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.     

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.     

Localization of Neuropeptide Y Y1 mRNA in the Human Brain: Abundant Expression in Cerebral Cortex and Striatum

Many neurobiological functions have been ascribed to the NPY Y1 receptor subtype, but autoradiographic analysis has failed to detect Y1 binding sites in most human brain areas, in contrast to the rat. We examined the regional distribution of Y1 mRNA-containing cells in the post-mortem human brain to clarify if there is a major species difference in terms of the existence of Y1 receptors in the human telencephalon, in particular the striatum and cortex. In situ hybridization experiments revealed widespread distribution of Y1 mRNA signals in all layers of most limbic and neocortical regions, predominantly in layer IV (most cortical regions) and layer VI. The striatum showed moderate Y1 receptor mRNA expression levels with intensely expressing cells localized to the nucleus accumbens. The highest Y1 receptor mRNA expression was apparent within the dentate gyrus, and the lowest in the subiculum, parahippocampal gyrus, cerebellum, and thalamus. In vitro autoradiography using [¹²⁵I]Leu³¹Pro³⁴-PYY and [¹²⁵I]PYY with NPY (13–36) or Leu³¹ Pro³⁴ NPY; confirmed the presence of low Y1–like binding in the human brain despite abundant Y1 mRNA expression. However, using a rat model of the human autopsy process, it was apparent that the inability to reveal high Y1– versus Y2–like receptors in the human brain was related in part to marked reductions of Y1–like, but not Y2–like, receptors within a 4 h post-mortem delay. Altogether, the results indicate that the Y1 receptor gene is abundant in the human brain and this receptor may have important roles in cognitive, limbic and motor function.     

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.     

High resolution micro-SPECT scanning in rats using 125I β-CIT: Effects of chronic treatment with carbamazepine

Purpose:  Carbamazepine (CBZ) is a first-line antiepileptic agent with mood-stabilizing effects in bipolar disorder. It has been reported to influence extracellular concentrations of serotonin and dopamine, suggesting an interaction with monoamine transporters. We have investigated this effect using in vivo single photon emission computed tomography (SPECT) in rats.
Methods:  Adult male rats received 3 mg/kg/h CBZ via mini-osmotic pump. After 14 days continuous treatment, animals underwent two consecutive SPECT scans, using ¹²⁵I β-CIT as a radiotracer to label serotonin transporter (SERT) and dopamine transporter (DAT) sites in the brain. Pharmacologic distinction was enabled by ¹²⁵I β-CIT SPECT imaging in rats acutely exposed to the serotonin and dopamine transporter inhibitors, fluoxetine and GBR12909. The interaction between CBZ and ¹²⁵I β-CIT binding to SERT and DAT was investigated using in vitro autoradiography.
Results:  Carbamazepine (10 μ m ) did not affect binding of ¹²⁵I β-CIT to isolated rat brain slices, thereby excluding a direct effect on ligand binding to SERT and DAT. SPECT studies with fluoxetine and GBR12909 highlighted SERT binding in thalamus, hippocampus, centromedial nuclei, and occipital cortex, and DAT binding in the caudate. Prolonged treatment with CBZ failed to influence ¹²⁵I β-CIT binding to either SERT or DAT in any of the brain regions examined.
Discussion:  This study employed the novel technique of small animal SPECT imaging to investigate the effects of CBZ on monoamine transporters in rat brain. Following prolonged treatment, the drug was without effect on SERT or DAT availability. The mechanism by which CBZ exerts its mood stabilizing effects remains elusive.     

Ontogenetic Development of 5-HT1D Receptors in Human Brain: An Autoradiographic Study

The pattern of pre- and postnatal appearance of 5-HT_1D receptors throughout the different areas of the human brain was studied by quantitative in vitro autoradiography, using [¹²⁵I]GTI (serotonin O -carboxymethyl-glycyl-[¹²⁵I]tyrosinamide) as a ligand. The anatomical distribution of 5-HT_1D receptors in neonatal, infant and children's brain was in good agreement with that observed in the adult, the basal ganglia and substantia nigra being the most intensely labelled areas. The development of these receptors throughout the human brain was mainly postnatal: low densities of [¹²⁵I]GTI binding sites were observed at the fetal/neonatal stage in most regions analyzed, in contrast with the high levels of labelling found in infant and children's brains. Indeed, in a number of regions, including the globus pallidus, substantia nigra and visual cortex, a peak of overexpression of 5-HT_1D receptors was observed in the first decade of life. Such overexpression could support a regulatory role for 5-HT_1D receptors in advanced periods of the CNS developmental process. Our results also indicate that the administration of drugs acting on 5-HT_1D receptors during the early postnatal period of life could result in modifications of their properties, as these receptors are already functional in this period.     

Evidence For Two Neurochemical Divisions in the Human Nucleus Accumbens

The neurochemical anatomy of the human nucleus accumbens was studied by comparing the distributional patterns of [³H]DAMGE (μ opioid receptor), [³H]bremazocine (κ opioid receptor), [³H]SCH-23390 (D₁-like dopamine receptor), [³H]7-OH-DPAT (D₃ dopamine receptor) binding, preproenkephalin mRNA and acetylcholinesterase activity in sections of post mortem human striatum. Our results demonstrate the presence of at least two neurochemically distinct divisions within the human nucleus accumbens, which may be homologous to the 'shell'and'core'divisions of the nucleus as found in the rat.     

Distribution of Nicotinic Receptors in the Human Hippocampus and Thalamus

Neuronal nicotinic acetylcholine receptors consist of different subunits, α and β, with different subtype arrangement corresponding to distinct pharmacological and functional properties. The expression of α3, α7 and β2 mRNA in the human brain was studied by in situ hybridization and compared to [³H]nicotine, [³H]cytisine and [¹²⁵l]α-bungarotoxin binding in contiguous sections. The β2 probe showed a strong hybridization signal in the granular layer of the dentate gyrus and in the CA2/CA3 region of the hippocampus and in the insular cortex, and a signal of lower intensity in the subicular complex and entorhinal cortex. The α3 probe showed strong hybridization in the dorsomedial, lateral posterior, ventroposteromedial and reticular nuclei of the thalamus, and a weak signal in the hippocampal region and in the entorhinal, insular and cingular cortex. The amount of α7 mRNA was high at the level of the dentate granular layer and the CA2/CA3 region of the hippocampus, in the caudate nucleus and in the pulvinar and ventroposterolateral nuclei of the thalamus. [³H]Nicotine and [³H]cytisine binding appeared to be identical in anatomical distribution and relative intensity. It was high in the thalamic nuclei, the putamen and in the hippocampal formation in the subicular complex and the stratum lacunosum moleculare. The level of [¹²⁵l]α-bungarotoxin binding was particularly high in the hippocampus and in the pyramidal cells of the CA1 region, but was relatively low in the subicular complex. Our data indicate that in the human brain nicotinic receptor subtypes have discrete distributions, which are in part different from those of other species.     

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.     

Melatonin Receptors are Present in Non-Optic Regions of the Brain of a Deep-Sea Fish Living in the Absence of Solar Light

Pineal melatonin hormonally transduces photoperiod to influence daily and seasonal cycles in most vertebrates (1,  2). Evidence of melatonin receptors throughout the brain of several fish species (3–5), particularly in retinorecipient structures, also indicates a role in visual processing. Despite the absence of solar light many deep-sea organisms show seasonality (6–8). The presence of central melatonin receptors was investigated by quantitative in vitro autoradiography in the deep-sea fish Coryphaenoides (Nematonurus) armatus . Specific, time-dependent, saturable, high affinity and guanine nucleotide sensitive, 2-[¹²⁵I]iodomelatonin binding was found over the mid-brain tegmentum and hindbrain. Competing ligand potency was iodomelatonin > melatonin 5-HT. Although C.(N.) armatus has well developed eyes no 2-[¹²⁵I]iodomelatonin binding occurred in optic tectum, cerebellum or hypothalamus. Thus melatonin involvement in processing of visual information and control of seasonal physiology via hypothalamic areas appears to be absent in this species. The presence of central G-protein coupled receptors indicates a function for melatonin unrelated to solar light.     

Transferrin receptors in the Parkinsonian midbrain

Several hypotheses have been put forward to explain the pathogenesis of Parkinson's disease (PD) and recently it has been suggested that alterations in iron homeostasis may be implicated. Because of the central role of the transferrin receptor in providing access of iron to cells, we have studied the distribution and density of transferrin receptors using [³H]–transferrin ([³H]–Tf) binding and tritium film autoradiography in the normal and PD midbrain. High levels of [³H]–Tf binding were found in the dorsal raphé, oculomotor nucleus and periaqueduc tal grey whilst lower levels of [³H]–Tf binding were found in the tegmentum, red nucleus and substantia nigra. Significant reductions in binding were found in the substantia nigra, red nucleus and oculomotor nucleus in PD, the reductions in [³H]–Tf binding being similar to the loss of nigral neurons in PD. The data suggest that the increased iron content of surviving nigral neurons may reflect a compensatory metabolic response rather than abnormal transferrin receptor expression.     

Differential Distribution of Thyroid Hormone Receptor Isoform in Rat Dorsal Root Ganglia and Sciatic Nerve in vivo and in vitro

Using autoradiographic techniques carried out under precise conditions we previously demonstrated that both sensory neurons and peripheral glial cells in dorsal root ganglia (DRG) or sciatic nerve, possess specific []> ¹²⁵I]-labeled T₃ binding sites. Thyroid hormone receptors (TR) include several isoforms (TRα₁, TRα₂, TRβ₁, TRβ_2...) The present study demonstrates that while sensory neurons and peripheral glial cells both possess functional TR, they express a differential expression of TR isoforms.     

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.     

Do dopamine agonists or levodopa modify Parkinson's disease progression?

During the past decade, in vivo imaging of the nigrostriatal dopaminergic system has been developed as a research tool to monitor progressive dopaminergic neuron loss in Parkinson's disease (PD) and to assess the effect of medication on imaging outcomes. Recently two similar studies compared the effect of initial treatment with a dopamine agonist (pramipexole (CALM-PD CIT) or ropinirole (REAL-PET)) or levodopa on the progression of PD as measured by [¹²³I] β -CIT or [¹⁸F]Dopa imaging. These two clinical imaging studies targeting dopamine function with different imaging ligands and technology both demonstrate slowing in the rate of loss of [¹²³I] β -CIT or [¹⁸F]Dopa uptake in early PD patients treated with dopamine agonists compared with levodopa. The relative reduction in the per cent loss from baseline of [¹²³I] β -CIT uptake in the pramipexole versus the levodopa group was 47% at 22 months, 44% at 34 months and 37% at 46 months after initiating treatment. The relative reduction of ¹⁸F-dopa uptake in the ropinirole group versus the levodopa group was 35% at 24 months. These results should be very cautiously interpreted with regard to the effect of dopamine agonists or levodopa on clinical disease progression. These data highlight the need to compare imaging outcomes of dopamine neuronal loss with multiple meaningful clinical endpoints of disease progression in placebo controlled, larger and long-term studies.     

Single photon emission computed tomography with [99Tc]-HM-PAO and [123I]-IBZM in Alzheimer's disease and dementia of frontal type: preliminary results

Dementia of frontal type (DFT) is a fairly common degenerative disease distinct from Alzheimer's disease (AD), whose reportedly distinctive instrumental feature is frontal lobe hypoperfusion on SPECT. We evaluated the cortical dopaminergic system in 6 AD, 5 DFT, and 6 control subjects with SPECT and both [⁹⁹Tc]-HM-PAO, a perfusion tracer, and [¹²³I]-IBZM, a D2 postsynaptic ligand. Both in AD and DFT patients, [⁹⁹Tc]-HM-PAO SPECT showed a relative frontal hypoperfusion. On the contrary, [¹²³I]-IBZM SPECT showed significantly reduced ligand uptake in superior frontal regions of DFT (0.89 ± 0.08 relative to control subjects) as compared to AD patients (0.97 ± 0.02; difference of means: 0.08, 95% Confidence Interval 0.004 to 0.156; p = 0.041). Results suggest more marked involvement of the frontal cortical dopaminergic system in DFT than in AD patients.     

Effects of the Two Somatostatin Variants Somatostatin-14 and [Pro2, Met13]Somatostatin-14 on Receptor Binding, Adenylyl Cyclase Activity and Growth Hormone Release from the Frog Pituitary

Two isoforms of somatostatin from frog brain have been recently characterized, namely somatostatin-14 (SS1) and [Pro², Met¹³]somatostatin-14 (SS2). The genes encoding for the precursors of these two somatostatin variants are expressed in hypothalamic nuclei involved in the control of the frog pituitary. The aim of the present study was to investigate the effect of SS1 and SS2 on adenohypophysial cells. Autoradiographic studies using [¹²⁵I-Tyr°, d -Trp⁸] SS1 as a radioligand revealed that somatostatin binding sites are evenly distributed in the frog pars distalis. The SS2 variant was significantly (P<0.01) more potent than SS1 in competing with the radioligand (IC₅₀=1.2±0.2 and 5.6±0.6  nM, respectively). Both SS1 and SS2 induced a modest but significant reduction in cAMP formation in dispersed distal lobe cells but did not affect spontaneous growth hormone (GH) release. Synthetic human GRF (hGRF) induced a significant increase in cAMP accumulation and GH release in this system. Both SS1 and SS2 inhibited the stimulatory effects of hGRF on cAMP formation and GH secretion. These data show that the SS1 and SS2 variants can regulate adenohypophysial functions. The fact that GH cells are exclusively located in the dorsal area of the frog adenohypophysis, while somatostatin receptors are present throughout the pars distalis, indicates that the two somatostatin isoforms may control the secretion of pituitary hormones additional to GH in amphibians.     

Somatostatin binds to murine macrophages through two distinct subsets of receptors

Somatostatin (SRIF) exerts anti-inflammatory effects, in part by deactivating monocytes/macrophages. Thus, the objective of this study was to characterize specific receptors for SRIF on these cells. Macrophages isolated from mouse peritoneal cells bound [125I]Tyr(0), D-Trp(8) SRIF(14) specifically. Scatchard analysis of saturation binding data revealed two classes of binding sites with an affinity of 0.44+/-0.13 and 2.58+/-0.56 nM, respectively. By sensitive and specific RT-PCR, the mRNAs for the five SRIF receptors (SSTR1 to SSTR5) could be detected. Evidence for the involvement of SSTR1 and SSTR2 in the binding of SRIF to the high and low affinity sites, respectively, was obtained by the demonstration that (1) only SSTR1 and SSTR2 subtype-specific agonists were active in competing for [125I]Tyr(0), D-Trp(8) SRIF(14) binding to high and low affinity sites, respectively, and (2) [125I]Tyr(0), D-Trp(8) SRIF(14) bound to high but not low affinity sites on macrophages isolated from SSTR2 knock-out mice. In conclusion, we have identified and characterized two different SRIF receptor subtypes in murine macrophages.