Imaging of caffeine-inducible release of intracellular calcium in cultured embryonic mouse telencephalic neurons

To gain a better understanding of Ca²⁺-induced Ca²⁺ release in central neurons, we have studied the increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by application of caffeine to cells cultured from embryonic mouse telencephalon (hippocampus or cortex). The magnitudes and distributions of changes in [Ca²⁺]_i in neuron somata were measured by quantitative video microscopy. We observed that application of caffeine to pyramidally shaped neurons typically initiated an increase in [Ca²⁺]_i in the cytoplasmic region between the nucleus and the base of a major dendrite. [Ca²⁺] in this region increased over a period of 3 to 6 s and was followed by, with a slight delay, a surge of Ca²⁺ that moved across the soma and into or over the nucleus. Similar Ca²⁺ that moved across the soma and into or over the nucleus. Similar Ca²⁺ responses to caffeine were observed in Ca²⁺-containing and nominally Ca²⁺-free external solutions, suggesting that caffeine was inducing Ca²⁺ release from intracellular stores. Ca²⁺ responses to caffeine were potentiated by inducing a tonic Ca²⁺ influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors activated by 0.3 μM glutamate and multiple responses to caffeine could be elicited by using this Ca²⁺ influx to refill the intracellular stores. Ryanodine inhibition of caffeine-induced Ca²⁺ release was use- and concentration-dependent; the median effective concentration EC₅₀ for ryanodine declined from 22 μM for the first application of caffeine to 20 nM for the fourth. We conclude, based on these responses to caffeine, that ryanodine-sensitive mechanisms of intracellular Ca²⁺ release are active in hippocampal and cortical neurons and may be involved in generation of directed Ca²⁺ waves that engulf the nucleus. © 1995 John Wiley & Sons, Inc.     

Involvement of the nitric oxide-cyclic GMP pathway in the desensitization of bradykinin responses of cultured rat sensory neurons.

Bradykinin (BK) excites a subset of dorsal root ganglion neurons by inducing an inward cation current (IBK) that strongly desensitizes and is accompanied by elevations in cGMP. We have examined the links between cGMP metabolism and IBK. The BK dose dependencies of IBK activation, desensitization, and cGMP production are comparable. Stimulation (with sodium nitroprusside [NP] or 8-bromo-cGMP [8Br-cGMP]) or inhibition (with methylene blue, hemoglobin, and nitric oxide synthase [NOS] inhibitors) of cGMP levels did not mimic or diminish IBK. However, desensitization was affected by the following agents: first, desensitization was enhanced by NP and reduced by NOS inhibitors. Second, the effects of NOS inhibitors could be overcome by 8Br-cGMP or L-arginine. Third, 8Br-cGMP modification of desensitization required receptor occupancy. We conclude that the NO-cGMP pathway affects a component of IBK desensitization at the receptor or G protein level.     

Apparent desensitization of NMDA responses in Xenopus oocytes involves calcium-dependent chloride current.

N-Methyl-D-aspartate (NMDA) receptors were expressed and studied in Xenopus oocytes injected with rat brain RNA. NMDA application elicits a rapid inward current that decays in several seconds to a relatively stable level. This decay is reportedly due to desensitization. However, we found the early transient component could be evoked more than once during a single application of NMDA, suggesting that the receptor did not actually desensitize. Removal of external Ca2+, replacement of Ca2+ with Ba2+, or intracellular injection of EGTA abolished the transient component. Furthermore, a variety of Cl- channel blockers nearly eliminated the transient component and inhibited the plateau current as well. We propose that a significant portion of the NMDA current recorded in oocytes is carried by a transient inward Cl- current triggered by Ca2+ influx through the NMDA receptor/channel.     

Activation of NMDA receptor partly involved in beta-bungarotoxin-induced neurotoxicity in cultured primary neurons

In this study, we demonstrated that a snake presynaptic toxin, beta-bungarotoxin (beta-BuTX), was capable of binding to NMDA receptors of the cultured primary neurons (cerebellar granule neurons, CGNs). We labeled beta-BuTX with fluorescent FITC (FITC-beta-BuTX) and showed that the binding of FITC-beta-BuTX was inhibited by unlabeled beta-BuTX and MK801 (an NMDA receptor antagonist). Meanwhile, the binding of [3H]-MK801 was also reduced by unlabeled MK801 and beta-BuTX. In addition, beta-BuTX produced a very potent neurotoxic effect on mature CGNs with the EC(50) of 3ng/ml (equivalent to 144pM), but was less effective in immature CGNs. We explored the signaling pathway of neuronal death and found that it was apparently due to the excessive production of reactive oxygen species (ROS) induced by beta-BuTX. MK801 and antioxidants (Vitamin C, N-acetylcysteine (NAC), melatonin, epigallocatechin gallate (EGCG), superoxide dismutase (SOD) and catalase) attenuated not only ROS production but also beta-BuTX-neurotoxicity. The downstream signaling of ROS was identified as the activation of caspase-3. Caspase inhibitor (z-DEVD-fmk) and antioxidants depressed both caspase-3 activation and neurotoxicity. Based on these findings and our previous reports, we conclude that the binding and activation of NMDA receptors by beta-BuTX was crucial step to produce the potent neurotoxic effect. The binding of NMDA receptors resulted in excessive Ca(2+) influx, followed by ROS production and activation of caspase-3. This snake toxin is considered not only to be a useful tool for exploring the death-signaling pathway of neurotoxicity, but also provides a model for searching neuroprotective agents.     

Dipyridamole is neuroprotective for cultured rat embryonic cortical neurons

The effects of a clinically useful cardiovascular agent, dipyridamole, were examined in a rodent tissue culture model of neuroprotection. Dipyridamole effectively protected rat embryonic day 18 (E18) cortical neurons from either 48 h trophic deprivation or 48 h exposure to the glutathione synthesis inhibitor, L-buthionine (R,S) sulfoximine. The neuron sparing actions of dipyridamole were time- and concentration-dependent and mimicked the actions of exogenously applied glutathione. These results demonstrate that dipyridamole protects primary neuronal cultures against either trophic or chemically mediated insults, and suggest that dipyridamole has a potent antioxidant ability that compensates for glutathione depletion in neuronal cultures.     

Single-channel currents from diethylpyrocarbonate-modified NMDA receptors in cultured rat brain cortical neurons

The role of histidine residues in the function of N-methyl-D-aspartate (NMDA)-activated channels was tested with the histidine-modifying reagent diethylpyrocarbonate (DEP) applied to cells and membrane patches from rat brain cortical neurons in culture. Channels in excised outside-out patches that were treated with 3 mM DEP for 15-30 s (pH 6.5) showed an average 3.4-fold potentiation in steady state open probability when exposed to NMDA and glycine. Analysis of the underlying alterations in channel gating revealed no changes in the numbers of kinetic states: distributions of open intervals were fitted with three exponential components, and four components described the shut intervals, in both control and DEP-modified channels. However, the distribution of shut intervals was obviously different after DEP treatment, consistent with the single-channel current record. After modification, the proportion of long shut states was decreased while the time constants were largely unaffected. Burst kinetics reflected these effects with an increase in the average number of openings/burst from 1.5 (control) to 2.2 (DEP), and a decrease in the average interburst interval from 54.1 to 38.2 ms. These effects were most likely due to histidine modification because other reagents (n- acetylimidazole and 2,4,6-trinitrobenzene 1-sulfonic acid) that are specific for residues other than histidine failed to reproduce the effects of DEP, whereas hydroxylamine could restore channel open probability to control levels. In contrast to these effects on channel gating, DEP had no effect on average single-channel conductance or reversal potential under bi-ionic (Na+:Cs+) conditions. Inhibition by zinc was also unaffected by DEP. We propose a channel gating model in which transitions between single- and multi-opening burst modes give rise to the channel activity observed under steady state conditions. When adjusted to account for the effects of DEP, this model suggests that one or more extracellular histidine residues involved in channel gating are associated with a single kinetic state.     

Synaptic activity-dependent developmental regulation of NMDA receptor subunit expression in cultured neocortical neurons

The biophysical properties of NMDA receptors are thought to be critical determinants involved in the regulation of long-term synaptic plasticity during neocortical development. NMDA receptor channel properties are strongly dependent on the subunit composition of heteromeric NMDA receptors. During neocortical development in vivo, the expression of the NMDA receptor 2A (NR2A) subunit is up-regulated at the mRNA and protein level correlating with changes in the kinetic and pharmacological properties of functional NMDA receptors. To investigate the developmental regulation of NMDA receptor subunit expression, we studied NR2 mRNA expression in cultured neocortical neurons. With increasing time in culture, they showed a similar up-regulation of NR2A mRNA expression as described in vivo. As demonstrated by chronic blockade of postsynaptic glutamate receptors in vitro, the regulation of NR2A mRNA was strongly dependent on synaptic activity. In contrast, NR2B mRNA expression was not influenced by activity blockade. Moreover, as shown pharmacologically, the regulation of NR2A mRNA expression was mediated by postsynaptic Ca(2+) influx through both NMDA receptors and L-type Ca(2+) channels. It is interesting that even relatively weak expression of NR2A mRNA was correlated with clearly reduced sensitivity of NMDA receptor-mediated whole-cell currents against the NR2B subunit-specific antagonist ifenprodil. Developmental changes in the expression of NR1 mRNA splice variants were also strongly dependent on synaptic activity and thus might, in addition to regulation of NR2 subunit expression, contribute to developmental changes in the properties of functional NMDA receptors. In summary, our results demonstrate that synaptic activity is a key factor in the regulation of NMDA receptor subunit expression during neocortical development.     

Catecholamine and indoleamine levels in chick cultured neurons and embryonic brain

Catecholamine and indoleamine levels were determined in cultured neurons from chick embryos and in the homologous embryonic cerebral hemispheres in order to study their neurotransmission systems. The seeding of a large number of cells resulted in a pure neuronal culture made of clusters interconnected by processes. Norepinephrine, which was absent from the starting material of the culture, appeared on the 2nd day and then decreased. A small amount of epinephrine was present on the 2nd day and decreased thereafter. Dopamine was not detected. In the cerebral hemispheres of chick embryos, dopamine appreared on the 10th day in ovo and increased steadily up to the 18th day. Epinephrine was also present in the cerebral hemispheres. Its level increased up to the 14th day and then decreased. Indoleamines were measured in the same material. The level of serotonin was markedly higher than that of catecholamines and it increased during cultivation. Tryptophan was already present in the starting material and its amount increased during cultivation. The level of 5-hydroxyindoleacetic acid changed like that of serotonin. In the embryonic cerebral hemispheres, the concentration of serotonin was highest on the 12th day after incubation and then decreased. Tryptophan level decreased steadily all during the embryogenesis. These results were discussed on the ground of differences in the synthesized neurotransmitters.     

Effects of concanavalin A on desensitization kinetics of GABA responses inAchatina fulica neurons

The effects of the lectin concanavalin A (Con A), on the kinetics of desensitization of the responses of voltage clampedAchatina fulica LP5 neuron to microperfused acetylcholine (ACh) and GABA were compared. Both ACh and GABA elicited increases in chloride conductance which decayed biphasically during prolonged applications of these agonists; an initial rapid decay was followed by a later slow decay. Con A (5 g/ml) accelerated both the fast and the slow decays of responses to ACh. Con A (5 g/ml) also accelerated the fast decay of responses to GABA, but the slow decay was unaffected, even by 20 g/ml or more of the lectin. It is suggested that, at least in the case of GABA receptor, the fast and slow decays involve distinct desensitization kinetics. The effects of Con A on the desensitization of the ACh and GABA responses were reversed byd-mannose, a competitive and specific inhibitor of Con A binding to membrane sugar residues. These results provide further evidence that receptor desensitization can be influenced by perturbing the sugar moieties associated with the subunits comprising these signalling macromolecules. The carbohydrate residues may play an important role in regulating desensitization of transmitter receptors.Abbreviations ACh acetylcholine - Con A concanavalin A     

Store Ca2+ depletion enhances NMDA responses in cultured human astrocytes.

NMDA produced whole-cell membrane currents in cultured human astrocytes. The currents were not inhibited by the selective NMDA receptor antagonist, APV, while they were partially inhibited by the broad G-protein inhibitor, GDPbetaS. NMDA-induced currents were enhanced by either the microsomal Ca2+/ATPase inhibitors, thapsigargin and cyclopiazonic acid, or the ATP-uncoupler, dinitrophenol (DNP). In the Ca2+ assay, NMDA increased intracellular calcium concentration. The increase was inhibited by 26% in Ca2+-free extracellular solution, and it was not inhibited by APV. The results of the present study suggest that NMDA responses in human astrocytes are regulated by store Ca2+ depletion-associated signal.     

Efficient differentiation of mouse embryonic stem cells into motor neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb9. Using this robust protocol, we achieved 51 ± 0.8% of differentiation efficiency (n = 3; p < 0.01, Student's t-test). Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.     

Guanine nucleotides inhibit NMDA and kainate-induced neurotoxicity in cultured rat hippocampal and neocortical neurons

Previous evidence suggests that guanine nucleotides can directly inhibit N-methyl-d-aspartate (NMDA) and AMPA/kainate receptors and antagonize a variety of cellular functions elicited by these glutamate receptor agonists. We investigated the possibility that the guanine nucleotides GTP, GDP, and GMP exert a neuroprotective effect on cultured rat hippocampal or neocortical neurons exposed to the excitotoxicants NMDA (30 microM) or kainate (300 microM). On co-application with NMDA all three nucleotides revealed a comparable rescue effect from 100 microM nucleotide concentrations onwards, with a higher inhibitory potential in hippocampal than in neocortical cultures. Similarly, kainate-induced neurotoxicity was inhibited by all three nucleotides but the inhibitory potential was lower than after application of NMDA. Guanosine had no effect on either culture system. GTP and GDP where hydrolyzed by hippocampal and cortical cultures with GMP accumulating in the medium, suggesting that hydrolysis of GTP had no effect on the effective nucleotide concentration. Our results show that GTP, GDP, and GMP inhibit NMDA- and kainate-mediated neurotoxicity in cultured hippocampal and neocortical neurons. They suggest that guanine nucleotides may be candidates for broadly antagonizing glutamate receptor-mediated neurotoxicity.     

Transgenic enrichment of mouse embryonic stem cell-derived progenitor motor neurons

Embryonic stem cells (ESCs) hold great potential for replacing neurons following injury or disease. The therapeutic and diagnostic potential of ESCs may be hindered by heterogeneity in ESC-derived populations. Drug selection has been used to purify ESC-derived cardiomyocytes and endothelial cells but has not been applied to specific neural lineages. In this study we investigated positive selection of progenitor motor neurons (pMNs) through transgenic expression of the puromycin resistance enzyme, puromycin N-acetyl-transferase (PAC), under the Olig2 promoter. The protein-coding region in one allele of Olig2 was replaced with PAC to generate the P-Olig2 cell line. This cell line provided specific puromycin resistance in cells that express Olig2, while Olig2⁻ cells were killed by puromycin. Positive selection significantly enriched populations of Olig2⁺ pMNs. Committed motoneurons (MNs) expressing Hb9, a common progeny of pMNs, were also enriched by the end of the selection period. Selected cells remained viable and differentiated into mature cholinergic MNs and oligodendrocyte precursor cells. Drug resistance may provide a scalable and inexpensive method for enriching desired neural cell types for use in research applications.     

Effect of protein phosphorylation on neurite outgrowth in cultured embryonic Xenopus spinal neurons

Intracellular signaling pathways involved in neurite outgrowth have been extensively studied in a variety of cell systems. While most of these studies utilized continuous neuronal-like cell lines, fewer studies have been conducted in primary neuronal culture. One primary culture system that has recently been used to dissect the signaling pathways involved in axon guidance consists of spinal neurons derived from embryonic Xenopus laevis. In this study, we used Xenopus to study neurite outgrowth by treating neuronal cultures with pharmacological agents that activate or inhibit various protein kinases or that inhibit protein phosphatases. We found that agents which affected signaling via cAMP-dependent protein kinase, calmodulin, cyclin-dependent kinase 5, or protein phosphatases had effects on Xenopus neurite outgrowth that were similar to those reported in other primary neurons or in neuronal-like cell lines. However, agents which affected protein kinase C signaling had effects on Xenopus neurite outgrowth that were distinct from those reported in neuronal-like cell lines. Although continuous cell lines have several advantages for the dissection of signaling pathways involved in neurodevelopment, these observations underscore the importance of also using primary neurons to examine these pathways.     

The regulation of M1 muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons

To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin. The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade. Inhibition of endogenous G protein-coupled receptor kinase 2 by transfection with the non-G(q/11)alpha-binding, catalytically-inactive (D110A,K220R)G protein-coupled receptor kinase 2 mutant, decreased the extent of M1 mACh receptor desensitization under all conditions. Pharmacological inhibition of protein kinase C (PKC) activity, or chronic phorbol ester-induced PKC down-regulation had no effect on agonist-mediated receptor desensitization in quiescent or spontaneously synaptically active neurons, but significantly decreased the extent of receptor desensitization in picrotoxin-treated neurons. MCh stimulated the translocation of diacylglycerol- sensitive eGFP-PKCepsilon, but not Ca2+/diacylglycerol-sensitive eGFP-PKCbetaII in both the absence, and presence of tetrodotoxin. Under these conditions, MCh-stimulated eGFP-myristoylated, alanine-rich C kinase substrate translocation was dependent on PKC activity, but not Ca2+/calmodulin. In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons.     

Quantitation of 3D ureteric branching morphogenesis in cultured embryonic mouse kidney

The growth and branching of the epithelial ureteric tree is critical for development of the permanent kidney (metanephros). Current methods of analysis of ureteric branching are mostly qualitative. We have developed a method for measuring the length of individual branches, and thereby the total length of the ureteric tree in 3 dimensions (3D). The method involves confocal microscopy of whole-mount immunostained metanephroi and computer-based image segmentation, skeletonisation and measurement. The algorithm performs semi-automatic segmentation of a set of confocal images and skeletonisation of the resulting binary object. Length measurements and number of branch points are automatically obtained. The final representation can be reconstructed providing a fully rotating 3D perspective of the skeletonised tree. After 36 h culture of E12 mouse metanephroi, the total length of the ureteric tree was 6103 +/- 291 microm (mean +/- SD), a four-fold increase compared with metanephroi cultured for just 6 h (1522 +/- 149 microm). Ureteric duct length increased at a rate of 153 microm/h over the first 30 h period and was maximal between 18 and 24 h at 325 microm/h. The distribution of branch lengths at the six time points studied was similar, suggesting tight control of ureteric lengthening and branching. This method will be of use in analysing ureteric growth in kidneys cultured in the presence of specific molecules suspected of regulating ureteric growth. The method can also be used to analyse in vivo kidneys and to quantify branching morphogenesis in other developing organs.     

Posttranslational modifications of tubulin in cultured mouse brain neurons and astroglia

Posttranslational modifications of tubulin were analyzed in mouse brain neurons and glia developing in culture. Purified tubulin was resolved by isoelectric focusing. After 3 weeks of culture, neurons were shown to express a high degree of tubulin heterogeneity (8 alpha and 10 beta isoforms), similar to that found in the brain at the same developmental stage. Astroglial tubulin exhibits a less complex pattern consisting of 4 alpha and 4 beta isoforms. After incubation of neuronal and glial cells with 3H-acetate in the presence of cycloheximide, a major posttranslational label was found associated with alpha-tubulin and a minor one with beta-tubulin. The acetate-labeled isotubulins of neurons were resolved by isoelectric focusing into as many as 6 alpha and 7 beta isoforms, while those of astroglia were resolved into only 2 alpha and 2 beta isoforms. The same alpha isoforms were also shown to react with a monoclonal antibody recognizing selectively the acetylated form(s) of alpha-tubulin. Whether acetate-labeling of alpha-tubulin in these cells corresponds to the acetylation of Lys40, as reported for Chlamydomonas reinhardtii, is discussed according to very recent data obtained by protein sequence analysis. Tubulin phosphorylation was analyzed by incubation of cell cultures with 32PO4. No phosphorylation of alpha-tubulin isoforms was detected. A single beta-tubulin isoform (beta'2), expressed only in neurons, was found to be phosphorylated. This isoform is similar to that previously identified in differentiated mouse neuroblastoma cells.     

Increased expression of NR2A subunit does not alter NMDA-evoked responses in cultured fetal trisomy 16 mouse hippocampal neurons

The trisomy 16 (Ts16) mouse is an animal model for human trisomy 21 (Down's syndrome). The gene encoding the NR2A subunit of the NMDA receptor has been localized to mouse chromosome 16. In the present study, western blot analysis revealed a 2.5-fold increase of NR2A expression in cultured Ts16 embryonic hippocampal neurons. However, this increase did not affect the properties of NMDA-evoked currents in response to various modulators. The sensitivity of NMDA receptors to transient applications of NMDA, spermine, and Zn(2+) was investigated in murine Ts16 and control diploid cultured embryonic hippocampal neurons. Peak and steady-state currents evoked by NMDA were potentiated by spermine at concentrations < 1 mM, and inhibited by Zn(2+) in a dose-dependent and voltage-independent manner. No marked difference was observed between Ts16 and control diploid neurons for any of these modulators with regard to IC(50) and EC(50) values or voltage dependency. Additionally, inhibition by the NR2B selective inhibitor, ifenprodil, was similar. These results demonstrate that NMDA-evoked currents are not altered in cultured embryonic Ts16 neurons and suggest that Ts16 neurons contain similar functional properties of NMDA receptors as diploid control neurons despite an increased level of NR2A expression.     

The development of hormonal responses of cultured embryonic chick limb mesenchymal cells

Cells obtained from stage 24 chick limb buds were cultured and assayed for their ability to respond to exogenously supplied parathyroid hormone (PTH) as monitored by analysis of cellular cyclic AMP (cAMP). After 3–4 days in culture, these cells developed a striking responsiveness to the hormone; 20 -to 50-fold elevations in cAMP were routinely observed upon exposure to 10^?8, M hormone for 2 min. This response was greatest in cells initially plated at low densities (1 × 10⁶ cells/35-mm dish) and was inversely correlated to the amount of cartilage which developed in such cultures. Cells obtained from limbs of stages 23–26 embryos developed a similar responsiveness to PTH after 3–4 days in culture, but cells obtained from limbs of stage 22 embryos showed no such responsiveness even after 6 days in culture. A response to calcitonin also was noted in cultures of stage 24 limb mesenchymal cells after 4–5 days in culture, but this was of much smaller magnitude than the PTH response. Of 12 other hormones tested, only β agonists elicited any cAMP response in the cultered stage 24 limb mesenchymal cells. Although cells initially plated at a high density and grown for 8 days in culture show no response to PTH, the presence of PTH-responsive cells in such cultures could be demonstrated by sequential digestion with collagenase and replating the extracellular matrix-free cells released by this treatment. Such replated cells then exhibited a responsiveness to PTH. Thus, the responsiveness of cultured limb mesenchymal cells depends on the developmental stage of the starting limb mesenchyme, the phenotypes which develop, and physical factors such as accessibility to exogenously supplied hormone.