A comparison of nerve transection and chronic application of β-bungarotoxin on acetylcholine receptor distribution and other nerve-muscle properties

β-Bungarotoxin (β-BuTX), a snake venom neurotoxin which acts presynaptically to inhibit acetylcholine (ACh) release at the neuromuscular junction, was applied to the rat phrenic nerve-diaphragm muscle preparation to determine its effectiveness to mimic denervation. The distribution of junctional and extrajunctional ACh receptors on the muscle were assayed biochemically by [¹²⁵I]α-bungarotoxin ([¹²⁵I]α-BuTX) binding and electrophysiologically by iontophoretic application of ACh. Spontaneous transmitter release and muscle membrane potential were measured under conditions of denervation, β-BuTX treatment, and bee venom phospholipase A₂ exposure. Within 7 days after treatment with a single dose (5μg/kg) of enzymatically active β-BuTX, extrajunctional [¹²⁵I]α-BuTX binding increased fivefold, and there was a decrease in miniature end-plate potential (MEPP) frequency and in resting membrane potential (RMP) to values less than those of control muscles but greater than those of denervated muscles. The same dose of enzymatically inactive β-BuTX or snake venom phospholipase A₂ was without effect, but a fivefold greater dose of enzymatically inactive β-BuTX resulted in changes in extrajunctional binding and RMP similar to those of muscles exposed to the enzymatically active toxin. However, unlike muscles treated with active toxin, those treated with inactive toxin had MEPP frequencies similar to control muscles and exhibited contraction elicited by phrenic nerve stimulation. These results taken together indicate that the nerve exerts a trophic influence on muscle independent of ACh and muscle activity.     

α-Bungarotoxin binding sites on sensory neurones and their axonal transport in sensory afferents

The autoradiographic localization of [¹²⁵I]α-bungarotoxin binding sites on primary sensory fibres was investigated. Nicotinic α-bungarotoxin binding sites were localized to a small sub-population of large dorsal root ganglion cells in the rat, monkey, cat and human dorsal root ganglia. Ligation of the sciatic nerve or dorsal root in the rat resulted in an anterograde accumulation of binding sites proximal to the dorsal root ganglion, and a small retrograde accumulation. Unilateral dorsal root section in the rat produced a loss of toxin binding sites mainly within lamina III of the dorsal horn. These results suggest that nicotinic α-bungarotoxin binding sites manufactured in large dorsal root ganglion cell bodies are transported both centrally to the spinal cord and also peripherally.     

α-Bungarotoxin binding to a high molecular weight component from lower vertebrate brain identified on dodecyl sulfate protein-blots

The binding of [¹²⁵I]iodo-α-bungarotoxin ([¹²⁵]α-BuTX) to the dissociated α-subunit of Torpedo acetylcholine receptor (AChR) can be readily demonstrated in a modified ‘protein-blot’ analysis utilizing electrophoretically transferred, dissociated subunits immobilized onto positively charged nylon membranes which are then incubated directly with [¹²⁵I]α-BuTX. We report here the use of the protein-blotting technique to detect the α-BuTX binding site present in the central nervous system of lower vertebrates and to characterize some of the physicochemical properties of the toxin binding site. High molecular weight (M200,000 and 120,000) α-BuTX-binding components can be readily demonstrated in avian and fish brain extracts upon protein-blotting with [¹²⁵I]α-BuTX following lithium dodecyl sulfate PAGE. Neither extensive reduction with dithiothreitol nor prior reduction followed by alkylation with iodoacetamide alter the mobility of the CNS-derived BuTX-binding sites. In contrast to our findings with Torpedo AChR or muscle AChR derived from a number of different species, no binding is observed in the molecular weight range of the α-subunit (M_r= 40,000) nor is any binding at any molecular weight observed in similar fractions prepared from adult, mammalian (rat, guinea pig) brain using this technique. These results demonstrated the existence in lower vertebrate brain of a BuTX binding site comparable in size to the AChR oligomeric complex of electric organ and muscle. They also suggest, however, striking structural differences between muscle AChR and the central neuronal BuTX-binding complex as well as a considerable difference between the neuronal BuTX-binding sites derived from lower and higher vertebrate brain.     

Muscarinic cholinergic receptors flow in the sciatic nerve

Muscarinic cholinergic receptors build up on the proximal side of a ligature placed on the rat sciatic and vagus nerves. These receptors appear to be flowing down the axons of at least a portion of the nerve fibers present in sciatic and vagus nerves. Most of the anterograde flowing muscarinic binding sites are displaceable with 10⁻⁴ M carbachol indicating that many are high affinity agonist binding sites. These receptors can also be localized in the ganglionic nerve cell bodies in the dorsal root ganglia which contribute fibers to the sciatic nerve. We hypothesize that muscarinic cholinergic receptors are synthesized in the ganglion cell bodies and transported distally in sensory nerve axons in the sciatic nerve.     

α-Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterisation, colocalisation with α7 subunits and up-regulation by chronic nicotine treatment

High density neuronal cultures from rat E18 hippocampus and cortex have been characterised with respect to cholinergic binding sites. No specific binding of [³H]nicotine or [³H]cyttine to live cells in situ was detected, although the limit for detection was estimated to be 30 fmol/mg protein. Muscarinic binding sites labelled with [³H]QNB were present at a density of 0.75 pmol/mg protein. [¹²⁵I]α-Bungarotoxin (αBgt) bound to hippocampal cultures with a B_max of 128 fmol/mg protein and a K_d of 0.6 nM; cortical cultures expressed five times fewer [¹²⁵I]α-Bgt binding sites. Fluorescence cytochemistry with rhodamine-α-Bgt indicated that 95% of hippocampal neurons were labelled, compared with only 36% of cortical neurons. Average densities of 4 × 10⁴ and 2 × 10⁴ binding sites/cell were calculated for hippocampal and cortical cultures, respectively. Double labelling experiments with mAb307 (which recognises the rat α7 nicotinic receptor subunit) and rhodamine-α-Bgt gave coincident labelling patterns, supporting the correlation between the α7 subunit and Bgt-sensitive neuronal nicotinic receptor. Treatment of hippocampal cultures with 10 μM nicotine for 14 days elicited a 40% increase in the numbers of [¹²⁵I]α-Bgt binding sites, mimicking the up-regulation observed in vivo studies. Primary cultures offer a useful in in vitro system for investigating the expression and regulation of brain α-Bgt-sensitive receptors.     

Axonal transport of polyamines: a reappraisal.

Axonal transport of putrescine and the polyamines in normal and transected motor axons of adult rat sciatic nerves was studied. [³H]Putrescine injected into the ventral horn region of the spinal cord was not transported distally along the course of the nerve as a function of time. l-[³H]ornithine, the immediate precursor of putrescine, when injected into the ventral horn of the spinal cord, was followed by rapid axonal transport of acid-insoluble material, but no transport of acid-soluble material, such as putrescine or the polyamines, was detected despite the demonstration that radioactive spermidine was synthesized at the site of the l-[³H]ornithine injection in the spinal cord. Furthermore, acid-soluble radioactive material failed to accumulate proximal to the site of a ligature of the sciatic nerve in acute and chronic experiments. Ligation of the sciatic nerve also failed to cause an accumulation of endogenous putrescine proximal to the ligature. The data showed no evidence for fast axonal transport of putrescine and the polyamines in normal and transected motor axons of the sciatic nerve in adult rats.     

Developments of α-bungarotoxin receptors in cultured chick ciliary ganglion neurons

We have maintained embryonic chick ciliary ganglion neurons in dissociated cell culture and studied the progressive appearance of surface receptors for [¹²⁵I]α-bungarotoxin. Cultures were established from 8-day-old embryos and fed a medium supplemented with 180 μg/ml of a soluble protein extract prepared from the eye, the target organ for the ciliary ganglion. Approximately 8064 neurons survived per ganglion and there was no evident loss of neurons through two weeks in culture. Binding of [¹²⁵I]α-bungarotoxin was determined at room temperature on intact cells still attached to their coverslips. Non-specific binding was less than 2% of the total. Specific binding of [¹²⁵I]α-bungarotoxin was saturable with respect to both time of incubation (20–30 min) and concentration of toxin (5–10 nM), with an apparent K_d = 1.0 nM. Binding sites for [¹²⁵I]α-bungarotoxin increased during the first week in culture from 1.8 fmol per 10⁴ neurons at 1 day in vitro (DIV) to 8.6 fmol per 10⁴ neurons at 7 DIV, after which the number of sites seemed to plateau. Light microscopic autoradiography was performed on cultures at 4 DIV and showed most of the grains associated with the surfaces of neuronal cell bodies, while scattered grains occurred over neuronal processes. When compared with previous reports on the in vivo development of α-bungarotoxin receptors in chick ciliary ganglia, the appearance of receptors in these cultured neurons followed a time course similar to, but at lower levels than, their in vivo counterparts. Nevertheless, this culture system should prove useful for the study of questions concerning the regulation, surface distribution and intracellular pathways of neuronal α-bungarotoxin receptors.     

Contrasting effect of a brain supernatant extract on neuronal vs neuromuscular α-bungarotoxin receptors

The effects of a rat brain supernatant extract and a partially purified supernatant preparation from bovine brain were determined on the binding of [¹²⁵I]α-bungarotoxin (α-BGT) to muscle membranes, as well as to membranes prepared from brain. In agreement with previous work, the supernatant preparations inhibited α-BGT binding to brain membranes in a dose-dependent fashion, (Brain Research, 245 (1982) 57–67); however, no significant effect of either of the preparations was observed on the binding of the toxin to muscle membranes. As well, the supernatant preparations did not affect binding of radiolabelled α-BGT to muscle cells in culture in competition binding experiments. The effect of long-term incubation of cells in culture with the supernatant preparations was subsequently determined. These studies showed that the binding of [¹²⁵I]α-BGT increased markedly (300%) in the presence of a crude rat brain supernatant preparation, while incubation of the muscle cells in the presence of the partially purified bovine supernatant extract had no significant effect on radiolabelled toxin binding. In contrast, both the rat and bovine supernatant preparations significantly decreased (up to 65%) radiolabelled toxin binding to a cultured neuronal cell population, adrenal medullary chromaffin cells. These results suggest that an endogenous factor(s), present in brain extracts, differentially regulates the neuronal as compared to the neuromuscular nicotinic α-bungarotoxin binding sites.     

Heterogeneity of brain melanocortin receptors suggested by differential ligand binding in situ

The existence of multiple brain melanocortin receptor types has been postulated, based on the complex pharmacology of intracerebrally administered melanocortin (melanocyte-stimulating hormone-related) peptides. In this study, this hypothesis was tested by determining whether different brain melanocortin receptor populations can be discriminated on a pharmacologic or neuroanatomic basis. The abilities of various pharmacologically active native melanocortins and structural analogs, as well as other test substances, to compete with biologically active [¹²⁵I]Nle⁴,d-Phe⁷-α-MSH([¹²⁵I]NDP-MSH) for binding to melanocortin receptors was determined, by in vitro binding and autoradiography in frozen rat brain tissue sections. We have previously shown that native melanocortins including α-MSH, γ-MSH and ACTH_1–39 compete with [¹²⁵I]NDP-MSH for binding to brain tissue sites. In the present studies, each of the melanocortin peptides α-MSH, des-acetyl-α-MSH, β-MSH and ACTH_1–24 when present at 1 μM virtually eliminated [¹²⁵I]NDP-MSH binding in each of a series of brain structures, including medial preoptic area, caudate putamen, olfactory tubercle, bed nucleus of the stria terminalis, ventral part of the lateral septal nucleus, hypothalamic periventricular and paraventricular nuclei, dorsal anterior amygdaloid area, substantia innominata and thalamic paraventricular nucleus; as well as in extraorbital lacrimal gland, a peripheral melanocortin target. In contrast, the behaviorally and neurotrophically active melanocortin analogs Met(O₂),d-Lys,Phe⁹-α-MSH_4–9 (Org2766), ACTH_4–9 and the antipyretic peptide α-MSH_11–13 did not affect [¹²⁵I]NDP-MSH binding at concentrations up to 100 μM, implying that the receptors or receptor binding sites which mediate the actions of these analogs must comprise additional types, distinct from those which bind [¹²⁵I]NDP-MSH. Binding of [¹²⁵I]NDP-MSH was also unaffected by the nonmelanotropic peptides ACTH_1–4, ACTH_34–39 and vasoactive intestinal polypeptide (VIP) and by the antipyretic drugs acetaminophen and lysine-salicylate. Although some of the brain structures are known to express mRNA encoding a γ-MSH-preferring melanocortin receptor type known as MC3, the relative order of binding affinities of melanocortins, determined in concentration-response studies, wasNdp-MSH≥ACTH_1–24α-MSH>γ-MSH>ACTH_4–10 probably account for most of the [¹²⁵I]NDP-MSH binding detectable in the brain. Furthermore, the potency relationships between these respective peptides and the binding activity ofD-Trp⁷,D-Phe^{10-α-MSH_6–11} amide, a synthetic α-MSH antagonist, varied considerably among the brain sites and peripheral (lacrimal and melanoma) tissues studied, suggesting some degree of heterogeneity of ligand binding properties among [¹²⁵I]NDP-MSH-binding melanocortin receptor populations in different regions of the brain. Considered together with the available data on the pharmacologic actions of melanocortins and the molecular biology of melanocortin receptors, the present results provide evidence for the existence of multiple, pharmacologically distinct classes of melanocortin receptors in the brain, potentially providing a basis for the pharmacological targeting of specific populations of central melanocortin receptors.     

Characterization of [I]HEAT binding to α1-receptors in human brain: assessment in aging and Alzheimer's disease

The binding of [¹²⁵I]2-(β-4-hydroxyphenylethylamino-ethyltetralone ([¹²⁵I]HEAT), an α₁-adrenergic receptor antagonist, to human brain membranes was characterized and the binding assessed in tissue from subjects with Alzheimer's disease (AD) and aging controls. Under Na⁺-K⁺ phosphate buffer conditions, [¹²⁵I]HEAT bound to a single class of binding sites in prefrontal cortex (Brodmann area 10) with a K_d of about 120 pM. High binding capacities of [¹²⁵I]HEAT were evident in the hippocampus and neocortex but were low in subcortical areas and cerebral microvessels comparable to the regional distribution of [³H]prazosin binding reported previously. Displacement of [¹²⁵I]HEAT by various adrenergic drugs was consistent with its binding to α₁-adrenergic receptors. The specific binding was not affected by postmortem delay between death and freezing of tissue at autopsy. There was no correlation of [¹²⁵I]HEAT binding with age of subjects. In AD subjects, the binding was significantly decreased in prefrontal cortex by about 25% but not changed in hippocampus, putamen or cerebellum compared to age-matched controls. The reduced binding of [¹²⁵I]HEAT in prefrontal cortex may reflect a region-specific change in α₁-adrenergic receptors associated with neuronal loss in AD.     

Distribution of an α-bungarotoxin-binding cholinergic nicotinic receptor in rat brain

Cholinergic nicotinic receptors in rat brain were demonstrated by the use of the potent nicotinic antagonist [¹²⁵I]α-bungarotoxin ([¹²⁵I]α-Btx). Biochemical studies on binding of [¹²⁵I]α-Btx to rat hippocampal homogenates revealed saturable binding sites which are protected by nicotine, d-tubocurarine and acetylcholine but not by atropine or oxotremorine. The hippocampus and hypothalamus displayed relatively high [¹²⁵I]α-Btx specific binding whereas the cerebellum was devoid of specific binding. Other regions displayed intermediate binding levels. Analysis of the regional distribution of [¹²⁵I]α-Btx binding by autoradiography of frontal brain sections revealed high labeling in the hippocampus, hypothalamic supraoptic, suprachiasmatic and periventricular nuclei, ventral lateral geniculate and the mesencephalic dorsal tegmental nucleus. It is suggested that the limbic forebrain and midbrain structures as well as sensory nuclei are the main nicotinic cholinoceptive structures in the brain.     

Retrograde axonal transport of β-adrenoreceptors in rat brain: Effect of reserpine

Retrograde axonal transport of β-adrenoreceptors was assessed by measuring the accumulation of binding sites for the β-receptor ligand [¹²⁵I]iodocyanopindolol ([¹²⁵I]ICP) distal to a unilateral 6-hydroxydopamine (6-OHDA) lesion placed in the ascending noradrenergic axons of the locus coeruleus. Accumulation of binding sites was linear over a 3 day period and was blocked by intracerebroventricular 6-OHDA given 1 day prior to sacrifice. A single dose of reserpine (5 mg/kg, i.p.) caused a long lasting (6–8 week) biphasic depletion of frontal cortex norepinephrine (NE) associated with increased frontal cortex binding of another β-receptor ligand. [³H]dihydroalprenolol ([³H]DHA), at 7–14 days, and again at 28 days post-reserpine. Unlike the changes in cortical β-receptors, retrograde transport of [¹²⁵I]ICP in presynaptic noradrenergic neurons was decreased or blocked completely at 7–14 days and at 6 weeks, and was increased to 470% and 240% of control at 21 days and 8 weeks after reserpine. Anterograde transport of [³H]DHA binding sites was measured by accumulation proximal to a 6-OHDA lesion in this pathway. This transport varied in a pattern similar to that seen for retrograde transport of [¹²⁵I]ICP binding sites. These data and others suggest that presynaptic β-receptors are regulated independently of frontal cortex β-receptors, which appear to be located primarily on postsynaptic cells. On the other hand, the regulation of both anterograde and retrograde transport appears to be interrelated since both types of transport were altered in a similar way in the face of long-term NE depletion by reserpine.     

Autoradiographic localization of the binding of calcium channel antagonist, [I]ω-agatoxin IIIA, in rat brain

The calcium channel antagonists ω-agatoxin IIIA (ω-Aga-IIIA) and ω-conotoxin GVIA (ω-CgTx) were radioiodinated and used to locate binding sites in the rat brain by receptor autoradiography. While patterns of regional binding to sagittal sections of rat brain were generally similar for the 2 toxins, notable differences in the cerebellum and hippocampus were observed. Specific [¹²⁵I]ω-Aga-IIIA binding was greatest in the granule cell layers of the cerebellum and of the dentate gyrus. In contrast, binding of [¹²⁵I]ω-CgTx was most intense in the molecular layers of these structures. Less than one-third of [¹²⁵I]ω-Aga-IIIA binding in rat brain slices was inhibited by pre-exposure to 250 nM ω-CgTx, while 40 nM ω-Aga-IIIA virtually eliminated the binding of [¹²⁵I]ω-CgTx under the same conditions. The P-type calcium channel antagonist ω-Aga-IVA blocked only a small fraction of [¹²⁵I]ω-Aga-IIIA and [¹²⁵I]ω-CgTx binding. These autoradiographic data are consistent with membrane binding experiments and indicate that the combined use of agatoxins and conotoxins may be useful in the characterization of separate types of neuronal calcium channels.     

Effects of gonadal steroids on the in vivo binding of [I]α-bungarotoxin to the suprachiasmatic nucleus

The radioligand [¹²⁵I]α-bungarotoxin (α-BTX) has been used to test receptor binding to putative nicotinic cholinergic receptors in the hypothalamus. Using light microscopic autoradiography following third ventricular infusion of the radioligand we have previously demonstrated that in normally cycling rats and in normal males, the suprachiasmatic nucleus (SCN) consistently binds the α-neurotoxin. In chronically (5 weeks) oophorectomized female, binding of [¹²⁵I]α-BTX to the SCN is markedly diminished. The present series of experiments were designed to test the effects of gonadal steroids on the binding of [¹²⁵I]α-BTX to the SCN. We first tested whether or not estradiol administered to ovariectomized females could duplicate the presence of the ovary. In females ovariectomized and immediately provided with a constant dose of estradiol-17β (E₂)—1.0 cm silastic capsules for 5 weeks, (n= 4), the binding of the neurotoxin to the SCN was maintained. In females ovariectomized for 3 weeks and replaced with E₂ for 2 weeks (n= 4), the binding of [¹²⁵I]α-BTX to the SCN was restored. In chronically (4 weeks) ovariectomized females receivingf E₂ for 6 dyas (n= 2), the binding of the neurotoxin was partially restored. We next tested the effect of chronic (5 weeks) castration (n= 100) and observed that binding of [¹²⁵I]α-BTX to the SCN of castrated males was like that of intact male controls (n= 6). We also tested the effect of neonatal adrogenization (1.25 mg testosterone proprionate, neonatal days 2 and 5) with subsequent adult oophorectomy (day 83) and observed that neonatally androgenized females with intact ovaries (n= 4) demonstrated [¹²⁵I]α-BTX binding to the SCN like that of non-adrogenized females, while neonatally androgenized females without ovaries (n= 4) had decreased binding of the neurotoxin to the SCN like that of oophorectomized adult females. These data demonstrate that: (1) the administration of estradiol can maintain and restore [¹²⁵I]α-BTX labeling of the SCN in ovariectomized rats; (2) binding of [¹²⁵I]α-BTX to the SCN is different in castrated males and ovariectomized females; and (3) the difference in α-BTX binding to the SCN receptor in adult castrated male and female rats may be dependent upon the presence of gonadal steroids at times other than the ‘critical’ postnatal period.     

Whole brain and regional [I]-α-bungarotoxin binding in developing rat

These experiments were conducted in order to determine if the total number of binding sites for [¹²⁵I]-α-bungarotoxin ([¹²⁵I]-α-BGT) in rat brain increases and then decreases during postnatal development as predicted by comparison with skeletal muscle, and, if so, to determine at approximately what age the peak in binding occurs in the brain as a whole. A further purpose was to investigate the time-course of development of the [¹²⁵I]-α-BGT binding sites in several brain regions.Specific binding for [¹²⁵I]-α-BGT was studied using the pellets from a 20 min, 14,000 × g centrifugation of rat brain homogenates from 4 or 5 postnatal ages. At least three binding assays were done per region and per age, on cerebral cortex, cerebellum, caudate-putamen, posterior hippocampus, pons-medulla and whole brain. In most regions, the [¹²⁵I]-α-BGT specific binding is measurable, but is low at day one, peaks at about 12–20 days and declines by adulthood. With a few exceptions, these data hold true whether binding is expressed as specific binding per mg protein, specific binding per gram wet tissue, or total specific binding per brain region. The absolute number of specifically bound [¹²⁵I]-α-BGT molecules is undistorted by simultaneous or non-linear growth of cells uninvolved with α-BGT binding and, thus, is the measurement most useful in determining developmental changes. Whole brain has the same age-related pattern as in the majority of the brain regions, i.e., compared to 19–20 days, the adult brain actually has fewer total binding sites.     

Radioligand binding and immunoautoradiographic evidence for a lack of toxicity to dopaminergic nerve terminals in human cocaine overdose victims

Radioligand binding to and immunolabeling of transport sites associated with monoamine-containing synaptic vesicles affords a novel approach for mapping the integrity of dopaminergic (DAergic) nerve terminals. The present study used [¹²⁵I]iodovinyltetrabenzine ([¹²⁵I]TBZ) and a fusion protein antibody directed at the large intraluminal loop of the neuronal vesicular monoamine transporter (hVMAT2-loop) as probes to assess the effects of chronic cocaine use on the integrity of DAergic nerve terminals in the striatum of cocaine fatalities. Visualization of [¹²⁵I]TBZ binding in human brain revealed a distinct pattern of labeling throughout the rostral-caudal extent of the striatum. Saturation binding of [¹²⁵I]TBZ in striatal membranes demonstrated a single high affinity site (K_d = 2.3 ± 0.9 nM and B_max = 55.5 ± 8.1 pmol/g tissue) with a pharmacological profile (tetrabenazine ≥ iodovinyltetrabenazine > ketanserin ≥ reserpine > haloperidol > GBR 12909) consistent with the specific labeling of hVMAT2. Quantitative in vitro autoradiography demonstrated no significant alteration in the density of [¹²⁵I]TBZ binding sites in the anterior and posterior sectors of the striatum in cocaine fatalities with and without preterminal excited delirium as compared to drug-free and age-matched control subjects. Similarly, the levels of hVMAT2-loop immunoreactivity were not significantly different across control and cocaine fatality groups. The results demonstrate the lack of an alteration in [¹²⁵I]TBZ binding sites and hVMAT2 protein in the striatum from a young cohort of cocaine fatalities. Since striatal VMAT2 is primarily associated with DAergic nerve terminals, these results suggest that chronic cocaine use failed to affect the integrity of striatal DAergic nerve terminals.     

Neuropeptide Y and Galanin Binding Sites in Rat and Monkev Lumbar Dorsal Root Ganalia and Spinal Cord and Effect of Peripheral Axotomy

Using monoiodinated peptide YY (PYY) and galanin as radioligands, and neuropeptide Y (NPY) fragments, the distribution of NPY binding sites and its subtypes Y1 and Y2, and of galanin binding sites, was investigated in rat and monkey lumbar (L) 4 and L5 dorsal root ganglia (DRG) and spinal cord before and after a unilateral sciatic nerve cut, ligation or crush. Receptor autoradiography revealed that [¹²⁵I]PYY bound to some DRG neurons and a few nerve fibres in normal rat DRG, and most of these neurons were small. NPY binding sites were observed in laminae I–IV and X of the rat dorsal horn and in the lateral spinal nucleus, with the highest density in laminae 1–11. [¹²⁵I]NPY binding was most strongly attenuated by NPY_13–36, a Y2 agonist, and partially inhibited by [Leu³¹,Pro³⁴]NPY, a Y1 agonist, in both rat DRG and the dorsal horn of the spinal cord. These findings suggest that Y2 receptors are the main NPY receptors in rat DRG and dorsal horn, but also that Y1 receptors exist. After sciatic nerve cut, PYY binding markedly increased in nerve fibres and neurons in DRG, especially in large neuron profiles, and in laminae III-IV of the dorsal horn, as well as in nerve fibres in dorsal roots and the sciatic nerve. Incubation with NPY_13–36 completely abolished PYY binding, which was also reduced by [Leu,³¹ Pro³⁴] NPY. However, the increase in PYY binding seen in laminae I–IV of the ipsilateral dorsal horn after axotomy was not observed after coincubation with [Leu³¹, Pro³⁴] NPY. NPY binding sites were seen in a few neurons in monkey DRG and in laminae I-II, X and IX of the monkey spinal cord. The intensity of PYY binding in laminae I-II of the dorsal horn was decreased after axotomy. Galanin receptor binding sites were not observed in rat DRG, but were observed in the superficial dorsal horn of the spinal cord, mainly in laminae I-II. Axotomy had no effect on galanin binding in rat DRG and dorsal horn. However, galanin receptor binding was observed in many neurons in monkey L4 and L5 DRG and in laminae I–IV and X of monkey L4 and L5 spinal cord, with the highest intensity in laminae I-II. No marked effect of axotomy was observed on the distribution and intensity of galanin binding in monkey DRG or spinal cord. The present results indicate that after axotomy the synthesis of NPY receptors is increased in rat DRG neurons, especially in large neurons, and is transported to the laminae I–IV of the ipsilateral dorsal horn and into the sciatic nerve. No such up-regulation of the NPY receptor occurred in monkey DRG after axotomy. The Y2 receptor seems to be the main NPY receptor in DRG and the dorsal horn of the rat and monkey spinal cord, but Y1 receptors also exist. The increase in NPY binding sites in laminae I–IV of the dorsal horn after axotomy partly represents Y1 receptors. In contrast to the rat, galanin binding sites could be identified in monkey lumbar DRG. No effect of axotomy on the distribution of galanin binding sites in rat or monkey DRG and dorsal horn was detected, suggesting their presence on local dorsal horn neurons (or central afferents).     

α1-Adrenergic receptors in the brain: characterization in astrocytic glial cultures and comparison with neuronal cultures

Binding of [¹²⁵I]HEAT to membranes prepared from primary cultures of astrocytic glial cells was time-dependent and 70–85% specific. Various adrenergic agonists and antagonists competed for [¹²⁵I]HEAT binding to the potencies of prazosin >, yohimbine , clonidine, norepinephrine (NE), and propranolol. Scatchard analysis showedB_max of 209 fmol/mg protein and a K_d of 184 pM for [¹²⁵I]HEAT binding by astrocytic glial membranes. Pretreatment of astrocytes with NE resulted in a dose-dependent downregulation of [¹²⁵I]HEAT binding sites with a maximal response observed after 8 h at 100 μM NE. Removal of NE from cultures after pretreatment resulted in a time- and protein synthesis-dependent recovery of binding sites to control levels within 120 h. Incubation of astrocytic glial cultures with NE stimulated phosphoinositide (PI) hydrolysis in a time- and dose-dependent manner with a maximal stimulation of 2-fold observed in 60 min by 100 μM NE.Clonidine expressed differential effects on α₁-adrenergic receptors of the neuronal and astrocytic glial cultures. Pretreatment with 10 μM clonidine caused a 40% decrease in the B_max of [¹²⁵I]HEAT binding without influencing the K_d value in neuronal cultures. This downregulatory effect of clonidine was associated with a reduction in the ability of NE to stimulate PI hydrolysis in clonidine pretreated cells. In contrast to neuronal cultures, clonidine neither downregulated [¹²⁵I]HEAT binding sites nor stimulated PI hydrolysis in glial cultures.     

Interleukin-1β, but not IL-1α, mediates nerve growth factor secretion from rat astrocytes via type I IL-1 receptor

In astrocytes, nerve growth factor (NGF) synthesis and secretion is stimulated by the cytokine interleukin-1β (IL-1β). In the present study, the role of IL-1 receptor binding sites in the regulation of NGF release was evaluated by determining the pharmacological properties of astroglially localized IL-1 receptors, and, by comparing the effects of both the agonists (IL-1α and IL-1β) and the antagonist (IL-1ra)—members of the IL-1 family on NGF secretion from rat neonatal cortical astrocytes in primary culture. Using receptor-binding studies, binding of [¹²⁵I] IL-1β to cultured astrocytes was saturable and of high affinity. Mean values for the K_D and B_max were calculated to be 60.7±7.4 pM and 2.5±0.1 fmol mg^-1 protein, respectively. The binding was rapid and readily reversible. IL-1 receptor agonists IL-1α (K_i of 341.1 pM) and IL-1β (K_i 59.9 pM), as well as the antagonist IL-1ra (K_i 257.6 pM), displaced specific [¹²⁵I] IL-1β binding from cultured astrocytes in a monophasic manner. Anti-IL-1RI antibody completely blocked specific [¹²⁵I] IL-1β binding while anti-IL-1RII antibody had no inhibitory effect. Exposure of cultured astrocytes to IL-1α and IL-1β revealed the functional difference between the agonists in influencing NGF release. In contrast to IL-1β (10 U/ml), which caused a 3-fold increase in NGF secretion compared to control cells, IL-1α by itself had no stimulatory action on NGF release. The simultaneous application of IL-1α and IL-1β elicited no additive response. IL-1ra had no effect on basal NGF release but dose-dependently inhibited the stimulatory response induced by IL-1β. We concluded that IL-1β-induced NGF secretion from cultured rat cortical astrocytes is mediated by functional type I IL-1 receptors, whereas IL-1α and IL-1ra, in spite of their affinity for IL-1RI, have no effect on NGF secretion from these cells. Type II IL-1R is not present on rat neonatal cortical astrocytes.     

Effects of ovariectomy on the binding of [I]-αbungarotoxin (2.2 and 3.3) to the suprachiasmatic nucleus of the hypothalamus: An in vivo autoradiographic analysis

α-Bungarotoxin (α-BTX) has been used to label receptor binding sites on neural membranes. α-BTX fractions 2.2 and 3.3 were purified from Bungarus multicinctus by the method of Ravdin and Berg (1979) and were iodinated. There was no difference between these two fractions in their binding affinity or specificity of binding with hypothalamic synaptosomes. [¹²⁵I]α-BTX 2.2S, 3.3 and commercially obtained [¹²⁵I]α-BTX were injected into the third ventricle of ovariectomized female rats (n = 22), normally cycling rats (n = 12) or normal male rats (n = 4) and autoradiographic examination performed. Saline injected hypothalami (n = 4) or hypothalami injected with unlabeled α-BTX in combination with radioligand (n = 2) did not show silver grain accumulation in the hypothalamus. Examination of serial sections from animals injected with each [¹²⁵I]α-BTX showed that the supraoptic, periventricular, arcuate, premamillary and mamillary nuclei were consistently labeled. While the suprachiasmatic nucleus (SCN) in the intact females and males both showed high densities of α-BTX binding, the SCN in ovariectomized females showed little or no α-BTX binding. Thus, the labeling of the SCN in females without ovaries and ovarian hormones was markedly different from that of the intact males and females. Labeling patterns in castrate and intact animals may contribute to our understanding of gonadal steroid regulation of hypothalamic function.