Immunocytochemical use of internally radiolabelled monoclonal antibodies against tyrosine hydroxylase, substance P and enkephalin.

An improved protocol for the internal radiolabelling of monoclonal antibodies with tritiated lysine is described. Hybridoma cell lines producing monoclonal antibodies against the biosynthetic enzyme tyrosine hydroxylase and the neuropeptides, substance P and enkephalin, were employed in this investigation. Immunocytochemical detection of the endogenous antigens with these internally labelled antibodies was performed and when used in immunocytochemistry, the subsequent data were in complete agreement with previous light and electron microscopic studies. These results indicate that the present internal radiolabelling procedure does not alter the ability of the monoclonal antibody to recognise the endogenous antigen. This study, therefore, supports the use of internally labelled antibodies with compatible immunocytochemistry techniques in double staining procedures.

Two distinct monoclonal antibodies raised against mouse beta nerve growth factor. Generation of bi-specific anti-nerve growth factor anti-horseradish peroxidase antibodies for use in a homogeneous enzyme immunoassay.

Two hybridomas producing monoclonal antibodies against mouse beta nerve growth factor (NGF) were obtained from the fusion of hyperimmune splenocytes from rats immunized with polymerized beta-NGF and Sp2/0.Ag mouse myeloma cells. The monoclonal antibodies coded IgG 24 and 30 produced and secreted by the hybrid cells are both of the IgG2a subclass. Both monoclonal antibodies are capable of recognizing native NGF coated on microassay plates as well as the denatured factor on Western blots. However, only IgG 30 has been found to block NGF-induced process outgrowth from the rat pheochromocytoma cell line (PC12) as well as NGF-induced increase in choline acetyltransferase activity in rat primary septal cell cultures. In addition, only IgG 30 was able to detect immunocytochemically NGF-immunoreactive sites in fixed tissue. And, finally, IgG 24 could not compete for IgG 30 binding to immobilized native NGF. Consequently, it appears that these antibodies are recognizing different epitopes on the NGF molecule. Neither monoclonal antibody displayed any crossreactivity with serum albumin, aprotinin, epidermal growth factor or insulin. A hybrid-hybridoma producing bi-specific anti-NGF anti-horseradish peroxidase (HRP) monoclonal antibodies was generated from the fusion of an azaguanine resistant anti-HRP hybridoma, coded RAP2.Ag and the anti-NGF IgG 30 hybridoma treated with emetine. The potential merits of using these bi-specific antibodies in combination with their mono-specific anti-NGF parent in a homogeneous sandwich immunoassay for the quantitation of NGF are discussed.

Transforming growth factor-alpha's effects on astroglial-cholinergic cell interactions in the medial septal area in vitro are mediated by alpha 2-macroglobulin.

We reported previously that two epidermal growth factor receptor ligands, epidermal growth factor and transforming growth factor-alpha, inhibit medial septal cholinergic cell phenotypic expression (choline acetyltransferase and acetylcholinesterase activities) in vitro indirectly via (a) soluble molecule(s) released from astrocytes [Kenigsberg R. L. et al. (1992) Neuroscience 50, 85-97; Kenigsberg R. L. and Mazzoni I. E. (1995) J. Neurosci. Res. 41, 734-744; Mazzoni I. E. and Kenigsberg R. L. (1996) Brain Res. 707, 88-99]. In the present study, we found that this response to transforming growth factor-alpha is mediated, for the most part, by alpha 2-macroglobulin, a potent protease inhibitor with a wide spectrum of biological activities. In this regard, the effects of transforming growth factor-alpha on cholinergic cells can be blocked with immunoneutralizing antibodies raised against alpha 2-macroglobulin. Furthermore, western blot analysis reveals that although alpha 2-macroglobulin is present in conditioned media from control septal cultures, it is more abundant in those treated with transforming growth factor-alpha. In addition, exogenous alpha 2-macroglobulin, both in its native and trypsin-activated forms, can mimic transforming growth factor-alpha's effects on septal cholinergic cell expression. However, while the native antiprotease can slightly but significantly decrease choline acetyltransferase activity, trypsin-activated alpha 2-macroglobulin, in the nanomolar range, induces as marked a decrease in this enzyme activity as that noted with transforming growth factor-alpha. Furthermore, trypsin-activated alpha 2-macroglobulin, like epidermal growth factor/transforming growth factor-alpha, decreases choline acetyltransferase activity by arresting its spontaneous increase that occurs with time in culture, does so in a reversible manner and is not neurotoxic. In addition, trypsin-activated alpha 2-macroglobulin, in the nanomolar range, can affect choline acetyltransferase in a dual manner, up-regulating it at low concentrations while down-regulating it at higher ones. These responses are identical in mixed neuronal-glial and pure neuronal septal cultures. Furthermore, when concentrations of trypsin-activated alpha 2-macroglobulin, which alone decrease choline acetyltransferase, are added simultaneously with nerve growth factor, they serve to potentiate the nerve growth factor-induced increase in enzymatic activity. As GABAergic cell expression is not affected by alpha 2-macroglobulin, it appears that the effects of this protease inhibitor on medial septal neuronal expression are neurotransmitter-specific. Finally, trypsin-activated but not native alpha 2-macroglobulin promotes a dose-dependent aggregation of the septal neurons. This change in morphology, however, is not related to those noted in choline acetyltransferase activity. In summary, these data suggest that the expression of alpha 2-macroglobulin in astroglia from the medial septal nucleus can be controlled by epidermal growth factor receptor ligands to impact the functioning of basal forebrain cholinergic neurons.

Microglia from the developing rat medial septal area can affect cholinergic and GABAergic neuronal differentiation in vitro.

The normal development of the central nervous system is regulated by glia. In this regard, we have reported that astrocytes, stimulated by epidermal growth factor or transforming growth factor alpha, suppress the biochemical differentiation of rat medial septal cholinergic neurons in vitro, as evidenced by a decrease in choline acetyltransferase activity. In this study, we found that, in contrast to astrocytes, microglia enhance rather than suppress this aspect of cholinergic cell expression. When in excess, microglia can revert the effects of epidermal growth factor on the septal cholinergic neurons without altering the astroglial proliferative response to this growth factor. In the absence of growth factors or other glial cell types, microglia increase choline acetyltransferase activity above control levels and thus, may be a source of cholinergic differentiating activity. The increase in enzyme activity induced by microglia is rapid in onset, detected as early as 2 h after their addition to the septal neurons and maintained up to six or seven days in vitro. Furthermore, in the absence or presence of other glial cell types, microglia also influence septal GABAergic neurons by significantly increasing glutamate decarboxylase activity. As microglia affect neither septal cholinergic nor GABAergic neuronal cell survival, they appear to enhance the biochemical differentiation of these two neuronal cell types. Specific immunoneutralizing antibodies were used to identify the microglia-derived factors affecting these two neuronal types. In this regard, we found that the microglia-derived cholinergic differentiating activity is significantly suppressed by antibodies raised against interleukin-3. Furthermore, interleukin-3 was detected in both conditioned media and cell homogenates from septal neuronal-microglial co-cultures by western blotting. Finally, although basic fibroblast growth factor and interleukin-3 significantly increase septal glutamate decarboxylase activity, neither appears to be implicated in the GABAergic cell response to the microglia. In conclusion, these results demonstrate that microglia can enhance the biochemical differentiation of developing cholinergic and GABAergic neurons in vitro.

Microinjection of calmodulin antibodies into cultured chromaffin cells blocks catecholamine release in response to stimulation.

Polyclonal monospecific antibodies raised in sheep against rat testis calmodulin demonstrated cross-reactivity with bovine adrenal medullary chromaffin cell calmodulin. This antibody immunoprecipitated a [35S]methionine-labelled protein from chromaffin cell extracts prepared from [35S]methionine prelabelled cells that comigrated on a sodium dodecylsulfate gel electrophoresis system with calmodulin. In addition, an excess of non-radioactive exogenous calmodulin was shown to readily compete with this labelled endogenous protein for the antibodies' binding sites. Erythrocyte ghosts were used as vehicles for microinjecting either preimmune immunoglobulin G or anti-calmodulin immunoglobulin G into chromaffin cells following a polyethylene glycol-induced cell fusion procedure. The efficiency of ghost cell fusion was monitored and found to be 43.6 +/- 1% (n = 33). Cell morbidity subsequent to fusion and microinjection was negligible (87.8 +/- 0.6% of the total cell population were viable cells; n = 33) as determined by the Trypan Blue exclusion test. The delivery of intact antibodies raised against calmodulin directly into the cytoplasm of cultured chromaffin cells by erythrocyte ghost-mediated microinjection, inhibited catecholamine output in response to stimulation by either acetylcholine (10(-4) M) or a depolarizing concentration of potassium (56 mM). However, under these conditions, the chromaffin cell's ability to accumulate exogenous catecholamines through a high affinity uptake system, as well as the kinetic parameters that characterize this uptake mechanism remained unaltered. Furthermore, microinjection of preimmune immunoglobulin G did not modify either catecholamine uptake or stimulation-induced amine release from chromaffin cells. It therefore appears that calmodulin may play a role in the process of stimulus-secretion coupling in the chromaffin cell in culture while it is of little significance to the high affinity amine uptake mechanism.

Thrombin indirectly affects cholinergic cell expression in primary septal cell cultures in a manner distinct from nerve growth factor.

The effects of thrombin were examined in primary cultures of dissociated medial septal cells from fetal (embryonic day 17) rat brains. Seven days of continuous exposure of these cultures to thrombin produced a dose-dependent increase in the activity of the enzyme choline acetyltransferase (EC 2.3.1.6) and no change in the number of acetylcholinesterase (EC 3.1.1.7)-positive cells. Maximal induction of choline acetyltransferase activity occurred around 1-2 nM thrombin and was first detected after five days of treatment. In addition, thrombin promoted neuronal cell aggregation, proliferation of the astroglia, and changes in astroglial cell morphology. Neuronal aggregation was first noted after 24 h of treatment, while the proliferative response of the astroglia was first apparent after four days of treatment, slightly prior to the increase in choline acetyltransferase enzymatic activity. In order to see if the induction of the enzyme choline acetyltransferase was dependent upon the astroglial cell response, we included the anti-mitotic agent 5-fluoro-2'-deoxyuridine, to find that astrocyte proliferation, as well as thrombin-induced increase in choline acetyltransferase, were both abolished. In contrast, the aggregation of neurons was not affected. Finally, thrombin-induced changes in choline acetyltransferase could not be antagonized by immunoneutralizing anti-nerve growth factor antibodies and when thrombin was added simultaneously with 100 ng/ml 2.5-S nerve growth factor, the increase in choline acetyltransferase activity was additive. In conclusion, it appears that thrombin affects cholinergic septal neurons indirectly via the responsive astrocytes in a manner distinct from nerve growth factor.

Trifluoperazine, a calmodulin inhibitor, blocks secretion in cultured chromaffin cells at a step distal from calcium entry.

Trifluoperazine, a calmodulin antagonist, inhibited the secretory response of cultured bovine adrenal medullary chromaffin cells to acetylcholine (10(-4) M) or a depolarizing concentration of [K+] (56 mM KCl) in a dose-related fashion. The ID50s of this effect were 2 x 10(-7) M and 2.2 x 10(-6) M for acetylcholine and high [K+], respectively. A decrease in external [Ca2+] concentration of the incubation medium from 4.4 to 0.275 mM resulted in an increase in the percentage of inhibition produced by trifluoperazine on the acetylcholine-evoked secretory response from 20.7 to 96.5%, respectively. However, trifluoperazine inhibited the acetylcholine-evoked catecholamine output by a similar absolute magnitude for all [Ca2+] concentrations tested with the exception of 4.4 mM [Ca2+]. Trifluoperazine, unlike the [Ca2+] channel blocker Ni2+, in concentrations (10(-6)-10(-5) M) that were found to inhibit significantly [K+]-induced amine output did not modify [K+]-induced 45Ca uptake or 45Ca efflux. However, trifluoperazine at a concentration of 2.5 x 10(-5) M was found to produce a small decrease in the 45Ca efflux curve and a decrease in the [K+]-evoked 45Ca uptake of 30 +/- 14% (n = 6). In addition, 2.5 x 10(-6) M trifluoperazine, a concentration which was found to suppress high [K+]-induced amine release by 64 +/- 5%, did not inhibit the 45Ca2+-Ca2+ exchange mechanism. These results demonstrate that trifluoperazine, an antipsychotic agent with anticalmodulin activity, blocks catecholamine release from cultured chromaffin cells at a step distal from calcium entry and, consequently, suggests a role for calmodulin in the secretory process of these cells.

Light and electron microscopic distribution of nerve growth factor receptor-like immunoreactivity in the skin of the rat lower lip.

Nerve growth factor receptor distribution in the skin of the adult rat was studied by immunocytochemistry with the use of the monoclonal antibody 192-IgG. Immunoreaction occurred in a patchy pattern in the epidermis and outer root sheaths of hair follicles, as well as in nerves and in capsulated and non-capsulated sensory receptors. Electron microscopic analysis revealed that the areas of patchy epithelial immunostaining corresponded to the plasma membrane of groups of keratinocytes, which were always associated with intra-epithelial nerve fibres. Immunostaining was also associated with Merkel cells, hair follicle receptors, and the capsular cells of capsulated receptors. In the nerves, immunostaining was detected in perineurial cells and axons. After sensory denervation of the skin, immunostaining decreased considerably in the epithelia, as well as in the small nerves, although short segments of increased staining were observed in the latter. In larger nerves, immunostaining was markedly increased and mainly associated with newly formed Schwann cell processes. After sympathetic denervation, the pattern of immunostaining was identical to controls, except for augmented immunoreactivity in short segments of degenerating nerves around blood vessels and smooth muscle fibres. From these observations, it is suggested that the epithelial and perineurial cells synthesize and express nerve growth factor receptors, while receptors present in nerve fibres originate from the nerve cell bodies in the gasserian ganglion.

Epidermal growth factor affects both glia and cholinergic neurons in septal cell cultures.

The effects of epidermal growth factor on high density primary cultures of fetal (embryonic day 17) rat septal cells were examined. Under serum-free conditions, the continuous exposure of these cultures to epidermal growth factor for seven days significantly decreased choline acetyltransferase (EC 2.3.1.6) activity in a dose-dependent manner. Maximal decreases were observed from 1 to 10 ng/ml epidermal growth factor. This effect was completely abolished by the addition of anti-epidermal growth factor antibodies. The epidermal growth factor-mediated decrease in choline acetyltransferase activity was culture-time dependent, being first detectable after five days of factor application and may likely represent an inhibition of the spontaneous increase in enzyme activity that occurs with time in culture. Concomitant with changes in enzyme activity, epidermal growth factor produced a significant and proportional decrease in the number of acetylcholinesterase-positive neurons. This decrease in acetylcholinesterase-positive cells did not reflect a decrease in cholinergic cell survival as nerve growth factor could restore the number of acetylcholinesterase-positive neurons in epidermal growth factor-treated cultures to control levels. Furthermore, in these high-density cultures, epidermal growth factor did not affect general neuronal survival, while it did produce an increase in the number and intensity of glial fibrillary acidic protein-immunoreactive astroglia as well as in the number of macrophage-like cells. The proliferative response of these non-neuronal cells to epidermal growth factor, as assessed by [3H]thymidine incorporation, was evident after three days of epidermal growth factor application, persisted thereafter, and could be antagonized by the inclusion of the antimitotic 5-fluorodeoxyuridine. Furthermore, 5-fluorodeoxyuridine completely blocked the epidermal growth factor-mediated decrease in choline acetyltransferase activity. However, when epidermal growth factor was tested in pure glial cultures, it only directly induced proliferation of astrocytes. These results suggest that the proliferative response of either one or both of these glial cell types in the mixed cultures may be indirectly affecting cholinergic cell expression.

Development of a bi-specific monoclonal antibody for simultaneous detection of rabbit IgG and horseradish peroxidase: use as a general reagent in immunocytochemistry and enzyme-linked immunosorbent assay.

We describe the development of bi-specific monoclonal antibodies (MAb) capable of simultaneous recognition of rabbit immunoglobulin G (IgG) and horseradish peroxidase (HRP) for use in a variety of immunobased techniques. This bi-specific antibody, named McC8, was produced by fusion of the aminopterin-sensitive mouse hybridoma MAP.Ag.1, which secretes MAb against HRP, and splenocytes from a mouse previously immunized with whole rabbit IgG. The resultant hybrid-hybridoma co-dominantly expresses and secretes the immunoglobulin chains, i.e., IgG1 and IgG2b, of its respective parents, as determined by radial immunodiffusion. The binding sites on rabbit IgG for McC8 were determined on Western blots and in competition solid-phase enzymatic immunoassays with the use of allotype-specific rabbit sera. Both these techniques demonstrated that McC8 recognizes the light chain of the rabbit IgG molecule with preferential binding to the B4 kappa light-chain allotype. McC8 was successfully used in two-step immunocytochemistry for localization of calcitonin gene-related peptide (CGRP) in fibers of the superficial layers of the spinal trigeminal nucleus of the rat, as well as for localization of glial fibrillary acidic protein (GFAP)-immunoreactive sites in primary rat septal cell cultures, thus demonstrating its potential as a general developing reagent in conventional immunocytochemistry. McC8 compared favorably with peroxidase-antiperoxidase immunocytochemistry with respect to sensitivity. However, the bi-specific developing reagent proved superior to the conventional peroxidase-antiperoxidase procedure when both were employed in a similar fashion in tissues prone to display high background staining. Finally, McC8 was also employed as a developing reagent in a competitive ELISA designed for quantitation of CGRP with the use of a rabbit anti-CGRP primary antibody. The sensitivity of this quantitative ELISA (190 pg or 50 fmol CGRP per well) renders this bi-specific antibody suitable for use in quantitative immunoassays for detection of relevant peptides in biological systems.

Monoclonal antibodies against ?-bungarotoxin.

Hybridomas secreting monoclonal antibodies against ?-bungarotoxin were produced from the fusions of mice lymphocytes from hyperimmune animals with two mice myeloma cell lines ((NSI/1 or Sp2/0). Several anti-?-bungarotoxin monoclonal antibodies were derived and characterized. One of these (spB57) belonged to the IgG(1) subclass and bound potently to ?-bungarotoxin in a radioimmunoassay. This effect was specific to the anti-?-bungarotoxin antibody; a control series of antibodies (against tyrosine hydroxylase, enkephalin, neurofilament and the nerve growth factor receptor) did not bind radiolabelled toxin. Furthermore, the anti-?-bungarotoxin antibody did not interact with other radiolabelled receptor ligands. Using autoradiographic techniques, spB57 was shown to block the binding of [(125)I]?-bungarotoxin to brain sections. Similarly, spB57 blocked radiolabelled toxin binding to brain membranes; again this was an effect specific to the anti-?-bungarotoxin antibody. The decrease in [(125)I]?-bungarotoxin binding suggested that spB57 specifically bound the toxin molecule such that it could no longer interact with its receptor. Since the ?-BGT site has the characteristics of a nicotinic receptor, the effect of the antibody was also tested on the inhibition of [(125)I]?-bungarotoxin binding by cholinergic ligands. SpB57 partially reversed the inhibition of ?-toxin binding observed with nicotinic agonists and d-tubocurarine, but not with other nicotinic antagonists nor with muscarinic receptor ligands. These effects appeared to be specific for spB57, as they occurred to a much lesser extent with two other anti-?-BGT mAbs, nsB8 and spB28. These results suggest that an antibody against the ?-toxin can affect the interaction of nicotinic receptor ligands at the ?-BGT site.

A technique for the microinjection of macromolecules into viable chromaffin cells in culture.

A technique is described by which macromolecules can be microinjected into chromaffin cells in monolayer culture. This technique employs erythrocyte ghosts as the vehicles for microinjection, phytohemagglutinin, a plant lectin, as an attachment agent, and polyethylene glycol as the fusagen. High erythrocyte ghost-chromaffin cell fusion indices have been obtained, with an average of 46.2 +/- 1.1% (n = 14) of the total chromaffin cell population efficiently injected. Cell viability is well maintained during and after fusion with an average cell loss of 12 +/- 0.4% (n = 14) of the total cell population. The functional parameters which characterize the chromaffin cells in culture are unaltered after fusion-induced microinjection. The endogenous catecholamine content, the uptake of exogenous catecholamines via the high-affinity uptake mechanism for catecholamines, as well as the cell's response to various secretagogues remain unchanged. This procedure which allows a large number of cultured cells to be injected rapidly without significant loss of cell viability will aid in the study of the molecular and cell biology in this system.

Transforming growth factor alpha differentially affects GABAergic and cholinergic neurons in rat medial septal cell cultures.

The effects of transforming growth factor alpha (TGF alpha) on low and high density cultures of fetal (embryonic day 17) rat medial septal cells were investigated and in some instances, compared to those of epidermal growth factor (EGF). In high density cultures, TGF alpha induces a significant increase in the number of astroglia and microglia. While the effects of TGF alpha on the astroglia are more pronounced when compared to EGF, those on the microglia are less notable. In addition, TGF alpha produces a time- and dose-dependent decrease in the activity of choline acetyltransferase (EC 2.3.1.6) and a proportional decrease in the number of acetylcholinesterase-positive neurons in these high density cultures. However, although both EGF and TGF alpha decreased choline acetyltransferase activity maximally at the same concentration (10 ng/ml), the latter was consistently more potent. TGF alpha does not affect cholinergic cell survival but the expression of their chemical phenotype and does so indirectly via the glial cells. On the other hand, TGF alpha directly induces a dose- and time-dependent increase in glutamic acid decarboxylase activity in these high density cultures without affecting the number of glutamic acid decarboxylase immunoreactive neurons. In low density cultures, TGF alpha acts as a general neuronal survival factor, affecting both cholinergic and GABAergic neurons. Here TGF alpha's neurotrophic activity is more evident than its effects on their chemical phenotype. These results suggest that TGF alpha exerts distinct and differential effects on the biochemical expression of two neuronal populations in the developing medial septum maintained in high density culture. Finally, as TGF alpha acts as a general neuronal survival factor in low density cultures, cell to cell interactions appear to be important in the ultimate response of these cells to this growth factor.

Localization and characterization of epidermal growth-factor receptors in the developing rat medial septal area in culture.

The presence and binding properties of epidermal growth-factor receptors (EGF-Rs) in different cell types purified from the rat medial septal area in culture were investigated. We report that astrocytes, oligodendrocytes and neurons from this area possess EGF-Rs while microglia do not. EGF-binding sites are detectable on astrocytes derived from the medial septum of both embryonic and neonatal rats. Scatchard analysis of the data for astrocytes from the fetal rats show that EGF specifically binds to both high- (Kd = 7.21 x 10(-10) M, Bmax = 3602 receptors/cell) and low-affinity (Kd = 3.99 x 10(-8) M, Bmax = 86,265 receptors/cell) receptors on these cells. On the other hand, astrocytes purified from neonatal tissue possess a greater number of high-affinity receptors (Bmax = 10,938 receptors/cell) when compared with the embryonic astroglia. With time in culture, the number of both types of receptors on neonatal astrocytes decreases. Oligodendrocytes also possess high- and low-affinity EGF-Rs with dissociation constants of 3.25 x 10(-10) M and 3.85 x 10(-8) M, respectively. The number of receptors on oligodendrocytes is significantly lower than those of neonatal astrocytes (Bmax = 1185 and 25,081 receptors/cell for high- and low-affinity binding sites, respectively). Finally, neurons from this area also exhibit two different EGF-R types with dissociation constants similar to those described for astrocytes. As the number of receptors/neuron (Bmax = 136 and 1159 receptors/cell for high- and low-affinity binding sites, respectively) appears to be extremely low, it is possible that EGF specifically binds only to a subpopulation of neurons from this area. These studies demonstrate which cell types in the developing medial septal area possess EGF-Rs and provide a detailed characterization of these binding sites. These EGF-R-bearing cells may be potential targets for this growth factor or for transforming growth factor alpha in this brain area.

Cholinergic cell expression in the developing rat medial septal nucleus in vitro is differentially controlled by GABAA and GABAB receptors.

The early appearance and relative abundance of GABAergic neurons in basal forebrain cholinergic nuclei like the medial septum suggest that the maturation of the later developing cholinergic neurons in these nuclei may be controlled by GABA. To examine this possibility, the effects of both exogenous GABA and specific GABA receptor agonists, as well as that of endogenous GABA on the phenotypic expression and survival of the cholinergic neurons in primary cultures from the fetal rat medial septum, were studied. Treatment of these cultures for six days with GABA significantly decreased the enzymatic activity of choline acetyltransferase (EC 2.3.1.6) (ChAT) in a dose-dependent manner. This response to exogenous GABA was blocked by bicuculline, mimicked by muscimol and slightly potentiated by saclofen. Consistent with this latter observation, the GABAB receptor agonist, baclofen, dose-dependently increased septal ChAT activity. However, while the effect of baclofen on cholinergic expression was lost in the absence of glia, the suppressive effects of GABA or muscimol were more marked. Acetylcholinesterase (EC 3.1.1.7) (AChE) expression in mixed neuronal-glial cultures, was, like ChAT activity, increased or decreased in intensity with the inclusion of baclofen or muscimol, respectively. Although the number of AChE positive neurons in muscimol-treated cultures was significantly lower than that in controls, no changes in neither neuronal nor general cell viability were noted. Finally, as GABAA or GABAB receptor antagonists bicuculline and picrotoxin or saclofen, when applied alone to mixed cultures, increased or decreased ChAT activity, respectively, it appears that endogenous GABA, tonically released in the developing septum, may, via specific receptor types, differentially control the biochemical maturation of the cholinergic neurons.

Identification of glial cell types involved in mediating epidermal growth factor's effects on septal cholinergic neurons.

We found previously that epidermal growth factor (EGF) decreases choline acetyltransferase (ChAT) activity in forebrain cholinergic neurons in vitro indirectly via glia (Kenigsberg et al.: Neuroscience 50: 85-97, 1992). However, which glial type(s) are implicated in this response remained to be determined. Here we report that in primary cultures from the fetal rat medial septal area the complete elimination of oligodendrocytes or partial elimination of microglia from these cultures does not change the cholinergic cell response to EGF. However, the elimination of astroglia in our cultures by alpha-aminoadipic acid treatment blocks EGF's effects on the cholinergic neurons. Co-culture experiments using pure neuronal and purified glial cells from the medial septum further demonstrate that the cholinergic cell response to EGF can be maintained in the presence of astroglia only. In addition, it appears that EGF regulates the release of soluble factors from pure astroglia cultures following their peak mitotic response to EGF that decreases ChAT enzymatic activity. This soluble cholinergic neuromodulatory activity found in conditioned media from EGF-treated astrocytes has a molecular weight greater than or equal to 10 kD and loses potency following multiple freeze-thaw cycles. These results suggest that a direct glial cell response to a specific glial growth factor like EGF may have an important impact on the expression of local neurons, like the cholinergic in the forebrain.

Production of a bi-specific monoclonal antibody recognizing mouse kappa light chains and horseradish peroxidase. Applications in immunoassays.

The production of a bi-specific monoclonal antibody that simultaneously recognizes mouse kappa light chains and horseradish peroxidase (HRP) for use as a general developing reagent in a wide variety of immunobased techniques is described. This antibody, named McC10, was produced by the fusion of an aminopterin-sensitive interspecies hybridoma which secretes rat monoclonal antibodies against HRP (RAP2.Ag) and splenocytes from a rat immunized with whole mouse immunoglobulin (Ig)G. The hybrid-hybridoma generated from this fusion expresses and secretes rat Igs of the IgG1 and IgG2a subclasses, as determined by radial immunodiffusion. In competitive binding solid-phase enzymatic assays, McC10 was found to cross-react with all four mouse IgG subclasses as well as mouse kappa light chains. In contrast, in this type of assay, McC10 did not appear to recognize mouse IgA, IgM or lambda light chains. However, IgM-bearing kappa light chains were recognized by immunocytochemistry. Epitope specificity of this bi-specific antibody was more clearly determined on immunoblots where McC10 was found to exclusively recognize mouse kappa light chains and display no cross-reactivity with mouse Ig heavy chains nor with kappa light chains from rat or rabbit. In addition, McC10 was used successfully in two-step immunocytochemistry (ICC) for the localization of enkephalin, nerve growth factor (NGF) receptor and paired helical filament-immunoreactive sites in rat brain, rat skin and human brain, respectively, using mouse IgG's and IgM's as primary antibodies. McC10 compared favourably with peroxidase-anti-peroxidase (PAP) ICC with respect to sensitivity but was markedly superior with respect to specificity when used in fixed human brain or rat skin.(ABSTRACT TRUNCATED AT 250 WORDS)

The production of a "universal developer" for the immunological detection of human IgG and its application in immunodiagnostics.

This study describes the development of a biospecific monoclonal antibody capable of the simultaneous recognition of horseradish peroxidase (HRP) and human IgG. This antibody, coded McC2, has been applied in a novel manner as a universal developing reagent for the detection of human IgG. McC2 cross-reacts with all human IgG subtypes and was found to recognise an epitope on the Fc portion of human IgG. McC2 does not cross-react with human IgM or IgA. This bi-specific antibody belongs to the mouse IgG1 subclass. McC2 was used for the detection of human IgG in a simple one step enzyme-linked immunosorbent assay (ELISA). Use of this bi-specific antibody in this assay resulted in an excellent signal to noise ratio with background in negative control wells virtually nonexistent. McC2 was also applied in a clinical diagnostic test for the detection of auto anti-nuclear antibodies in patient sera. McC2 was substituted, in a blind study, for a HRP-conjugated second antibody supplied with the test kit. All sera were tested both with the kit's second antibody and McC2. When using McC2, we obtained no false positive results whereas five false positives were obtained when using the kit's second antibody. However, one false negative result was obtained with the use of McC2 as a developing reagent while none were noted with the use of the kit's second antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Gangliosides potentiate in vivo and in vitro effects of nerve growth factor on central cholinergic neurons.

The effects of nerve growth factor beta (beta-NGF) and ganglioside GM1 on forebrain cholinergic neurons were examined in vivo and in vitro. Following unilateral decortication of rats, GM1 (5 mg/kg per day) administered intracerebroventricularly could protect forebrain cholinergic neurons of the nucleus basalis magnocellularis from retrograde degeneration in a manner comparable to beta-NGF. Administered in combination with beta-NGF, GM1 produced a significant increase in choline acetyltransferase activity in the nucleus basalis magnocellularis and remaining cortex ipsilateral to the lesion. Concentrations of GM1 that were ineffective when administered alone in this lesion model, when given with beta-NGF, potentiated beta-NGF effects in both of the above brain areas. In dissociated septal cells in vitro, an increase in choline acetyltransferase activity was noted at beta-NGF concentrations as low as 0.1 pM and reached a plateau at 1 nM. A moderate (up to 35%) stimulation of choline acetyltransferase activity was observed with 10 microM GM1. The application of beta-NGF in combination with 10 microM GM1 or 0.1 microM GM1, a concentration that is ineffective in these cultures, produced a much greater increase in choline acetyltransferase activity than did beta-NGF alone. These observations support the idea that exogenously applied gangliosides can elicit trophic responses in cholinergic neurons of the central nervous system. That GM1 increases and even potentiates beta-NGF effects suggests that some of the trophic actions of this compound may be mediated through endogenous trophic factors.