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.     

Nicotinic acetylcholine receptors in rat cochlear nucleus: [125I]-α-bungarotoxin receptor autoradiography and in situ hybridization of α7 nAChR subunit mRNA

The cochlear nucleus (CN) is the first site in the central nervous system (CNS) for processing auditory information. Acetylcholine in the CN is primarily extrinsic and is an important neurotransmitter in efferent pathways thought to provide CNS modulation of afferent signal processing. Although muscarinic acetylcholine receptors have been studied in the CN, the role of nicotinic receptors has not. We examined the distribution of one nicotinic acetylcholine receptor subtype, the α-bungarotoxin receptor (αBgt), in the CN. Quantitative autoradiography was used to localize receptors and in situ hybridization was used to localize α7 mRNA in CN neurons that express the αBgt receptor. Binding sites for αBgt are abundant in the anterior ventral, posterior ventral, and dorsal divisions of the CN, and receptor density is low in the granule cell layer and interstitial nucleus. Heterogeneity in CN subregions is described. Four distinct patterns of αBgt binding were observed: (1) binding over and around neuronal cell bodies, (2) receptors locally surrounding neurons, (3) dense punctate binding in the dorsal CN (DCN) not associated with neuronal cell bodies, and (4) diffuse fields of αBgt receptors prominent in the DCN molecular layer, a field underlying the granule cell layer and in the medial sheet. The perikaryial receptors are abundant in the ventral CN (VCN) and are always associated with neurons expressing mRNA for the receptor. Other neurons in the VCN also express α7 mRNA, but without αBgt receptor expression associated with the cell body. In general, αBgt receptor distribution parallels cholinergic terminal distribution, except in granule cell regions rich in cholinergic markers but low in αBgt receptors. The findings indicate that αBgt receptors are widespread in the CN but are selectively localized on somata, proximal dendrites, or distal dendrites depending on the specific CN subregion. The data are consistent with the hypothesis that descending cholinergic fibers modulate afferent auditory signals by regulating intracellular Ca²⁺ through αBgt receptors. J. Comp. Neurol. 397:163–180, 1998. © 1998 Wiley-Liss, Inc.     

Distribution of [3H]QNB and [125I]α-bungarotoxin binding and acetylcholinesterase activity in visual system and hippocampal structures of eleven mammalian species

This study assessed interspecies differences in regional brain distribution of [³H]QNB binding, [¹²⁵I]α-bungarotoxin binding and acetylcholinesterase activity, by autoradiographic and histochemical methods. Eleven mammalian species were examined, including carnivores (cat, dog), a lagomorph (rabbit), and rodents (squirrel, guinea pig, gerbil, hamster, vole, lemming, rat, mouse). Comparisons were based on primary visual system structures (superior colliculus, lateral geniculate nucleus, primary visual cortex) and the hippocampal formation. The two radioligands differed greatly in the degree of interspecies variation: while the pattern of [³H]QNB binding was quite similar across species, [¹²⁵I] α-bungarotoxin showed striking interspecies diversity. This contrast was most obvious in laminar patterns of the visual cortex and hippocampal formation. Regional distributions of acetylcholinesterase staining were fairly diverse, and were unlike the patterns of either [³H]QNB or [¹²⁵I]α-bungarotoxin. The two ligands showed more consistency in overall levels across species than did acetylcholinesterase. Possible correlates of the differences in interspecies diversity are discussed. © 1993 Wiley-Liss, Inc.     

Sexual dimorphism in α-bungarotoxin binding capacity in the mouse amygdala

A sex difference in α-bungarotoxin binding capacity in the mouse amygdala has been demonstrated by quantitative light microscopic autoradiography. The difference persisted even under widely different steroid-hormonal environment. In addition, it was observed that the binding capacities in both sexes were reversibly activated by administration of either testosterone or estradiol. Neonatal castration, on the other hand, permanently altered the toxin binding capacity in the adult male mouse. These data suggest the possibility that neonatal sex steroids irreversibly modify the cholinergic nicotinic mechanism in the developing mouse amygdala, while the hormones reversibly modulate the mechanism when applied in adulthood.     

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.     

Immunologic similarities between the hypothalamic α-bungarotoxin receptor and theTorpedo californica nicotinic cholinergic receptor

The solubilized rat central nervous system (hypothalamic) nicotinic cholinergic receptor and the Torpedo nicotinic cholinergic receptors are immunologically similar technique. Antibodies to the Electrophorus and Torpedo receptors also decrease the rate of α-bungarotoxin binding to these membraneous receptors. It is concluded that the Torpedo and hypothalamic nicotinic receptors are immunologically similar and that receptor binding sites for α-bungarotoxin and antibodies are physically close. These studies indicate that α-bungarotoxin can be used to study the nicotinic cholinergic receptor of the rat hypothalamus.     

PET imaging of acetylcholinesterase inhibitor‐induced effects on α4β2 nicotinic acetylcholine receptor binding

Acetylcholinesterase inhibitors (AChEIs) are drugs that increase synaptic acetylcholine (ACh) concentrations and are under investigation as treatments for symptoms accompanying Alzheimer's disease. The goal of this work was to use PET imaging to evaluate alterations of in vivo α4β2 nicotinic acetylcholine receptor (nAChR) binding induced by the AChEIs physostigmine (PHY) and galanthamine (GAL). The α4β2 nAChR‐specific radioligand [¹⁸F]nifene was used to examine the effects of 0.1–0.2 mg/kg PHY, 5 mg/kg GAL, and saline in three separate experiments all performed on each of two rat subjects. A 60‐min bolus‐infusion protocol was used with drug administered after 30 min. Data from the thalamus and cortex were analyzed with a graphical model accounting for neurotransmitter activation using the cerebellum as a reference region to test for transient competition with bound [¹⁸F]nifene. Significant [¹⁸F]nifene displacement was detected in both regions during one PHY and both GAL studies, while no significant competition was observed in both saline studies. This preliminary work indicates the viability of [¹⁸F]nifene in detecting increases in synaptic ACh induced by AChEIs. Synapse 67:882–886, 2013. © 2013 Wiley Periodicals, Inc.     

Postmortem stability of α-bungarotoxin binding sites in mouse and human brain

The postmortem stability of α-bungarotoxin binding sites was examined in the brains of mice handled under conditions designed to simulate the handling of human autopsy material. No significant changes in the concentration of binding sites were evident up to 24 h after death. No correlation between the number of binding sites and the delay period between death and autopsy was found in studies of frontal cortex or mid-temporal gyrus from normal humans or cases of dementia of the Alzheimer type. Samples of mid-temporal gyrus from demented patients show a significant reduction in the number of binding sites.     

Presence of an endogenous factor which inhibits binding of α-bungarotoxin 2.2 to its receptor

Cerebral cortical membranes and supernatant from rat were prepared by centrifugation of tissue homogenates at 45,000 g for 10 min. The supernatant fraction thus obtained was found to significantly inhibit α-bungarotoxin binding to the membrane preparation. After a 3 min incubation period, the supernatant inhibited toxin binding by approximately 65%, while the inhibition declined to about 40% after 30 min of incubation, presumably due to the slow reversility of α-bungarotoxin binding. The choice of buffer was found to be an important determinant of the degree of inhibition observed, with 10 mM Tris pH 7.4 providing the most effective condition. This inhibition of toxin binding to cortical membranes by the 45,000 g supernatant was shown not to be due to adsorption of the radiolabeled compound to soluble or residual particulate material in the supernatant fraction. Specificity of the supernatant for the α-bungarotoxin site was demonstrated; a supernatant fr action could be prepared which inhibited α-bungarotoxin binding by 50% but had no effect on [³H]spiroperidol (DA₂ and 5-HT₂), [³H]prazosin, (α₁-adrenergic), [³H]5-hydroxytryptamine (5-HT₁) and [³H]quinuclidinylbenzilate (muscarinic cholinergic) binding. The inhibition of toxin binding also occurred in several other CNS regions including hippocampus, brainstem, spinal cord and cerebellum with an 80 to 90% inhibition of binding occuring in the latter two regions. In addition, the 45,000 g cortical supernatant completely prevented the binding of α-bungarotoxin to extrajunctional neuromuscular receptors and inhibited the binding to junctional receptors by 50%. Supernatants prepared from heart, liver and kidney or bovine serum albumin, at a concentration similar to the supernatant fraction, did not alter radiolabeled toxin binding to cortical membranes, while supernatant prepared from striated muscle tissue was effective. These results suggest there may be an endogenous ligand for the α-bungarotoxin 2.2 binding site in tissues which receive nicotinic cholinergic innervation.     

Autoradiographic localization of extrastriatal D2-dopamine receptors in the human brain using [125I]epidepride

Epidepride is a benzamide with high affinity for central D₂- and D₃-dopamine receptors. The anatomical distribution of [¹²⁵I]epidepride binding was examined by autoradiography, using postmortem human whole-hemisphere cryosections. The density of [¹²⁵I]epidepride binding sites was high in caudate nucleus and putamen. [¹²⁵I]epidepride also labeled receptors in extrastriatal region such as in the pallidum, some thalamic nuclei, the neocortex, and the substantia nigra. The neocortical binding was heterogeneously distributed. In most cortical regions, binding sites were located in superficial layers (I-II). However, in basal levels of the occipital cortex, [¹²⁵I]epidepride binding was located in a deeper layer, probably corresponding to layer V. Competition studies indicated that most of the [¹²⁵I]epidepride binding represented predominantly D₂-dopamine receptors, in striatal as well as in extrastriatal regions. The presence of extrastriatal D₂-dopamine receptor populations is of particular interest for research on schizophrenia and antipsychotic drug action. © 1996 Wiley-Liss, Inc.     

Axonal transport of α-bungarotoxin binding sites in rat sciatic nerve

[¹²⁵I]α-Bungarotoxin (α-BuTX) binding sites accumulate both proximal and distal to a ligature positioned around the sciatic nerve of rats. [¹²⁵I]α-BuTX binding sites, localized using quantitative receptor autoradiography, were found to accumulate at nerve ligatures at a relatively constant rate which suggests that they undergo both anterograde and retrograde axonal transport. [¹²⁵I]α-BuTX binding to sections of ligated sciatic nerve was saturable with apparent dissociation constants of 0.97 nM proximal and 0.53 nM distal to the ligature.d-Tubocurarine, nicotine, decamethonium and atropine displaced [¹²⁵I]α-BuTX from sciatic nerve sections with affinities comparable to those previously reported for the toxin binding component of rat brain. These data indicate that [¹²⁵I]α-BuTX binding sites pharmacologically similar to those of rat brain are transported in sciatic nerve. Axonally transported toxin binding sites may correspond to those previously localized to the plasma membrane of peripheral nerve axons and on the terminals of motor neurons.     

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.     

Comparing α7 nicotinic acetylcholine receptor binding,amyloid‐β deposition,and mitochondria complex‐I function in living brain: A PET study in aged monkeys

This study was aimed to assess the correlations among α7 nicotinic acetylcholine receptor (α7‐nAChR) binding, amyloid‐β (Aβ) deposition, and mitochondrial complex I (MC‐I) activity in the brain of aged monkeys (Macaca mulatta). Positron emission tomography (PET) measurements with [¹¹C](R)‐MeQAA, [¹¹C]PIB, and [¹⁸F]BCPP‐EF were conducted in monkeys in a conscious condition. [¹¹C](R)‐MeQAA binding was analyzed by a simplified reference tissue model to calculate nondisplaceable binding potential (BP_ND), [¹¹C]PIB uptake was calculated by standard uptake value ratio (SUVR), and [¹⁸F]BCPP‐EF binding was determined by Logan graphical analysis to calculate total distribution volume (V_T) with arterial blood sampling. Higher brain uptake was determined in the thalamus, hippocampus, striatum, and cortical regions for [¹¹C](R)‐MeQAA, while being lower in the cerebellum. Significant age‐related reduction of [¹¹C](R)‐MeQAA binding to α7‐nAChR was determined only in the occipital cortex. The plot of Vt of [¹⁸F]BCPP‐EF against BP_ND of [¹¹C](R)‐MeQAA indicated a significant negative correlation in the hippocampus and cortical regions in aged animals. Plotting of SUVR of [¹¹C]PIB against BP_ND of [¹¹C](R)‐MeQAA showed a positive correlation. The in vivo binding of [¹¹C](R)‐MeQAA could reflect the upregulation of α7‐nAChR induced by neurodegenerative damage determined by Aβ deposition as well as impaired MC‐I activity in living brain. Synapse 69:475–483, 2015. © 2015 Wiley Periodicals, Inc.     

[3H]Neurotensin receptor densities in human postmortem brain tissue obtained from normal and schizophrenic persons. An autoradiographic study

Summary. [³H]Neurotensin binding and autoradiographic techniques were used to determine the distribution and density of neurotensin receptors in normal and schizophrenic postmortem brain tissue. Coronal hemi-brain blocks of tissue were cut at the level of the caudate and hippocampus from frozen brain tissue obtained from normal individuals with no known psychiatric or neurologic illnesses and from schizophrenic subjects off- or on-antipsychotic drugs at the time of death. Each hemi-block was further divided, sectioned, thaw mounted on to slides, incubated with [³H]neurotensin and apposed to film. Digitized images were analyzed for binding densities. Areas of intense binding include the substantia nigra, the entorhinal cortex, superficial layers of the cingulate, middle frontal, and insular cortices; and with moderate binding in nucleus accumbens, and caudate. Schizophrenic patients off- (3 months or more) or on-antipsychotic drugs at the time of death were tested; all patients showed a reduced level of neurotensin receptors in the caudate (68% of normals), cingulate (34%) and prefrontal cortices (25%). Accepted February 2, 1998; received October 8, 1997     

β‐Amyloid activates presynaptic α7 nicotinic acetylcholine receptors reconstituted into a model nerve cell system: involvement of lipid rafts

Beta amyloid (Aβ) plays a central role in the pathogenesis of Alzheimer’s disease. Aβ is the major constituent of senile plaques, but there is a significant presence of Aβ in the brain in soluble forms. The results of functional studies indicate that soluble Aβ interacts with the α7 nicotinic acetylcholine receptor (nAChR) complex with apparent high affinity. However, conflicting data exist as to the nature of the Aβ–α7 nAChR interaction, and whether it is the result of specific binding. Moreover, both agonist‐like and antagonist‐like effects have been reported. In particular, agonist‐like effects have been observed for presynaptic nAChRs. Here, we demonstrate Aβ_1‐42‐evoked stimulatory changes in presynaptic Ca²⁺ level via exogenous α7 nAChRs expressed in the axonal varicosities of differentiated hybrid neuroblastoma NG108‐15 cells as a model, presynaptic system. The Aβ_1‐42‐evoked responses were concentration‐dependent and were sensitive to the highly selective α7 nAChR antagonist α‐bungarotoxin. Voltage‐gated Ca²⁺ channels and internal Ca²⁺ stores were both involved in Aβ_1‐42‐evoked increases in presynaptic Ca²⁺ following activation of α7 nAChRs. In addition, disruption of lipid rafts by cholesterol depletion led to substantially attenuated responses to Aβ_1‐42, whereas responses to nicotine were largely intact. These results directly implicate the nicotinic receptor complex as a target for the agonist‐like action of pico‐ to nanomolar concentrations of soluble Aβ_1‐42 on the presynaptic nerve terminal, including the possible involvement of receptor‐associated lipid rafts. This interaction probably plays an important neuromodulatory role in synaptic dynamics.