Autoradiographic quantitation of β-adrenergic receptors on neural cells in primary cultures. II. Comparison of receptors on various types of immunocytochemically identified cells

We have developed a microcomputer-based video method to quantify neurotransmitter receptors on single, immunocytochemically labeled cultured cells. This method has been applied to determine whether β-adrenergic receptors are more numerous on neurons, astroglia, oligodendroglia or fibroblasts in primary neural cell cultures, and to assess the heterogeneity of receptor expression within a single cell type. Dissociated cells from perinatal rat cerebral cortex were grown in very sparse cultures on polylysine-coated glass slides. The cultured cells were fixed and permeated, then stained with fluorescently labeled immunocytochemical markers for astroglia (glial fibrillary acidic protein), fibroblasts (fibronectin), oligodendroglia (galactocerebroside) or neurons (A2B5). β-Adrenergic receptors were labeled with [¹²⁵I]pindolol or [¹²⁵I]cyanopindolol, and dry-mount autoradiography was carried out on the fixed cells. Cells were identified according to their morphology and cell-type specific staining, then autoradiographic grains associated with the defined cells were visualized by reflected polarized light microscopy and counted with a microcomputer-based video digitizing system. Using this technique, we have determined that fibroblasts have less than 15% of the number of β-adrenergic receptors expressed by polygonal astroglia, whereas oligodendroglia and neurons had no detectable binding of¹²⁵I-labelled ligands. This suggests that in these mixed neural cell cultures, the great majority of β-adrenergic receptors are associated with astroglia. Furthermore, we determined that process-bearing astroglia have less than 5% of the number of β-adrenergic receptors expressed by polygonal astroglia. Since process-bearing astroglia are thought to be derived from polygonal astroglia, these results suggest that the β-adrenergic receptor is lost from this population of astroglia during development. This method provides a novel approach to the problem of measuring cell-type specific, membrane-associated receptors on individual, well-characterized cells in mixed primary neural cultures.     

Identification of nerve growth factor receptors in primary cultures of chick neural crest cells

Primary cultures of chick neural crest cells obtained from explanted neural tubes have binding sites for radioiodinated nerve growth factor ([125I]NGF) but not for radioiodinated epidermal growth factor ([125I]EGF). The binding of [125I]NGF was shown to be a specific and saturable process with a high affinity (Kd = 0.3 nM) for the ligand. Despite the expression of these NGF binding sites, incubation of the neural crest cultures with nerve growth factor did not induce neurite outgrowth; no morphological alterations were observed. This was not due to an inability of the cells to express a neuronal phenotype, since the neural crest cells spontaneously differentiated into neurite-bearing cells. However, the nerve growth factor binding sites do appear to be functional receptors, since nerve growth factor could produce a modest induction of ornithine decarboxylase. The quantity of nerve growth factor binding sites seemed to be independent of the phenotype expressed by the neural crest cells, since both pigmented cells and neuron-like neural crest cells exhibited binding. These findings suggest that the differentiation of neural crest cells into mature nerve growth factor-responsive neurons may involve the coupling of nerve growth factor receptors to cellular responses important in the expression of the neuronal phenotype.     

Excitatory amino acid receptors in primary cultures of glial cells from the retina.

Binding of 3H-L-aspartate to membranes from retinal glial cells in primary culture was characterized. Binding kinetics showed a saturable, reversible binding to three populations of sites with KB = 40, 200, and 1,300 nM. The first two were present at 1 day in vitro (DIV), whereas the latter two were observed at 12 DIV. The possibility of the 40 nM site being neuronal cannot be discarded, since some neurons are present at 1 DIV. In 12 DIV cultures, the presence or absence of sodium determined two different pharmacological patterns, comparable to those described for electrogenic glutamate transport in Müller cells, and QA metabotropic receptors in astrocytes, respectively. Results suggest that, as has been shown for some receptors in nerve tissue, the properties of glial cell receptors undergo age-dependent changes. In turn, this could be related to changes in the function of neurotransmitter substances during development.     

Mesencephalic dopaminergic neurons in primary cultures express functional neurotensin receptors.

The cellular distribution and functional aspects of neurotensin (NT) binding sites in rat mesencephalic cells in primary culture were investigated by an original approach combining anatomical and biochemical studies. Using a double-labeling protocol combining 125I-NT receptor radioautography and tyrosine hydroxylase (TH) immunocytochemistry, we obtained the first direct visualization of NT binding sites on TH-immunoreactive neurons. Eighty percent of the TH neurons were endowed with NT binding sites, which can be observed on both cell bodies and processes. TH-immunoreactive neurons were characterized as dopaminergic neurons by their ability to take up dopamine in a benztropine- and nomifensine-sensitive manner. In the mesencephalic cultures, NT increased potassium-evoked release of tritiated dopamine, and the relative potencies of various NT-related peptides to increase dopamine release were in good agreement with their abilities to bind to NT sites. These results show for the first time that cultured rat mesencephalic dopaminergic cells express functional NT receptors. Finally, the specificity and distribution of NT receptors on dopaminergic neurons in primary culture are quite similar to what was observed in the adult rat brain using pharmacological and radioautographic approaches. These data indicate that NT can influence the activity of dopaminergic neurons at very early stages of the rat brain development.     

Ontogeny of the nerve growth factor receptor in primary cultures of neural crest cells

Neural crest cells maintained as primary cultures for 4-7 days demonstrated specific binding of radioiodinated nerve growth factor ([125I]NGF); occasionally, significant binding of ([125I]NGF could be detected in 3-day cultures. Parallel cultures processed for indirect immunofluorescence revealed that the NGF receptor-positive neural crest cells represented a subpopulation of the total cells in culture. Cultures aged 4-7 days demonstrated fluorescent cells which were often grouped as aggregates. Some 3-day cultures contained faintly fluorescent cells. One- and two-day cultures were non-fluorescent. Melanocytes did not appear to bind NGF. Preliminary flow cytofluorometric analysis indicated that ca. 28% of neural crest cells in 5-day cultures possessed specific receptors for NGF.     

Coexistence of GABA receptors and GABA-modulin in primary cultures of rat cerebellar granule cells

GABA-modulin (GM), a basic polypeptide purified from rat brain synaptosomes, which is an allosteric inhibitor of GABA recognition sites, has been detected in primary cultures of cerebellar interneurons enriched in granule cells by immunohistochemistry, using a specific antibody raised in rabbit injected with GM purified from rat brain synaptosomes. In these cultures, GM is expressed by the granule cells, which are postsynaptic to GABAergic interneurons, but not by glial cells. In rat cerebellar sections anti-GM antiserum intensely strains the granular cell layer and Purkinje cell dendrites and cell bodies. GM has been purified from the cerebellar granule cell cultures and appears to be identical under biochemical, immunological, and functional criteria to authentic GM purified from rat brain synaptosomes. Granule cell cultures devoid of GABAergic neurons contain the GABA/BZ/Cl- receptor complex; in fact, intact cell monolayers, incubated in physiological buffer at 25 degrees C, express 3H-muscimol and 3H-flunitrazepam binding sites, which are comparable to the sites detected in cell membrane preparations and which modulate each other reciprocally. It is concluded that GM might participate in the supramolecular organization of the GABA receptor complex, perhaps functioning as a modulator of this receptor protein.     

Selective expression of high-affinity nerve growth factor receptors on tyrosine hydroxylase-containing neuron-like cells in neural crest cultures

A subpopulation of cultured neural crest cells undergoing differentiation have receptors for nerve growth factor (NGF) that exhibit a binding constant similar to that of the low-affinity NGF binding site (3.2 nM). Recent studies have shown that NGF receptors are not present on neuron-like cells immunoreactive for tyrosine hydroxylase (TH), serotonin, or vasoactive intestinal polypeptide. Since tissues innervated by sympathetic neurons in vivo produce NGF, we sought to determine whether NGF deficits in the tissue culture microenvironment may be one parameter preventing the expression of NGF receptors on TH-containing neuron-like cells. Neural crest cultures were therefore grown in complete tissue culture medium (15% fetal bovine serum and 5% chicken embryo extract), with or without exogenous NGF (50 ng/ml). Examination of light-microscopic radioautographs following incubation with 125I-NGF revealed that, if the cultures were supplemented with NGF for 7 d, approximately 33% of neuron-like cells exhibiting TH-like immunoreactivity possessed NGF receptors. There were no obvious morphological differences between TH-containing cells that did or did not have NGF receptors. Scatchard analysis of cultures grown under these conditions again demonstrated the sole presence of the low-affinity form of the NGF receptor (Kd, 3.4 nM). Embryonic catecholaminergic sympathetic neurons exhibit both high- and low-affinity forms of the NGF receptor, raising the possibility that the Scatchard analysis may not have been sensitive enough to detect the high-affinity form of the receptor on a relatively small population of cells. Therefore we used a morphological approach that took advantage of the different dissociation rates of the 2 receptor types.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitochondrial glutathione and methyl iodide-induced neurotoxicity in primary neural cell cultures.

The status of both cytosolic and mitochondrial glutathione was studied in primary cultured cerebrocortical cells from fetal mice using the selective membrane-solubilizing properties of digitonin and after exposure to the monohalomethane methyl iodide. A correlation was found between cell injury (assessed by lactate dehydrogenase leakage 24 hr after exposure) and early loss of mitochondrial glutathione (2 hr after exposure), while cell death did not appear directly dependent on cytosolic glutathione depletion. The antioxidants BW 755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline) and DPPD (N,N'-diphenyl-p-phenylenediamine), and the glutathione precursor N-acetyl-L-cysteine were used to modify cellular responses to methyl iodide. Prevention of cell injury by these reagents was obtained only under conditions where at least 50% of the normal level of mitochondrial glutathione was preserved after methyl iodide exposure. Mitochondrial metabolic activity (reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, MTT) was affected by exposure to methyl iodide and correlated with mitochondrial glutathione depletion and cytotoxicity. These findings indicate that the mitochondrial glutathione pool and mitochondrial functions may be the most significant intracellular targets of methyl iodide in neural cultures. Moreover, the present work exemplifies the dependence of neural cell viability on the status of mitochondrial functions and suggests that, as in the liver, mitochondrial glutathione is an important component of cellular homeostasis in nervous tissue.     

Excitatory amino acid receptors coupled with guanylate cyclase in primary cultures of cerebellar granule cells

Primary cultures of cerebellar granule cells have been used in pharmacologically and functionally characterizing excitatory amino acid recognition sites coupled with guanylate cyclase. When granule cells were incubated in physiological culture conditions (Locke's solution, pH 7.4), only kainate and, to a lesser extent, L-glutamate increased cyclic GMP (cGMP) levels. Under these conditions, L-aspartate, N-methyl-D-aspartate (NMDA), and quisqualate were inactive. When granule cells were incubated in the absence of extracellular Mg2+ or in the presence of the depolarizing agent veratrine, L-glutamate, L-aspartate, and NMDA became as effective as kainate in enhancing cGMP formation. The action of kainate was preferentially antagonized by 2,3-cis-piperidindicarboxylate, whereas the action of L-glutamate was preferentially antagonized by (+/-)2-amino-5-phosphonovalerate. These data suggest that 2 different excitatory amino acid recognition sites (activated by kainate or by L-glutamate, L-aspartate, and NMDA, respectively) are coupled with guanylate cyclase in primary cultures of cerebellar granule cells: While the coupling of the recognition site for kainate with guanylate cyclase operates under resting conditions and in the presence of Mg2+, the coupling of the recognition site for L-glutamate, L-aspartate, and NMDA with guanylate cyclase requires depolarizing conditions or the absence of extracellular Mg2+.     

Morphologic effects of ammonia on primary astrocyte cultures. II. Electron microscopic studies

Light microscopic studies of primary astrocyte cultures following exposure to ammonia have shown several alterations. To determine the nature and significance of these changes, electron microscopic studies were performed. Ultrastructural changes consisted of proliferation, pleomorphism and swelling of mitochondria, condensation of the mitochondrial matrix, cytoplasmic lucency and vacuolization, disaggregation of polyribosomal clusters, an initial increase followed by degranulation of rough endoplasmic reticulum, proliferation of smooth endoplasmic reticulum, an accumulation of dense bodies and a loss of intermediate glial filaments. The early alterations appeared reactive and perhaps reflected ammonia detoxification. Some changes were degenerative and support the view that ammonia exerts a direct toxic effect on astrocytes. It is postulated that these changes may interfere with critical astroglial functions and thereby play a key role in the neurologic dysfunction seen in hyperammonemia.     

Catecholaminergic cells and support cell precursors in neural crest cultures differentially express nerve growth factor receptors

Long-term neural crest cultures grown in the continuous absence of exogenous nerve growth factor (NGF) contain a subpopulation of cells with NGF receptors exclusively of the low affinity subtype (Kd of approximately 3.2 nM). The current studies combined immunocytochemistry, using GIN1 (a support cell marker) or tyrosine hydroxylase antibodies, with radioautography following exposure to iodinated nerve growth factor (125I-NGF). The majority of cells specifically binding 125I-NGF were found to be immunoreactive for GIN1, indicating that the primary cell phenotype expressing receptors for NGF appear to be support cell precursors, at least under these conditions. These cells are likely to be responsive to and/or dependent upon NGF; the nature of this response or dependency remains to be determined. Some cells exhibiting silver grains were not immunoreactive for GIN1, suggesting that other cell phenotypes in neural crest cultures also have NGF receptors. In addition, some neural crest cells were found that stained with GIN1 and lacked 125I-NGF binding. Tyrosine hydroxylase-like immunoreactive cells apparently did not bind 125I-NGF under these culture conditions. Catecholaminergic sympathetic and sensory neurons from embryonic ganglia, derived from the neural crest, express both the high and low affinity forms of the NGF receptor. In order to determine whether the microenvironment played a role in the type of catecholaminergic cells appearing in culture, neural crest cells were grown in the continuous presence of exogenous NGF. Under these conditions, many tyrosine hydroxylase-like immunoreactive cells were found that specifically bound 125I-NGF. In addition, silver grains were still detected on these cells following a chase with nonradioactive NGF, designed to eliminate 125I-NGF bound to low affinity sites. Therefore, the catecholaminergic cells possess both the low and high affinity forms of the receptor. NGF's ability to modulate tyrosine hydroxylase activity, as it does in mature catecholaminergic neurons, was tested in this system. Surprisingly, there was no statistically significant difference in tyrosine hydroxylase activity in cultures grown in the absence or presence of exogenous NGF. This raises the possibility that embryonic catecholaminergic cells are unable to respond to NGF in this specific way, even though the receptors for the factor are present.     

Multiple fluorescent ligands for dopamine receptors. II. Visualization in neural tissues

Selective dopamine receptor ligands, (R,S)-5-(4'-aminophenyl)-8-chloro-2,3,4, 5-tetrahydro-3-methyl-[1H]-3-benzazepin-7-ol, the 4'-amino derivative of the high affinity D1 receptor antagonist SCH 23390, the high affinity D2 receptor antagonist N-(p-aminophenethyl)-spiperone or NAPS, and the D2 selective agonist, 2-(N-phenethyl-N-propyl)-amino-5-hydroxytetralin or PPHT were chemically coupled to the fluorescent compounds, Bodipy, Cascade blue, coumarin, fluorescein, rhodamine, or Texas red. The utility of the 6 fluorescent moieties linked to the 3 dopamine receptor binding ligands for anatomical study of regional and cellular distribution patterns of the two dopaminergic receptor subtypes has been assessed in frozen sections of the rat striatum and compared to our previous report using the rhodamine-labeled antagonists. The regional staining for the two dopaminergic receptor binding sites supports previous work using in vitro receptor autoradiographic analyses; the D1 receptor binding was more robust than that of D2 receptors in the caudate nucleus. The cellular element which most frequently expressed striatal D1 binding sites had a medium-diameter cell body. Medium-sized cells also exhibited fluorescence for the D2 binding site, as did a much larger diameter element; potentially the cholinergic interneuron of the caudate nucleus. The pharmacological specificity for each of the different D1 fluorescent antagonist ligands in the tissues was determined by competition with 100-fold excess of unlabeled SCH 23390 (non-specific binding), spiroperidol (binding selectivity), the stereoactive paired isomers of butaclamol, and the serotonin 5-HT2 receptor antagonist ketanserin. The same criteria were used to assess the different D2 fluorescent agonist and antagonist ligand derivatives. The anatomical efficacy of these novel ligands was determined using selective dichroic filters to stimulate the fluorescent moieties in the optimal excitation wavelength, and the amount of fluorescent dopamine receptor binding was photographically measured and contrasted for each of the newly synthesized fluoroprobes. Using the most pharmacologically specific and anatomically efficient of these novel fluoroprobes, we determined the localization pattern of the D1 and D2 dopamine receptor binding sites in tissues reported to exhibit both subtypes of the receptor. The cellular distribution of the dopamine receptor binding sites was determined concurrently using fluoroprobes in the forebrain, mesencephalon, pituitary, retina, and superior cervical ganglion of the rodent, and bovine adrenal medullary chromaffin cells were examined using the rhodamine-labeled antagonists.     

Autoradiographic localisation of substance P (NK1) receptors in human primary visual cortex

This study utilised autoradiography to examine []-Bolton Hunter substance P (BHSP) binding in postmortem human visual cortex. In the primary visual area, layers I–III, IVC and VI exhibited low levels of BHSP binding, while high levels were observed in layers IVB and V. Because cells in layers IVB and V are known to be involved in processing direction-specific stimuli, it is possible that SP plays a role in modulating this visual process.     

Two types of acetylcholine receptors on the soma of primary afferent neurons

Acetylcholine (ACh) caused two types of depolarizations of the soma membrane of bullfrog primary afferent neurons (dorsal root ganglion cells); the one, a rapid transient depolarization, was nicotinic and other, a long-lasting one, as muscarinic in nature, respectively. The rapid transient depolarization was due to a simultaneous increase in sodium and potassium conductance, whereas the slow one was caused by a decrease in membrane potassium conductance. These results indicate that the soma of bullfrog primary afferent neuron is endowed with nicotinic and muscarinic receptors.     

Autoradiographic localization of hormone-concentrating cells in the brain of an amphibian, Xenopus laevis. II. Estradiol.

Autoradiographic techniques for light microscopic examination of sex steroid retention were applied to the brains of male and female Xenopus laevis, and anuran amphibian, after 3H-estradiol administration. Estrogen was concentrated by cells in three telencephalic areas (the ventral striatum, the ventral-lateral septum and the amygdala), the anterior preoptic area, the ventral thalamus, the ventral infundibular nucleus, and in the torus semicircularis. The anterior preoptic area and the ventral infundibular nucleus contained the greatest number of labelled cells. The topography of estrogen-concentrating cells was the same in male and female brains. This fact and comparisons of 3H-estradiol with 3H-testosterone retention in Xenopus suggest that the sex steroid itself, and not the genetic sex of Xenopus determines the pattern of uptake by cells in the brain. The distribution of hormone-concentrating cells in Xenopus has many similarities to that found in birds and mammals. Preoptic, hypothalamic (tuberal), limbic forebrain and specific mesencephalic sites in all these forms contain labelled cells following radioactive sex steroid administration. Findings in Xenopus add to the argument for a phylogenetically stable system of hormone-concentrating nerve cells in limbic, hypothalamic and mesencephalic structures.     

Pharmacological characteristics of diazepam receptors in neurons and astrocytes in primary cultures

Benzodiazepine binding sites in mouse astrocytes and neurons in primary cultures were labeled with [3H]diazepam (1.8 nM), and their inhibition by 14 different benzodiazepines and 3 benzodiazepine antagonists was studied. RO 5-4864, RO 7-3351, and, especially, the antagonist PK 11195 were much more potent in astrocytes than in neurons, whereas the opposite was true for the agonists alprazolam, clonazepam, flurazepam, RO 11-3128, and chlordiazepoxide, and, especially, the antagonists CGS-8216 and RO 15-1788. Flunitrazepam, diazepam, midazolam, RO 11-6893, and RO 5-2181 were about equipotent in the two cell types. The neuronal, but not the astrocytic, binding site showed stereospecificity. In astrocytes most of the drugs had pseudo-Hill coefficients close to one, whereas the pseudo-Hill coefficients in neurons, except for RO 5-4864 and PK 11195, were distinctly lower than one. Thus, the benzodiazepine binding sites had profoundly different pharmacological characteristics in neurons and in astrocytes.     

Metabolism and toxicity of methyl iodide in primary dissociated neural cell cultures

The metabolism and the toxicity of methyl iodide (Mel) has been studied in primary dissociated neuronal and glial murine cell cultures to further characterize the mechanisms of monohalomethane neurotoxicity. Measurement of intracellular glutathione (GSH) concentrations in cerebellar and cerebral cultures revealed GSH levels (21.6 +/- 1.9 and 29.1 +/- 1.9 nmol/mg protein, respectively) close to brain GSH levels measured in vivo. A GSH-depleting effect of Mel was demonstrated, with an ED50 for a 5 min exposure of 0.2 and 0.5 mM for glial and mixed (neurons + glia) cultures, respectively. Mel-induced GSH depletion was correlated with its neurotoxicity as the two powerful protective agents of monohalomethane toxicity, 3-amino-1-[m-(trifluoromethyl) phenyl]-2-pyrazoline (BW 755C, 1 mM) and nordihydroguaiaretic acid (NDGA, 10 microM) provided a 20-fold protection against depletion of GSH levels following Mel exposure. When glia and neurons from cerebral cultures were exposed in suspension to increasing concentrations of Mel for 30 min at 37 degrees C, a concentration-dependent increase in the production of formaldehyde resulted. Formaldehyde appeared to be an indicator of Mel metabolism as its production was decreased by sulfasalazine, a compound which was shown to be an inhibitor of the glutathione-S-transferases in this culture system. Since BW 755C and NDGA had no effect on formaldehyde production, while sulfasalazine as well as semicarbazide, a protective agent against formaldehyde-producing toxicants, failed to protect the cells against Mel toxicity, mechanism(s) of Mel neurotoxicity appeared independent of the GSH-mediated metabolism of this compound. It is concluded that GSH-mediated metabolic biotransformation is not necessary for the neurotoxicity of the monohalomethanes, that GSH depletion may act as a starting point in the chain of events leading to neural cell death, and that glia may be more sensitive than neurons to this primary effect. Moreover, these results demonstrate the value of primary dissociated neuronal cell cultures for studies of biochemical mechanisms of neurotoxicity.     

Morphology and distribution of neurons and glial cells expressing beta-adrenergic receptors in developing kitten visual cortex.

The morphology and distribution of cells expressing beta-adrenergic receptors has been studied in developing kitten visual cortex using a monoclonal antibody which recognizes both beta-1 and beta-2 adrenergic receptors. We found specific populations of neurons and glial cells which express beta-adrenergic receptor immunoreactivity in the kitten visual cortex. In adult animals, the receptors are most concentrated in the superficial and deep cortical layers (layers I, II, III and VI). About 50% of the stained neural cells in adult cat visual cortex are glial cells. Most of the immunoreactive neurons in layers III and V are pyramidal cells while those in layers II and IV are more likely to be nonpyramidal cells. In neonatal kittens, staining is weaker than that in adult cats and it appears to be concentrated in neurons of the deep cortical layers and in the subcortical plate and white matter. Only a few immunoreactive glial cells were found at this age. Receptor numbers increase after birth and by 24 days of age, the laminar distribution of beta-adrenergic receptors approaches that of adult animals. Immunoreactive glial cells in the white matter show a progressive increase in number throughout postnatal development.