GAD and GABA in an enriched population of cultured GABAergic neurons from rat cerebral cortex

To study various aspects of GABAergic metabolism in an easily accessible system, dissociated cells from postnatal rat cerebral cortex were cultured in a serum-based medium and characterized morphologically and biochemically. The majority (70–90%) of the neurons were GABAergic as determined by three double-labeling procedures. The specific activity of glutamine synthetase in the cultures was 4–5% of the levels in rat astrocyte cultures and intact rat brain, indicating that glia were a minor component. The developmental increase of GABA levels preceded the increase of GAD activity in both immunocytochemical and biochemical experiments. GABA turnover rates also increased with culture age and were 20–30% of GAD activity. Four anti-GAD antibodies, which recognize GAD subunits with differing molecular masses to varying degrees, were used to stain cultured neurons and make immunoblots. Immunoblots showed that the neurons contained two major subunits of GAD which differed in mass by 2 kDa. All four antibodies immunostained both neuronal perikarya and neurites but one antibody, which on the immunoblots predominantly labeled the GAD protein with the lower molecular weight, showed a somewhat more pronounced punctate staining, possibly indicating a principal localization to neurites.     

Cell differentiation in the hippocampus of the fetal rat in cell culture

Cell cultures from hippocampus of 16 and 17 days old embryonal rats were cultivated up to 4 weeks. After 24 hours in vitro on 18.4 percent of cells and after 5 days in vitro on 70 percent of cells processes could be recognized. These are neuroblasts. The cells reaggregated. Nerve fibers after 4 weeks in vitro are 200 to 300 mum long. Small and big neurons with 12 mum to 26 mum diameters of perikarya, bi- and multipolar neurons after 4 weeks in vitro were observed. In cultures and meningothel-monolayer developed. Maintenance and differentiation of cultures are possible only by sowing in at least 60,000 cells/ml medium. The advantage of cell culture opposite to organ culture exists in experiments with immediate selective influence.     

GABA neurones in retinas of different species and their postnatal development in situ and in culture in the rabbit retina

Summary The localisation of GABA immunoreactive neurones in retinas of a variety of animals was examined. Immunoreactivity was associated with specific populations of amacrine neurones in all species examined, viz. rat, rabbit, goldfish, frog, pigeon and guinea-pig. All species, with the exception of the frog, possessed immunoreactive perikarya in their retinal ganglion cell layers. These perikarya are probably displaced amacrine cells because GABA immunoreactivity was absent from the optic nerves and destruction of the rat optic nerve did not result in degeneration of these cells. GABA immunoreactivity was also associated with the outer plexiform layers of all the retinas studied; these processes are derived from GABA-positive horizontal cells in rat, rabbit, frog, pigeon and goldfish retinas, from bipolar-like cells in the frog, and probably from interplexiform cells in the guinea-pig retina.The development of GABA-positive neurones in the rabbit retina was also analysed. Immunoreactivity was clearly associated with subpopulations of amacrine and horizontal cells on the second postnatal day. The immunoreactivity at this stage is strong, and fairly well developed processes are apparent. The intensity of the immunoreactivity increases with development in the case of the amacrine cells. The immunoreactive neurones appear fully developed at about the 8th postnatal day, although the immunoreactivity in the inner plexiform layer becomes more dispersed as development proceeds. The immunoreactive horizontal cells become less apparent as development proceeds, but they can still be seen in the adult retina.The GABA immunoreactive cells in rabbit retinas can be maintained in culture. Cultures of retinal cells derived from 2-day-old animals can be maintained for up to 20 days and show the presence of GABA-positive cells at all stages. In one-day-old cultures the GABA immunoreactive cells lacked processes but within three days had clearly defined processes. After maintenance for 10 days a meshwork of GABA-positive fibres could also be seen in the cultures.     

Localization of insulin-like immunoreactivity in the neurons from primary cultures of rat brain

Brains from 1-day-old rats were dissociated with trypsin and the cells were maintained in culture for 3-4 weeks. These primary cultures contained insulin-like immunoreactivity in the limited populations of neurons. Typically, the fluorescent staining pattern observed in the soma was homogeneous and varicosity-like structures were observed on the neurites of the majority of insulin-like immunoreactive neurons. Serum deprivation of brain cell cultures did not reduce immunoreactivity, whereas cycloheximide caused approx. 80% decrease in the number of insulin-like immunoreactive neurons. Incubation of these cultures with [3H]valine resulted in the incorporation of radioactivity into immunoprecipitable insulin. These results suggest that insulin-like immunoreactivity present in the Central Nervous System (CNS) neurons may be synthesized by brain cell cultures.     

Uptake of GABA by neuronal and nonneuronal cells in dispersed cell cultures of postnatal rat cerebellum.

A study was made of the time course and kinetics of [3H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [3H]GABA, or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent Kmvalues for GABA neurons for high and low affinity uptake were 0.33 X 10(-6) M and 41.8 X 10(-4) M, respectively. For nonneuronal cells, the apparent Km for high affinity uptake was 0.29 X 10(-6) M. The apparent Vmax values for GABA neurons for high and low affinity uptake were 28.7 X 10(-6) mol/g DNA/min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent Vmax for high affinity uptake was 0.06 X 10(-6) mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 X 10(-9) M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [3H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by noneuronal cells was only slightly decreased. Most (75-85%) of the [3H]GABA (4.4 X 10(-6) M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent.     

Tissue effects on the expression of serotonin, tyrosine hydroxylase and GABA in cultures of neurogenic cells from the neuraxis and branchial arches

The phenotypically diverse neurones of the enteric nervous system are developmentally derived from precursors that migrate to the bowel from the vagal and sacral regions of the neuraxis. In order to gain insight into the generation of enteric neuronal diversity, we examined the expression of serotonin (5-HT), tyrosine hydroxylase and GABA in vitro. In the mature avian intestine, intrinsic neurones contain 5-HT or GABA but not tyrosine hydroxylase. These markers were demonstrated immunocytochemically, singly or simultaneously. All three phenotypic markers developed in cultures of cranial, vagal or truncal neural crest when the cultures were grown in enriched medium, containing horse serum and chick embryo extract; however, 5-HT and GABA, but not tyrosine hydroxylase-immunoreactive cells, also developed in cultures that were grown in partially defined medium. Tyrosine hydroxylase immunoreactivity was seen when partially defined medium was supplemented with nerve growth factor (NGF). Cultures of branchial arches (III and IV) contained cells that displayed tyrosine hydroxylase immunoreactivity, but not that of 5-HT- or GABA-; however, 5-HT immunoreactivity was seen when branchial arches were cocultured with aneuronal hindgut (from 4-day chick embryos). Cultures of cells from chick gut dissociated at 7 days contained tyrosine hydroxylase as well as 5-HT and GABA immunoreactivities; however, no cultures of bowel dissociated at 8 days or later expressed tyrosine hydroxylase immunoreactivity. When neuraxial cells were cocultured with branchial arches or heart instead of gut, no 5-HT-immunoreactive cells were seen; nevertheless, the further addition of explants of gut to the heart/crest cocultures did permit the expression of 5-HT immunoreactivity. These results are consistent with the hypotheses that precursors with the potential to give rise to cells that express 5-HT, GABA and tyrosine hydroxylase are found at several levels of the neuraxis; however, the ability to express these phenotypes may be suppressed either while the crest cells are migrating (for example, 5-HT and GABA expression by crest cells passing through the branchial arches) or in their final destination (for example, tyrosine hydroxylase in the gut). This suppression may be transient and reversed by the microenvironment of the target organs.     

Release of acetylcholinesterase by cultured spinal cord cells

The release of acetylcholinesterase from neurons was studied using cultured chick-embryo spinal-cord cells. Cells dissociated from 12-day-old chick-embryo spinal cords were grown in culture for 10-12 days. Numerous well differentiated spinal neurons were found after 7-10 days in culture. Acetylcholinesterase activity per dish increased by 60-fold from days 2-12. Acetylcholinesterase was released into the surrounding media by the cells when they were incubated either in the standard culture medium or the serum-free medium. Acetylcholinesterase release was significantly reduced when protein synthesis and microtubules were disrupted by cycloheximide and colchicine, respectively. Histochemical localization of acetylcholinesterase indicated that the synthesis and relase of acetylcholinesterase are attributable to neurons. Cultured chick-embryo brain and neuroblastoma cells also released acetylcholinesterase into the media. These results are discussed with regard to possible physiological roles for acetylcholinesterase secretion from neurons.     

Differential expression of 240 kDa ConA-binding glycoprotein in vitro and in vivo detected by immunochemical methods

The expression of the 240 ConA-binding glycoprotein (240 kDa), a marker of synaptic junctions isolated from the rat cerebellum, was studied by immunocytochemical techniques in forebrain and cerebellum from rat and chicken, and in chick dorsal root ganglia. Parallel studies were carried out either on tissue sections or in dissociated cell cultures. In all cases non neuronal cells were not immunostained. The tissue sections of cerebellum from rat and chick exhibited 240 kDa glycoprotein immunoreactivity, especially in the molecular layer, while the forebrain sections from rat and chick did not show any significant immunostaining. In contrast, in dissociated forebrain cell cultures, all neuronal cells expressed 240 kDa glycoprotein immunoreactivity, while glial cells remained totally unlabelled. In tissue sections of dorsal root ganglion (DRG), sensory neurons expressed the 240 kDa only after the embryonic day (E 10). A large number of small neurons in the dorsomedial part of DRG were immunostained with 240 kDa glycoprotein antiserum, whereas only a small number of neurons in the ventrolateral part of the ganglia displayed 240 kDa immunoreactivity. In dissociated DRG cells cultures (mixed or neuron-enriched DRG cell cultures) all the neuronal perikarya but not their processes were stained. These studies indicate that 240 kDa glycoprotein expression is completely modified in cultures of neurons of CNS or PNS since the antigen becomes synthetized in high amount by all cells independent of synapse formation. This demonstrates that the expression of 240 kDa is controlled by the cell environment.     

Striatal interneurons in dissociated cell culture

In addition to the well-characterized direct and indirect projection neurons there are four major interneuron types in the striatum. Three contain GABA and either parvalbumin, calretinin or NOS/NPY/somatostatin. The fourth is cholinergic. It might be assumed that dissociated cell cultures of striatum (typically from embryonic day E18.5 in rat and E14.5 for mouse) contain each of these neuronal types. However, in dissociated rat striatal (caudate/putamen, CPu) cultures arguably the most important interneuron, the giant aspiny cholinergic neuron, is not present. When dissociated striatal neurons from E14.5 Sprague–Dawley rats were mixed with those from E18.5 rats, combined cultures from these two gestational periods yielded surviving cholinergic interneurons and representative populations of the other interneuron types at 5 weeks in vitro. Neurons from E12.5 CD-1 mice were combined with CPu neurons from E14.5 mice and the characteristics of striatal interneurons after 5 weeks in vitro were determined. All four major classes of interneurons were identified in these cultures as well as rare tyrosine hydroxylase positive interneurons. However, E14.5 mouse CPu cultures contained relatively few cholinergic interneurons rather than the nearly total absence seen in the rat. A later dissection day (E16.5) was required to obtain mouse CPu cultures totally lacking the cholinergic interneuron. We show that these cultures generated from two gestational age cells have much more nearly normal proportions of interneurons than the more common organotypic cultures of striatum. Interneurons are generated from both ages of embryos except for the cholinergic interneurons that originate from the medial ganglionic eminence of younger embryos. Study of these cultures should more accurately reflect neuronal processing as it occurs in the striatum in vivo. Furthermore, these results reveal a procedure for parallel culture of striatum and cholinergic depleted striatum that can be used to examine the function of the cholinergic interneuron in striatal networks.     

Demonstration of extensive GABA synthesis in the small population of GAD positive neurons in cerebellar cultures by the use of pharmacological tools

Cultures of dissociated cerebella from 7-day-old mice were maintained in vitro for 1-13 days. GABA biosynthesis and degradation were studied during development in culture and pharmacological agents were used to identify the enzymes involved. The amount of GABA increased, whereas that of glutamate was unchanged during the first 5 days and both decreased thereafter. The presence of aminooxyacetic acid (AOAA, 10 microM) which inhibits transaminases and other pyridoxal phosphate dependent enzymes including GABA-transaminase (GABA-T), in the culture medium caused an increase in the intracellular amount of GABA and a decrease in glutamate. The GABA content was also increased following exposure to the specific GABA-T inhibitor gamma-vinyl GABA. From day 6 in culture (day 4 when cultured in the presence of AOAA) GABA levels in the medium were increased compared to that in medium from 1-day-old cultures. Synthesis of GABA during the first 3 days was demonstrated by the finding that incubation with either [1-(13)C]glucose or [U-(13)C]glutamine led to formation of labeled GABA. Synthesis of GABA after 1 week in culture, when the enzymatic machinery is considered to be at a more differentiated level, was shown by labeling from [U-(13)C]glutamine added on day 7. Altogether the findings show continuous GABA synthesis and degradation throughout the culture period in the cerebellar neurons. At 10 microM AOAA, GABA synthesis from [U-(13)C]glutamine was not affected, indicating that transaminases are not involved in GABA synthesis and thus excluding the putrescine pathway. At a concentration of 5 mM AOAA GABA labeling was, however, abolished, showing that glutamate decarboxylase, which is inhibited at this level of AOAA, is responsible for GABA synthesis in the cerebellar cultures. In conclusion, the present study shows that GABA synthesis is taking place via GAD in a subpopulation of the cerebellar neurons, throughout the culture period.     

GABA immunoreactivity in the human cerebellar cortex: a light and electron microscopical study

The distribution of gamma-aminobutyric acid (GABA) in surgical samples of human cerebellar cortex was studied by light and electron microscope immunocytochemistry using a polyclonal antibody generated in rabbit against GABA coupled to bovine serum albumin with glutaraldehyde. Observations by light microscopy revealed immunostained neuronal bodies and processes as well as axon terminals in all layers of the cerebellar cortex. Perikarya of stellate, basket and Golgi neurons showed evident GABA immunoreactivity. In contrast, perikarya of Purkinje neurons appeared to be negative or weakly positive. Immunoreactive tracts of longitudinally- or obliquely-sectioned neuronal processes and punctate elements, corresponding to axon terminals or cross-sectioned neuronal processes, showed a layer-specific pattern of distribution and were seen on the surface of neuronal bodies, in the neuropil and at microvessel walls. Electron microscope observations mainly focussed on the analysis of GABA-labelled axon terminals and of their relationships with neurons and microvessels. GABA-labelled terminals contained gold particles associated with pleomorphic vesicles and mitochondria and established symmetric synapses with neuronal bodies and dendrites in all cortex layers. GABA-labelled terminals associated with capillaries were seen to contact the perivascular glial processes, basal lamina and endothelial cells and to establish synapses with subendothelial unlabelled axons.     

Astroglial cells provide a template for the positioning of developing cerebellar neurons in vitro

Indirect immunocytochemical staining with antisera raised against purified glial filament protein and a neurofilament polypeptide was used to study cell interactions between astrocytes and neurons dissociated from embryonic and early postnatal cerebellum. Staining with antibodies raised against purified glial filament protein revealed that greater than 99% of all processes present in cerebellar cultures during the 1st wk in vitro were glial in origin. After 1 wk in culture, unstained processes that were presumably neuronal were observed. Stained astroglial processes formed a dense network that served as a template for cerebellar neurons, identified by indirect immunocytochemical localization of tetanus toxin. More than 90% of neurons from postnatal days 1 or 7 were positioned within one cell diameter of a glial process. In contrast, less than 40% of the neurons dissociated from early embryonic cerebellum were located adjacent to a glial process. Staining with antibodies raised against purified glial filament protein also revealed differences in astroglial morphology that were under developmental regulation. Astroglial cells from embryonic cerebellum were fewer in number and had thick, unbranched processes. Those from postnatal day 1 were more slender, branched, and stellate. Those from postnatal day 7 were highly branched and stellate. Some veil-like astroglial processes were also observed in cells from postnatal animals. These morphological changes were also observed when cells from embryonic day 13 were maintained for a week in vitro. No specific staining of embryonic or postnatal cerebellum cells was observed with antibodies raised against purified neurofilament polypeptides.     

Development of a high-affinity GABA uptake system in embryonic amphibian spinal neurons

High-affinity uptake systems for amino acid neurotransmitter precursors have been highly correlated with the use of the particular amino acid or its derivative as a transmitter. We have found interneurons in the Xenopus embryo spinal cord which accumulate GABA by a high-affinity uptake system. They originate near the end of gastrulation and their ability to accumulate GABA first appears at the early tail bud stage. By position and appearance they are comparable to some of the embryonic interneurons described by A. Roberts and J. D. W. Clarke (1982, Phil. Trans. R. Soc. London Ser. B 296, 195-212). GABA-accumulating neurons also develop in dissociated cell cultures made from the presumptive spinal cord of neural plate stage Xenopus embryos. GABA accumulation in cultured neurons, as in cells in vivo, occurs via a high-affinity uptake system; GABA-accumulating cells have the same time of origin as the cells in vivo, and the ability to accumulate GABA in the population of cultured neurons appears at a time equivalent to that observed in intact sibling embryos. Thus it seems likely that the population of GABA-accumulating neurons developing in cell culture corresponds to the GABA-accumulating interneurons in vivo. The development of these neurons in dissociated cell cultures permits perturbation experiments that would be difficult to perform in vivo. We have examined the development of high-affinity GABA uptake in conditions that permit no electrical impulse activity in the cultures. The onset and extent of development of GABA accumulation in the neuronal population are normal under these conditions.     

Colocalization of GABA, enkephalin and neuropeptide Y in the tectum of the green frog Rana esculenta.

The colocalization of GABA, enkephalin and neuropeptide Y immunoreactivity in neurons in the pretectal area and in the mesencephalic tectum of the green frog (Rana esculenta) was studied. Several Met-enkephalin immunoreactive perikarya were found in layer 6 of the tectum and every third of these neurons showed GABA-ir as well. Colocalization of GABA and NPY could also be shown in half of the neuropeptide Y immunopositive cells in the 6th layer of the tectum, but only a few cells were double stained in layers 9 and 4. In the pretectal area no colocalization of the investigated peptides and GABA was found.     

Tubulin immunohistochemistry

Summary We report here the results observed when tubulin fluorescence immunohistochemistry is performed upon dissociated cultures of nervous tissue, principally of chick embryo spinal cord. When fixation includes nonpolar solvents or detergents, a uniform fluorescence is seen in neuron perikarya (with the exception of their nuclei), and the processes to which they give rise. Fixation with formaldehyde or glutaraldehyde alone, however, results in a discontinuous staining of neurites, and a less regular staining of their perikarya. The former pattern of response can be elicited if aldehyde fixation is followed by exposure to non-polar solvents. Such results are obtained both in thinly spread regions of the cultures, where neurons and their processes can easily be seen, and in the cell aggregates that also characterise them. Possible interpretations of these results are discussed.     

Toxicity of 1-Methyl-4-Phenylpyridinium for Rat Dopaminergic Neurons in Culture: Selectivity and Irreversibility

Cultures of dissociated embryonic rat mesencephalic cells were exposed to 10 microM 1-methyl-4-phenylpyridinium (MPP+), a concentration shown earlier to result in loss of greater than 85% of tyrosine hydroxylase (TH)-positive neurons without affecting the total number of cells observed by phase-contrast microscopy. To characterize better the selectivity of the toxic action of MPP+, other parameters were measured reflecting survival and function of dopaminergic or nondopaminergic neurons. Exposure of cultures to 10 microM MPP+ for 48 h reduced TH activity to 11% of control values without reducing protein levels. [3H]Dopamine uptake was reduced to less than 4% of control values, whereas the uptake of gamma-[3H]aminobutyric acid ([3H]GABA) was not affected in these cultures. This same treatment failed to reduce the number of cholinergic cells visualized in septal cultures and did not affect either choline acetyltransferase activity or high-affinity choline uptake. To assess for possible recovery of dopaminergic neurons, cultures were exposed to 10, 1.0, or 0.1 microM MPP+ for 48 h and then kept for up to 6 days in MPP(+)-free medium. After exposure to 10 microM MPP+, the number of TH-positive neurons, their neurite density, TH activity, and [3H]dopamine uptake remained at constant, reduced levels throughout the period of observation after termination of exposure, whereas GABA uptake remained normal. Treatment with lower concentrations of MPP+, i.e., 1.0 and 0.1 microM, induced less pronounced dopaminergic toxic effects. However, no recovery was seen after posttreatment incubation in toxin-free medium. These findings provide evidence that MPP+ treatment results in highly selective and irreversible toxicity for cultured dopaminergic neurons.     

Characteristics of choline acetyltransferase and cholinesterases in two types of cultured cells from embryonic chick brain

Cells that were mechanically dissociated from the brains of 7-day-old chick embryos were grown in culture for 7–8 days. Two major cell populations were observed: (1) cells that aggregated and sent out processes, (2) flat cells that proliferated rapidly and formed a confluent layer by day 4 of culture. Many of the cells of the first type had the morphological, histochemical and biochemical attributes of neurons. They possessed choline acetyltransferase (ChAc) and acetylcholinesterase (AChEs) activities. The flat cells possessed neither of the activities, but did have butyrylcholinesterase (BuChEs) activity and a choline independent acetylase activity (CIA) that may be carnitine acetyltransferase.The activities of ChAc and AChEs in the cultured neurons increased approximately 9-fold and 5-fold, respectively, over an 8-day period. The patterns of change of these enzymes were not unlike those seen in vivo in intact developing chick brain.The addition of thyroxine (10^?6M) to these cultures increased the activities of neuronal AChEs and flat cell BuChEs by 30–70%.     

Two forms of cerebellar glial cells interact differently with neurons in vitro

Specific interactions between neurons and glia dissociated from early postnatal mouse cerebellar tissue were studied in vitro by indirect immunocytochemical staining with antisera raised against purified glial filament protein, galactocerebroside, and the NILE glycoprotein. Two forms of cells were stained with antisera raised against purified glial filament protein. The first, characterized by a cell body 9 microns diam and processes 130-150 microns long, usually had two to three neurons associated with them and resembled Bergmann glia. The second had a slightly larger cell body with markedly shorter arms among which were nestled several dozen neuronal cells, and resembled astrocytes of the granular layer. Staining with monoclonal antisera raised against purified galactocerebroside revealed the presence of immature oligodendroglia in the cultures. These glial cells constituted approximately 2% of the total cell population in the cultures and, in contrast to astroglia, did not form specific contacts with neurons. Staining with two neuronal markers, antisera raised against purified NILE glycoprotein and tetanus toxin, revealed that most cells associated with presumed astroglia were small neurons (5-8 microns). After 1-2 d in culture, some stained neurons had very fine, short processes. Nearly all of the processes greater than 10-20 micron long were glial in origin. Electron microscopy also demonstrated the presence of two forms of astroglia in the cultures, each with a different organizing influence on cerebellar neurons. Most neurons associated with astroglia were granule neurons, although a few larger neurons sometimes associated with them. Time-lapse video microscopy revealed extensive cell migration (approximately 10 microns/h) along the arms of Bergmann-like astroglia. In contrast, cells did not migrate along the arms of astrocyte-like astroglia, but remained stationary at or near branch points. Growth cone activity, pulsating movements of cell perikarya, and ruffling of the membranes of glial and neuronal processes were also seen.     

Long-term effects of dexamethasone and nerve growth factor on adrenal medullary cells cultured from young adult rats

Summary Normal postnatal rat chromaffin cells and rat pheochromocytoma cells are known to show extensive Nerve Growth Factor (NGF)-induced process outgrowth in culture, and this outgrowth from the postnatal chromaffin cells is abolished by the corticosteroid dexamethasone. To determine whether adult rat chromaffin cells respond to NGF and dexamethasone, dissociated adrenal medullary cells from 3-month-old rats were cultured for 30 days in the presence or absence of these agents. Such cultures contained typical chromaffin cells, chromaffin cells with processes, and neurons. Fewer than 2 % of normal adult chromaffin cells formed processes under any of the conditions studied, and statistically significant changes in this proportion were not detectable in the presence of NGF or dexamethasone. Adrenal medullary neurons, however, were observed only in the presence of NGF, in cultures with or without dexamethasone, and thus appear to be previously unreported NGF targets which require NGF for survival or process outgrowth. Dexamethasone markedly increased total catecholamine content, total content of epinephrine, and tyrosine hydroxylase activity in cultures with or without NGF. In contrast, postnatal rat chromaffin and rat pheochromocytoma cells which have been studied in culture do not produce epinephrine under any of these conditions. It is concluded that rat adrenal chromaffin cells undergo age-related changes in both structural and functional plasticity. The in vitro characteristics of rat pheochromocytoma cells more closely resemble those of postnatal than of adult rat chromaffin cells, but may not entirely reflect the properties of the majority of chromaffin cells in either age group.