     

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.     

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.     

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.     

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.     

Glucocorticoid regulation of hippocampal oxytocin receptor binding

The effects of glucocorticoid hormones on oxytocin receptors in rat hippocampus were investigated. Oxytocin receptor autoradiography (using 0.1 and 1.2 nM concentrations of [¹²⁵I]OVTA) revealed a significant (P < 0.02) decrease in oxytocin receptor binding in adrenalectomized animals 7 days after the surgery. Corticosterone replacement at the time of adrenalectomy prevented the decrease in oxytocin binding. The findings were significant in hippocampus and subiculum. These findings suggest regulation of oxytocin receptors, and possibly oxytocin-regulated behaviors by glucocorticoids.     

Regulation of neurotensin-containing neurons in the rat striatum. Effects of unilateral striatal lesions with quinolinic acid and ibotenic acid on neurotensin content and its binding site density

Recently, we reported bilateral increases in striatal neurotensin (NT) levels following unilateral 6-hydroxydopamine lesion of the nigrostriatal dopaminergic pathway. In the present study, the effect of unilateral striatal lesions with quinolinic acid (QA, 300 nmol) or ibotenic acid (IBO, 130 nmol) on striatal NT levels and binding site densities were analyzed in order to investigate other possible regulations of NT systems. QA and IBO injection decreased γ-aminobutyric acid (GABA) levels and [¹²⁵I]iodosulpride (a specific D2 receptor antagonist) binding site densities in the lesioned striatum, indicating degeneration of striatal intrinsic neurons. Striatal dopaminergic terminals were not altered by QA as shown by the lack of changes in [³H]dihydrotetrabenazine ([³H]TBZOH, a specific ligand of the vesicular monoamine transporter) binding site densities. Moreover, QA lesion induced an increase in NT levels and a decrease in NT binding sites in the lesioned striatum without any change in the contralateral structure. In contrast to QA, IBO might destroy a certain proportion of dopaminergic terminals in the lesioned striatum, as shown by a 54% decrease in [³H]TBZOH binding. Furthermore, IBO lesion enhanced striatal NT levels bilaterally, while NT binding sites decreased in the lesioned striatum and increased in the contralateral side. The present results suggest that not only dopaminergic neurons but also striatal intrinsic neurons may control NT systems in the striatum.     

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).     

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.     

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.     

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.     

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.     

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     

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.     

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.     

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.     

Levodopa or D2 agonist induced dyskinesia in MPTP monkeys: correlation with changes in dopamine and GABAA, receptors in the striatopallidal complex

Dopamine D1 and D2 receptors as well as the GABA/benzodiazepine receptor complex in the striatum and the globus pallidus (internal: GPi and external: GPe) were studied by autoradiography using [³H]SCH 23390, [³H]spiperone, and [³H]flunitrazepam ([³H]FNZ) respectively, in five groups of cynomolgus monkeys. These included (i) untreated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-monkeys; (ii) MPTP monkeys treated chronically with levodopa injections; (iii) MPTP monkeys treated chronically with injections of the novel D2 agonist U91356A; (iv) MPTP monkeys treated chronically with U91356A delivered through an osmotic mini-pump; and (5) naive controls. Animals treated in a pulsatile mode with U91356A or levodopa injections showed progressive sensitization to their respective drug and developed choreic dyskinesia. In contrast, animals treated in a continuous mode with U91356A showed behavioral tolerance but did not develop dyskinesia. A trend for a down-regulation of putaminal D2 receptors was observed following D2 agonist stimulation with U913356A. Striatal [³H]FNZ binding was significantly decreased only in animals treated in a continuous mode with U91356A. The dopamine receptor decrease in the striatum could be implicated with the development of tolerance but cannot explain the appearnce of dyskinesia. Denervation by MPTP was associated with a decrease of the GPe/GPi [³H]FNZ binding ratio which reflects an imbalance of striatal output pathways; this ratio was not reversed by any of the treatments although changes were observed in the GPe and GPi. Indeed, pulsatile U91356A treatment restored the decreased [³H]FNZ binding in the GPe near control values and levodopa showed a similar tendency. A significant increase of [³H]FNZ binding in the GPi only of dyskinetic monkeys, namely those treated with pulsatile U91356A or levodopa was seen compared to untreated MPTP or naive controls. This GABA_A receptor up-regulation might lead to a supersensitive state of the GPi to gabaergic input which may be involved in the mechanism underlying the development of dopaminomimetic-induced dyskinesia.     

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.     

Properties of the phencyclidine (PCP) receptors

1. 1. [³H]Phencyclidine (PCP, Angel Dust) receptors have been characterized using a rat brain binding section technique.

2. 2. [³H]PCP labels a single class of site in rat brain (k_d = 46 nM; B_max = 10.5 fold/slice). Ligand selectivity pattern strongly suggests that [³h]pcp binds to sites relevant for its pharmacological actions.

3. 3. Chronic PCP treatment (10 mg/kg/day for 14 days) decreases the number of sites (B_max) for [³H]PCP and [³H]spiperone binding but not for [³H]dihydromorphine. These modifications could be related to the development of tolerance and dependence to PCP.

4. 4. Visualization of [³H]PCP binding sites shows high densities of receptors in cortical areas and hyppocampus. Lower densities are observed in caudate-putamen, nucleus accumbens, and amygdala. Negligible quantities of receptors are seen in brain stem and over white matter.

5. 5. The presence of specific [³h]pcp binding sites in rat brain suggests the possible existence of an endogenous ligand for this unique receptor.

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.