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.     

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.     

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.     

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.     

Tachykinin receptor (NK(1), NK(2), NK(3)) binding sites in the rat caudal brainstem following neonatal capsaicin administration

Binding of [125I]-Bolton-Hunter substance P ([125I]-BHSP), [125I]-neurokinin A and [3H]-senktide to tachykinin NK(1), NK(2) and NK(3) receptors, respectively, was examined in caudal brainstem sections of 10-week-old rats pretreated as neonates (P2) with capsaicin (50 mg/kg, s.c.) or vehicle. [125I]-BHSP binding was localised to the nucleus of the solitary tract (NTS), hypoglossal nucleus and inferior olivary complex, whereas [125I]-neurokinin A and [3H]-senktide binding were confined to the NTS. The distribution and density of binding sites were similar in vehicle- and capsaicin-pretreated rats, suggesting that sensory neuron ablation by neonatal capsaicin does not affect tachykinin receptor numbers in the rat caudal brainstem.     

In Vivo Modulation of Sigma Receptor Sites by Calcitonin Gene-related Peptide in the Mouse and Rat Hippocampal Formation: Radioligand Binding and Electrophysiological Studies

Possible interactions between sigma (σ) receptor sites and calcitonin gene-related peptides (CGRP) were investigated using receptor subtype-related analogues and fragments in in vivo [³H](+)SKF 10 047/σ binding in the hippocampus, and electrophysiological recording of the N -methyl-D-aspartate (NMDA)-induced activation of CA3 pyramidal neurons, two well-established σ assays. In both paradigms, CGRP and the agonist [Cys(ACM)^2,7]hCGRPα modulated σ systems. In vivo binding experiments demonstrated that CGRP and [Cys(ACM)^2,7]hCGRPα inhibited 25–40% of specific [³H](+)SKF 10 047 labelling in the mouse hippocampal formation while the purported antagonist hCGRP_8–37 was inactive. The specificity of this modulation was demonstrated further by the lack of effect of other vasoactive peptides, including the atrial natriuretic peptide, substance P, and its N-terminal fragment, substance P_1–7. In the CA3 subfield of the rat dorsal hippocampus, hCGRPα decreased (up to 61%) the NMDA-induced activation of the pyramidal neurons. Conversely, the linear analogue [Cys(ACM)^2,7]hCGRPα enhanced (by 85%) the NMDA-induced activation of CA3 pyramidal neurons, while the antagonistic fragment hCGRP_8–37 had no effect. Haloperidol, a high-affinity σ receptor ligand, inhibited by 90% the in vivo [3H](+)SKF 10 047 labelling, and prevented the modulation of the NMDA-induced activation by hCGRPα and [Cys(ACM)^2,7]hCGRPα. It thus appears that CGRP can modulate σ-related systems in the hippocampal formation.     

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.     

Reserpine Affects Differentially the Density of the Vesicular Monoamine Transporter and Dihydrotetrabenazine Binding Sites

We have studied the effect of a single injection of reserpine (5 mg/kg, s.c.) on the synaptic vesicle monoamine transporter (VMAT) density in the rat striatum, using two labelling procedures: radioimmunolabelling with an antibody against VMAT, and binding of the specific ligand [³H]dihydrotetrabenazine ([³H]TBZOH). In the rostral and medial striatum, the distribution of VMAT immunoreactivity displayed the highest density in the lateral subregions. In the caudal part of the striatum, VMAT immunoreactivity showed increasing density from dorsal to ventral subregions. The VMAT immunoreactivity was not altered 2 and 30 days after the reserpine injection, whereas [³H]TBZOH binding site density, measured on adjacent slices, showed a dramatic decrease at day 2 and a moderate recovery at day 30, suggesting that despite a persistent blockade of [³H]TBZOH binding sites, VMAT protein density was unchanged.     

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.     

Melatonin Receptors in the Brain and Pituitary Gland of Hypothalamo-Pituitary Disconnected Soay Rams

In Soay rams in which the pituitary gland has been surgically separated from the hypothalamus, blood prolactin concentrations vary in response to changes in photoperiod and the administration of melatonin, as in intact animals, providing evidence that melatonin acts within the pituitary gland to control prolactin secretion. In this study the presence of potentially functional melatonin receptors in the pars tuberalis and zona tuberalis (PT/ZT) of hypothalamo-pituitary disconnected (HPD) Soay rams is confirmed using both in vitro autoradiography with the ligand 2-(¹²⁵I)- iodomelatonin and in situ hybridization for the melatonin receptor. There was no effect of the HPD operation on the pattern and quantity of 2-(¹²⁵I)iodomelatonin binding in the brain demonstrating that this binding is independent of hypothalamic regulation. The possibility that melatonin may control prolactin secretion directly via specific receptors on lactotrophs was investigated using dual in situ hybridization with a (³⁵S) labelled probe for the ovine melatonin receptor (Mel1a_b) and a Digoxigenin labelled probe for ovine prolactin. Melatonin receptor gene expression was observed in the PT/ZT in both intact and HPD rams, however, there was no colocalization with prolactin gene expression; only in the ZT was there a close association between cells expressing the melatonin receptor and lactotrophs. The results provide strong support for the view that melatonin acts via the PT/ZT to mediate the effects of photoperiod on the seasonal cycle in prolactin secretion.     

Plasticity of &mgr; and delta Opioid Receptors in the Superficial Dorsal Horn of the Adult Rat Spinal Cord Following Dorsal Rhizotomies: A Quantitative Autoradiographic Study

The aim was to study the regulation of μ and δ opioid binding sites in the superficial layers (laminae I–II) of the dorsal horn of the adult rat spinal cord 1, 2, 4 and 12 weeks after unilateral dorsal rhizotomies of various extents. Using quantitative autoradiography and highly selective tritiated opioid ligands, we have shown that the decrease in [³H]Tyr*- d -Ala-Gly-NMe-Phe-Gly-ol ([³H]DAMGO) (μ sites) and [³H]Tyr*- d -Thr-Gly-Phe-Leu-Thr ([³H]DTLET) (δ sites) binding in the side ipsilateral to the lesion as compared to the intact side is related to the number of dorsal roots cut. In the segment central to the lesion, 1 week after the lesion, ipsilateral/contralateral side binding ratios for [³H]DAMGO were 0.70, 0.49, 0.36 and 0.25 when 1, 3, 5 and 7 roots respectively were sectioned. For [³H]DTLET, the ratios were 0.71, 0.54, 0.42 and 0.39. The time-related analysis of binding ratios showed that, in partially deafferented spinal segments after long-term deafferentation (12 weeks postlesion) there were greater numbers of μ and δ binding sites than in cases of short-term deafferentation (1–2 weeks). By contrast, in spinal segments considered as completely deafferented, there was no difference in the remaining μ and δ binding sites at 12 weeks as compared to 1 week postlesion. Consequently, it is deduced that the partial recovery of μ and δ binding observed after long-term partial deafferentation could be associated with neuronal plasticity (probably collateral sprouting) of fine diameter primary afferent fibres arising from intact dorsal roots.     

Daily and Circadian Variations in 2-[125I]-Iodomelatonin Binding Sites in the Pike Brain (Esox Iucius)

The fish pineal organ, through its 24  h rhythmic release of melatonin, acts as a transducer of the photoperiod, influencing different physiological functions (e.g. reproduction, growth). We have investigated the binding of 2-[¹²⁵I]iodomelatonin to whole brain membrane preparations from pikes ( Esox lucius L., teleost) maintained for 24–48  h under different photoperiodic conditions. Specific binding was stable, reversible, saturable and sensitive to the presence of a GTP analogue. Scatchard analysis revealed one class of binding sites. Displacement experiments suggested the presence of two components with affinities in the femtomolar and nanomolar range of concentrations, respectively. The B_max exhibited monophasic nycthemeral variations, with higher values at the light-to-dark transition (34.0±4.5  fmol/mg protein) and low values during the second half of night (10.0±1.0  fmol/mg protein). Under the same conditions, the K_D exhibited biphasic variations: values were low during daytime and at the middle of the dark phase (approximately 100  pM); they were high at the beginning (approximately 225  pM) and at the end (approximately 330  pM) of the night. These variations were maintained under constant light (LL) and constant darkness (DD). Thus, the variations in the number and affinity of the melatonin binding sites were controlled by circadian oscillators, synchronized by the photoperiod. The nature of these oscillators is not known. Therefore, in fish, we suggest that the photodependent effects of melatonin result from the circadian variations of both its production by the pineal and its binding sites in the brain.     

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.     

Temporal cortex dopamine D2/3 receptor binding in major depression

The aim of this study was to assess the dopamine function of the temporal cortex in major depressive disorder using [¹²³I]epidepride to image D_2/3 receptor binding sites. Ten major depressives and 10 healthy controls were selected from a general population sample for single-photon emission computed tomography imaging. Among the major depressives there was a strong bilateral correlation between the scores on the 21-item Hamilton Depression Rating Scale and D_2/3 receptor binding. Dopaminergic abnormalities may be present in the temporal cortices of major depressives.     

[3H]Nitrendipine Binding to Rabbit Colonic Smooth Muscle

We used [³H] nitrendipine binding to isolated smooth muscle cells and isometric tension studies of muscle strips to characterize the calcium channels from rabbit proximal and distal colon. At 25°C [³H] nitrendipine binding was rapid, saturable, reversible, specific, and linearly proportional to cell number. The affinity of the ligand for its receptor was similar in proximal and distal colon (K_D 129 ± 21 pM and 124 ± 17 pM, respectively). In the proximal colon there were 68,000 receptors per cell, compared to 58,000 receptors per cell in the distal colon (p > .1). The Hill coefficient for nitrendipine was close to unity, suggesting binding to a single receptor. Although nitrendipine and nifedipine competitively inhibited [³H]nitrendipine binding, verapamil did not alter [³H] nitrendipine binding, suggesting the presence of at least two discrete, noninteracting sites for the binding of drugs that block calcium channels. In studies with muscle strips nitrendipine competitively inhibited isometric tension stimulated by both bethanechol and high extracellular potassium concentration. There were no significant differences in response from proximal and distal colon. These results suggest that calcium antagonist binding characteristics to calcium channels are similar in proximal and distal colon, and do not explain previously observed differences in the function of muscle in these tissues.     

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.     

Consequences of Transient Focal Cerebral Ischaemia for Second Messenger and Neurotransmitter Binding in the Rat: Quantitative Autoradiographic Analysis of Forskolin, Dopamine D1 Receptor Binding and Cerebral Blood Flow Changes

In order to study the consequences of reperfusion for ischaemic brain injury, quantitative ligand binding autoradiography was carried out in a model of reversible focal cerebral ischaemia. Endothelin-1 applied to the abluminal surface of the middle cerebral artery in anaesthetized Sprague-Dawley rats induced severe focal ischaemia and subsequent reperfusion (assessed by blood flow tracers [^99mTc]HMPAO and [¹⁴C]iodoantipyrine respectively) by 2 h after insult. Ligand binding autoradiography on consecutive sections demonstrated these blood flow changes to be associated with a significant reduction in forskolin binding throughout the middle cerebral artery territory (e.g. 25% in parietal cortex, 11% in dorsolateral caudate nucleus). The most marked losses in forskolin binding were in areas where ischaemia was severe and reperfusion was poor. However, the same changes in cerebral blood flow had no significant effect on D₁ dopamine receptor binding (e.g. >2% reduction in the caudate nucleus). These data demonstrate that ligand binding characteristics are significantly affected as early as 2 h after insult, with evidence of differential sensitivity for forskolin and D₁ dopamine binding. With regard to the consequences of reperfusion, comparison with our previous study of 2 h maintained ischaemia demonstrates reperfusion-related salvage of dopamine and forskolin binding in the caudate nucleus but possible exacerbation of forskolin binding loss in the cortex.