Uptake of [3H]serotonin in the abdominal ganglion of Aplysia californica. Further studies on the morphological and biochemical basis of presynaptic facilitation

Sensitization of the gill-withdrawal reflex in Aplysia california is mediated, in part, by a group of identified neurons, the L29 cells, which produce presynaptic facilitation of transmitter release from siphon sensory neurons. Physiological and pharmacological studies have provided indirect evidence that the L29 cells are serotonergic. In the present study we have used the specific uptake [3H]serotonin ([3H]5-HT) and electron-microscopic autoradiography in combination with horseradish peroxidase-labeling of identified neurons to characterize the fine structure of Aplysia serotonergic terminals and to examine more directly the transmitter biochemistry of the L29 neurons. Abdominal ganglia were incubated for 2 h in 10(-6) M [3H]5-HT and thick and thin plastic sections examined with the light and electron microscope. L29 varicosities, identified by labeling with HRP, were found to accumulate [3H]5-HT. In addition, [3H]-5-HT was localized to unidentified varicosities within the neuropil as well as to vesicle-filled terminals that formed axosomatic contacts in the cortical regions of the ganglion. The processes that accumulated [3H]5-HT contained conspicuous dense core vesicles identical in morphology to those previously described for L29. Some processes were found to make contact with HRP-labeled varicosities of sensory neurons. Comparison with results obtained from ganglia exposed to [3H]5-HT in the presence of either non-radioactive 5-HT or non-radioactive dopamine indicate that the uptake process is transmitter-specific. These studies provide additional evidence that the L29 cells are serotonergic and are consistent with the notion that aminergic neurons may be preferentially involved in modulatory synaptic actions.     

Distribution of buccalin-like immunoreactivity in the central nervous system and peripheral tissues of Aplysia californica.

The neuropeptide buccalin A was originally purified and sequenced from a nerve-muscle system used in feeding-related behaviors of Aplysia californica in which it has been proposed that it acts as a modulatory cotransmitter. The distribution of buccalin-like immunoreactivity in the central ganglia and in peripheral tissues of Aplysia californica was examined by whole mount immunohistochemical techniques. Immunoreactive material was located in specific cell bodies and clusters of neurons in each of the ganglia. Immunoreactive fibers were present in each of the connectives between ganglia, in tracts coursing through the ganglia, and in the majority of the peripheral nerves. Most fibers were smooth in contour, but some had regularly spaced swellings. Varicosities containing immunoreactive material were located on specific neuronal somata and on certain tissues associated with the feeding, circulatory, digestive, and reproductive systems. The specific and widespread distribution of buccalin-like immunoreactivity supports the hypothesis that members of the buccalin peptide family act as neuromodulators or neurotransmitters in a variety of central and peripheral circuits in Aplysia.     

Localization of myomodulin-like immunoreactivity in the central nervous system and peripheral tissues of Aplysia californica.

The distribution of myomodulin-like peptides in the nervous system of Aplysia californica was examined by using immunocytochemical techniques. Neurons and cell clusters containing immunoreactive material were located in each of the major central ganglia. Myomodulin-like immunoreactivity was also present in fibers in each of the connectives between the ganglia and in peripheral nerves. Varicosities containing immunoreactive material were located on specific regions of peripheral tissues associated with the feeding, digestive, cardiovascular, and reproductive systems. Double-labeling experiments were used to demonstrate myomodulin-like immunoreactivity in two identified neurons, the motor neuron B16 in the buccal ganglion and the widely acting interneuron L10 in the abdominal ganglion. Structures in the eye and cerebral ganglion that may correspond to the optic circadian pacemaker system were also stained. The central and peripheral distribution of myomodulin-like immunoreactivity indicates that this family of neuropeptides is present in specific efferent, afferent, and interneuronal elements that participate in a diversity of neural circuits in Aplysia.     

Heterogeneity of cholecystokinin/gastrin-like immunoreactivity in the nervous system of the nematode Ascaris suum

A wholemount immunocytochemical method was used for the localization of cholecystokinin (CCK8)-like and gastrin-like immunoreactivity in Ascaris. The patterns of specific neuronal staining given by two antisera and four monoclonal antibodies made against CCK8, and one antiserum made against gastrin were investigated. Preabsorption of these antibodies with CCK8 or gastrin 17 resulted in complete loss of immunoreactivity in almost all of the neurons (two antisera also contained nonspecific antibodies), suggesting that all of the antibodies recognize epitopes, in Ascaris neurons, that include some or all of the C-terminal five amino acids that are identical in CCK8 and gastrin 17. However, the seven different antibodies showed immunoreactivity in different subpopulations of neurons, implying that there are at least seven different species of CCK-like molecules in Ascaris. Fractionation of Ascaris peptide extracts by high performance liquid chromatography (HPLC), monitoring fractions with a CCK8 radioimmunoassay (RIA), also shows heterogeneity of molecules immunologically related to CCK8. © 1996 Wiley-Liss, Inc.     

Distribution of membrane glycoproteins among the organelles of a single identified neuron of aplysia. I. association of a [H]glycoprotein with vesicles

[³H]-acetylgalactosamine injected into the cell body of R2, the giant cholinergic neuron in the abdominal ganglion, is rapidly incorporated into membrane glycoprotein and glycolipid. Incorporation, which is localized to the injected cells, occurs at a constant rate for approximately 15 h. By that time, 83% of the labeled macromolecules are associated with membranes. Quantitative electron microscopic radioautography of the cell body shows that labeling of membranous organelles is selective: the Golgi apparatus, endoplasmic reticulum, and lucent and compound vesicles are labeled, while the nucleus, end-stage lysosomes, and mitochondria are not. SDS-polyacrylamide gel electrophoresis of the total membranes from more than 40 R2s examined individually resolves reproducibly 5 major labeled glycoprotein components. In order to determine which of these are associated with vesicles, we isolated a labeled vesicle fraction from R2 using a combination of differential centrifugation and filtration on a column of glass beads with 200 nm pores. This fraction was consistently enriched in [³H]glycoproteins I (180,000 Daltons) and V (90,000 Daltons) relative to those fractions containing larger organelles. These experiments suggest that different organelles contain characteristic membrane components.     

Comparative localization of two serotonin receptors and sensorin in the central nervous system of Aplysia californica

Aplysia californica is a powerful model for understanding the cellular and molecular mechanisms underlying modulation of neuronal plasticity and learning. In the central nervous system of Aplysia, serotonin is associated with various behaviors. For example, it induces short-, intermediate-, and long-term synaptic changes in sensory neurons during learning and inhibits the afterdischarge of the bag cells that initiate egg-laying behavior. Little is known about the nature and contribution of serotonin receptors involved in the numerous serotonin-mediated physiological responses in Aplysia. Recently, two G(i)-coupled serotonin receptors (5-HT(ap1) and 5-HT(ap2)) were cloned. We now report that, by using in situ hybridization to express the profile of these receptors, we are able to gain critical insight into their roles in the behavior of Aplysia. We compared their distribution to that of sensorin-A, a peptide specifically found in sensory neurons. We wished to determine their involvement in some simple forms of behavioral modifications. 5-HT(ap1) and 5-HT(ap2) mRNAs are expressed in all ganglia of the Aplysia central nervous system. Stronger signal was observed with the 5-HT(ap2) antisense probe than with the 5-HT(ap1) antisense probe. Notably, mRNA coding for the receptors was found in several identified neurons, in the bag cells, in characterized serotonergic neurons, and in neurons of the mechanosensory clusters that expressed sensorin. We also observed heterogeneity of receptor expression between R2 and LPl1 and among neurons of a single cluster of sensory neurons. These results suggest that 5-HT(ap1) and 5-HT(ap2) receptors may regulate the response to serotonin and/or its release in several neurons.     

Biochemical and immunocytological localization of molluscan small cardioactive peptides in the nervous system of Aplysia californica

High pressure liquid chromatography (HPLC) followed by bioassay on isolated snail hearts were used to locate two related peptides, termed small cardioactive peptides A and B (SCPA and SCPB) in each of the central ganglia of Aplysia. The peptides are most concentrated in the buccal ganglia, the ganglia involved in the control of feeding movements. Immunocytology with antisera raised to conjugated SCPB stained three groups of neurons in the buccal ganglia. One group consisted of relatively small neurons that were tightly clustered. The second group was comprised of larger neurons that were more scattered. The third group was made up of several neurons including the two largest in the ganglia, identified cells B1 and B2. B1 and B2 and other neurons in this group innervate the gut by way of the esophageal nerve. HPLC-bioassay of single, individually dissected B1 or B2 neurons demonstrated that the two peptides are present in a single cell. For B2, but not B1, choline injected into the cell body was converted to the conventional transmitter, acetylcholine. This indicates that, in addition to the two peptides, B2 also contains choline acetyltransferase, and raises the possibility that acetylcholine and the SCPs may act as co-transmitters in B2. Strong immunocytological staining of fibers and varicosities was observed in the neuropilar region of the cerebral, pleural, pedal, and abdominal ganglia. In addition to the buccal ganglia, immunoreactive neurons were observed in all of the other central ganglia. The high concentration of the SCPs and the relatively large number of immunoreactive neurons in the buccal ganglion suggest a particularly important role of these peptides specifically in feeding behavior. However, the widespread occurrence of the SCPs in fibers and neuronal cell bodies throughout the nervous system suggests that these peptides also may have additional behavioral functions in Aplysia.     

Catecholamine-containing cells in the central nervous system and periphery of Aplysia californica.

Previous studies have suggested the presence of numerous catecholamine-containing cells in both the central ganglia and peripheral tissues of Aplysia, but they often offered conflicting or incomplete accounts of numbers, locations, and morphologies. The current study combines aldehyde-induced histofluorescence and tyrosine hydroxylase-like immunoreactivity together with confocal microscopy to provide details of these cells. Approximately 35-50 neurones in the cerebral ganglia, 4-8 neurones in the pedal ganglia, 5 neurones in the buccal ganglia, and numerous small fibres in various nerve trunks exhibited both immunoreactivity and aldehyde-induced fluorescence. Approximately 20 cells in the pedal ganglia and 4 cells in the buccal ganglia exhibited only immunoreactivity whereas 15-20 neurons in the cerebral ganglia exhibited only aldehyde-induced fluorescence. No somata in the pleural or abdominal ganglia exhibited aldehyde-induced fluorescence or immunoreactivity. Both aldehyde-induced histofluorescence and immunoreactivity also labelled what appeared to be two classes of catecholamine-containing cells in the gill, siphon, oesophagus, rhinophore, tentacle, and reproductive organs. The more numerous, but smaller cells had subepithelial somata and processes penetrating the overlying body wall, thus suggesting a sensory function. Another class of neurones had larger somata, often located more deeply within the tissue, and occasionally appeared to be multipolar. Processes from these various peripheral cells appeared to comprise the major component of afferent fibres and to form an extensive peripheral plexus, often associated with various muscles. The morphologies of the peripheral cells thus suggest involvement in both local and centrally mediated reflexes and responses, but additional studies must test such hypothesised functions and determine the sensory modalities that the cells mediate.     

The enteric nervous system in tissue culture. III. Studies on neuronal survival and the retention of biochemical and morphological differentiation

The maintenance of differentiated properties and survival rates of enteric neurons, grown in explant cultures for periods of up to 3 weeks, was studied. Using catecholamine fluorescence, immunohistochemistry and autoradiography, it was found that adrenergic neurons, VIP-containing neurons and putative GABAergic neurons, which constitute small subpopulations of guinea pig myenteric neurons in vivo, were all represented in plexus explants after maintenance in culture for 2–3 weeks. The pattern of expression of the transmitter-related enzymes, acetylcholinesterase and monamine oxidase, paralleled that found in in situ studies. Investigation of neuronal structure by intracellular injection of horseradish peroxidase revealed that the cultured neurons continue to express the wide diversity in gross morphology which characterizes these cells in vivo. Employing autoradiography following uptake of [³H]GABA to label putative GABAergic neurons, their survival rate from days 1 to 15 of culturing was determined. No neuronal death was detected between days 1 and 5, while the number of neurons decreased between days 5 and 15. These observations suggest that enteric neurons maintained in explant cultures survive well and maintain to a high degree their histochemical and morphological properties.     

Segmental differentiation in the leech central nervous system: proposed segmental homologs of the heart accessory neurons.

As part of an on-going study of segmental differentiation in the central nervous system (CNS) of the leech Hirudo medicinalis, a search was made for putative segmental homologs of the heart accessory (HA) neurons, which exist exclusively as a bilateral pair in the ganglia of the fifth and sixth body segments. As it is not yet feasible to obtain adequate cell lineage information in H. medicinalis, potential homologs of the HA neurons were determined using morphological, immunohistochemical, and electrophysiological criteria. Among cells in other body ganglia with somata in the same locations as HA neurons, a pair was found having extensive morphological and physiological similarities to HA neurons. These we have called HA-like (HAL) neurons. Adult HA and HAL neurons have closely related patterns of primary branching, in terms of shape, intraganglionic pathways taken, and extraganglionic projections. The number, location, and relative thickness of branches are also similar among these cells. In embryos 10 to 11 days old, HA and HAL neurons have virtually identical branching patterns, with primary and secondary branches of nearly uniform caliber. Differences in branch thickness develop gradually; by embryonic day 20, they resemble those found in adult neurons. Two features found to differ between HA and HAL neurons were the cell body diameter (larger for the HA cells) and the expression of antigens recognized by the monoclonal antibody Laz1-1 (absent at a detectable level in the HA neurons). At a physiological level, the HA and HAL neurons showed action potentials of similar size and shape, as well as inhibitory synaptic inputs from a common source, the heart interneurons (HN). The observations presented here suggest that there is a common developmental origin for the HA and HAL neurons, and hence that their fates are positionally determined by as yet unknown factors.     

Localization and characterization of gastrin/cholecystokinin-like immunoreactivity in the central nervous system of Aplysia californica

Gastrin/cholecystokinin-like immunoreactivity (G/CCK-LI) was localized by immunocytochemistry in neurons in the central nervous system of Aplysia californica. Neuronal cell bodies were specifically immunostained in the buccal, cerebral, pedal, and abdominal ganglia but not in the pleural ganglia. Neural G/CCK-LI processes were observed in the neuropil of all ganglia and connectives and in some but not all of the peripheral nerves examined. Somata containing G/CCK-LI ranged from 15 to 200 micron in diameter. Ganglionic G/CCK-LI was most efficiently extracted in neutral or basic solutions and consisted mainly of a substance eluting in a volume corresponding to a peptide of between 8 and 17 amino acid residues on gel filtration. This G/CCK-LI diluted in parallel to mammalian gastrin in radioimmunoassays using two antisera differing in their specificities for the bioactive COOH-terminal region of mammalian G/CCK. We conclude that G/CCK-LI is distributed widely in the central and peripheral nervous systems of Aplysia californica and that this immunoreactivity probably represents a small peptide which is similar but not identical to mammalian gastrins and cholecystokinins at the functionally critical COOH terminus.     

Localization of GABA-like immunoreactivity in the central nervous system of Aplysia californica.

Gamma-aminobutyric acid (GABA) is present in the central nervous system of Aplysia californica (Gastropoda, Opisthobranchia) where its role as a neurotransmitter is supported by pharmacological, biochemical, and anatomical investigations. In this study, the distribution of GABA-immunoreactive (GABAi) neurons and fiber systems in Aplysia was examined by using wholemount immunohistochemistry and nerve backfill methods. GABAi neurons were located in the buccal, cerebral, and pedal ganglia. Major commissural fiber systems were present in each of these ganglia, whereas more limited fiber systems were observed in the ganglionic connectives. Some of the interganglionic fibers were found to originate from two unpaired GABAi neurons, one in the buccal ganglion and one in the right pedal ganglion, each of which exhibited bilateral projections. No GABAi fibers were found in the nerves that innervate peripheral sensory, motor, or visceral organs. Although GABAi cells were not observed in the pleural or abdominal ganglia, these ganglia did receive limited projections of GABAi fibers originating from neurons in the pedal ganglia. The distribution of GABAi neurons suggests that this transmitter system may be primarily involved in coordinating certain bilateral central pattern generator (CPG) systems related to feeding and locomotion. In addition, the presence of specific interganglionic GABAi projections also suggests a role in the regulation or coordination of circuits that produce components of complex behaviors.     

Serotonin immunoreactivity in the central nervous system of the marine molluscs Pleurobranchaea californica and Tritonia diomedea

The central nervous systems of the marine molluscs Pleurobranchaea californica (Opisthobranchia: Notaspidea) and Tritonia diomedea (Opisthobranchia: Nudibranchia) were examined for serotonin-immunoreactive (5-HT-IR) neurons and processes. Bilaterally paired clusters of 5-HT-IR neuron somata were distributed similarly in ganglia of the two species. In the cerebropleural ganglion complex, these were the metacerebral giant neurons (both species), a dorsal anterior cluster (Pleurobranchaea only), a dorsal medial cluster including identified neurons of the escape swimming network (both species), and a dorsal lateral cluster in the cerebropleural ganglion (Pleurobranchaea only). A ventral anterior cluster (both species) adjoined the metacerebral giant somata at the anterior ganglion edge. Pedal ganglia had the greatest number of 5-HT-IR somata, the majority located near the roots of the pedal commissure in both species. Most 5-HT-IR neurons were on the dorsal surface of the pedal ganglia in Pleurobranchaea and were ventral in Tritonia. Neither the buccal ganglion of both species nor the visceral ganglion of Pleurobranchaea had 5-HT-IR somata. A few asymmetrical 5-HT-IR somata were found in cerebropleural and pedal ganglia in both species, always on the left side. The clustering of 5-HT-IR neurons, their diverse axon pathways, and the known physiologic properties of their identified members are consistent with a loosely organized arousal system of serotonergic neurons whose components can be generally or differentially active in expression of diverse behaviors. J. Comp. Neurol. 395:466–480, 1998. © 1998 Wiley-Liss, Inc.     

Distribution of membrane glycoproteins among the organelles of a single identified neuronm of aplysia. II. isolation and characterization of a glycoprotein associated with vesicles

Glycoprotein-I (mol.wt. 180,000) is associated with a vesicle fraction from the cytoplasm of R2, the giant cholinergic neuron in the abdominal ganglion. Electron microscopy has shown that R2 contains both lucent and compound vesicles. We have used anisomycin, an inhibitor of protein synthesis, to provide evidence that Component-I is a constituent of somatic compound vesicles. In the presence of the drug, [³H]N-acetylgalactosamine injected into R2 is incorporated almost exclusively into Component-I. Quantitative electron microscopic radioautography of treated cells shows a marked increase in the proportion of silver grains over compound vesicles and a decrease in labeling of other organelles compared with untreated cells. Analysis of Component-I, isolated using the chaotropic agent lithium diiodosalicylate, shows it to contain fucose, N-acetylgalactosamine, and N-acetylglucosamine. Proteolytic digestion with pronase yields a complex pattern glycopeptides. The proportion of [³H]N-acetylgalactosamine in these glycopeptides is altered in the presence of anisomycin. These results together with radioautographic analyses suggest that large carbohydrate chains are elaborated within the endoplasmic reticulum and that smaller chains are added in the Golgi region or on the membrane of the compound vesicles.     

Interleukin-2 inhibits the GABA-induced Cl- current in identified Aplysia neurons.

The effects of extracellularly applied recombinant human interleukin-2 (rhIL-2) on the gamma-aminobutyric acid (GABA)-induced Cl- current recorded from identified neurons (R9 and R12) of Aplysia kurodai were investigated with conventional voltage-clamp and pressure ejection techniques. Bath-applied rhIL-2 (10-40 U/ml) reduced the GABA-induced current in the neurons without affecting resting membrane conductance and the holding current. The suppressing effect of rhIL-2 on the current was completely reversible. Heat-inactivated rhIL-2 was without effect. These results suggest that the immunomodulator IL-2 can modulate the GABA-induced response in the nervous system.     

Programmed cell death in the nervous system of an adult insect

The tobacco hornworm moth, Manduca sexta, shows extensive degeneration of abdominal neurons after the adult moth emerges (ecloses) from the old pupal skin. Death of interneurons begins about 2 hours before eclosion and continues through the next 30 hours. Motorneuron degeneration starts slightly later, commencing at about 8 hours after eclosion. Analysis of the death of identified motorneuons showed that there was a precise spatial and temporal program of neuronal death. Particular cells invariably either continued to live or died and, among the latter, the time of death varied from cell to cell but for a given cell was constant. This program of cell death was somewhat flexible in that the fates of certain cells could be adaptively modified by behaviors that might naturally occur in the life of the insect.     

Postembryonic development of serotoninlike immunoreactivity in the central nervous system of the snail, Lymnaea stagnalis

Posthatching growth in the pond snail Lymnaea stagnalis involves approximately a 20-fold increase in the linear dimensions of the ganglia composing the central nervous system. Developmental change within the population of neurons exhibiting serotoninlike immunoreactivity (SLIR) was examined in order to explain this growth in cellular terms. The study indicates that at least two factors contribute to the growth of the nervous system. First, SLIR cells approximately double in number from the 200-250 cells in hatchlings to the complement found in animals approaching sexual maturity. Much of this increase in cell number occurred within identifiable discrete clusters of neurons with different clusters adding cells at different rates and at different times. The number of SLIR cells also increased in more diffuse populations, particularly along the medial aspects of the paired pedal and the right parietal ganglion. No identified cells were added postembryonically. In addition to the increases in neuron numbers, posthatching development in Lymnaea also involves the growth of individual cells. All cells examined showed continuous somatic growth during posthatching development, but different identified cells and different cell clusters were characterized by different rates of relative growth. Together, the results highlight the complexity of postembryonic development in the snail by indicating the temporal and spatial specificity for both cell addition and cell growth within the nervous system.     

Aplysia synaptosomes. I. Preparation and biochemical and morphological characterization of subcellular membrane fractions

We prepared and characterized subcellular membrane fractions from the CNS of Aplysia californica that are enriched in isolated nerve terminals (synaptosomes). Ganglia were homogenized in 1.1 M sucrose and fractionated on a 2-step sucrose gradient, yielding 50 micrograms protein/animal in the synaptosomal fraction (P3), which was enriched 3-fold in plasma membrane as compared with the initial homogenate. Quantitative morphometry of electron micrographs revealed that P3 contained 25% intact synaptosomes, a 5-fold enrichment over the homogenate. Although fractionation on a 5-step sucrose gradient reduced the yield of protein in the synaptosomal fraction to 40 micrograms/animal, this fraction (the 0.35 M/0.75 M interface) was more enriched in plasma membrane than P3 and was less contaminated by lysosomes and free mitochondria. By electron microscopy, the 0.35 M/0.75 M interface contained up to 50% synaptosomes. Synaptosomal fractions contained cAMP-, Ca2+/calmodulin-, and Ca2+/phospholipid-dependent protein kinase activities and were enriched in a Mr 40,000 pertussis toxin substrate, Gi/o. In the accompanying paper, we show that these synaptosomes retain the ability to release transmitters.     

Localization of immunoreactive alpha-bag-cell peptide in the central nervous system of Aplysia

The bag cells of the marine mollusc Aplysia are well-characterized neuroendocrine cells that initiate egg laying, but the natural stimulus triggering bag-cell activity has not been determined. As a first step toward identifying central neurons that might provide synaptic or neurohormonal input onto the bag-cell network, antibodies specific for alpha-bag-cell peptide (alpha-BCP) were generated. This peptide belongs to a small family of structurally related peptides that can elicit bag-cell activity in vitro. Antibody specificity was established by immunodot assay and preabsorption studies: immunocytochemical labeling was abolished in each ganglion when the antibodies were preincubated with either alpha-BCP-thyroglobulin conjugate or alpha-BCP-(1-8) but was not affected by preincubation with thyroglobulin or thyroglobulin-thyroglobulin conjugate. The antibodies specifically labeled the bag cells in the abdominal ganglion and ectopic bag cells in both the abdominal and right pleural ganglia. The ectopic bag cells were similar to conventional bag cells in size and morphology, but varied in number and location among preparations. In the cerebral ganglion, the antibodies labeled a bilaterally symmetrical pair of cell clusters, containing approximately ten cells each, on the dorsal surface of the ganglion. The cerebral cells were smaller than bag cells, were constant in location, and sent their processes into the neuropil rather than the connective tissue sheath. Immunoreactive processes were observed in the neuropils of the cerebral, pleural, and pedal ganglia and among the axons of the cerebropedal, cerebropleural, and pleurovisceral connectives. No immunoreactive cell bodies were observed in the buccal or pedal ganglia. Identical patterns of labeling were observed in Aplysia californica, A. brasiliana, and A. dactylomela. The distribution of immunoreactive cell bodies within the circumesophageal ganglia of all three species thus parallels the distribution of receptive sites for the in vitro induction of bag-cell activity by atrial gland peptide B, a peptide structurally related to alpha-BCP. These observations suggest that the immunoreactive cells identified in these studies, or a subset of them, may be involved in the physiological induction of bag-cell activity. Since low doses of alpha-BCP have additional inhibitory actions on the bag cells, however, it is possible that the identified cells could play a more complex role in the regulation of bag-cell activity.