Differentiation and survival of rat olfactory epithelial neurons in dissociated cell culture

Olfactory epithelial neurons in mammals are unique in that they continue to differentiate from precursor cells in the adult. These neurons extend long axons into the olfactory bulb. Previous attempts to grow these cells in dissociated cell cultures at low density, have not been entirely satisfactory. We report that when plated at very low density, rat olfactory epithelial neurons will differentiate morphologically and biochemically when cultured on astrocytes, but not on non-cellular substrata, such as polylysine, laminin or fibronectin. We demonstrate with antibodies, that these olfactory epithelial neurons require N-CAM, N-cadherin and L1 for neurite extension. Furthermore, synthetic cadherin-peptides containing the tripeptide HAV which is found in the first extracellular domain of N-cadherin, as well as the amino acids flanking this region, appear to be important in cadherin-mediated neurite growth on astrocytes. Astrocytes also appear to enhance the survival and differentiation of olfactory epithelial neurons from embryonic day 15 and 4-5 week post-natal rats, but this effect is not sustained beyond 5 days in cultures of postnatal epithelium.     

In vivo electrophysiological maturation of neurons derived from a multipotent precursor (embryonal carcinoma) cell line

The multipotent embryonal carcinoma (EC) P19 cell line differentiates into neurons, glia and smooth muscle following exposure to retinoic acid (RA). RA-induced differentiation is irreversible and the neurons that develop are abundant, post-mitotic, and survive for prolonged periods in culture or when grafted into the CNS of adult rats. Striatal slices containing grafted P19 cells were studied with intracellular recording and labelling techniques to examine the development of electrophysiological and morphological properties of P19-derived neurons over a period of 6 to 120 days after grafting into ibotenic acid lesioned striatum. Cells from 1-week-old grafts had a range of immature electrophysiological characteristics including unstable resting membrane potentials (RMP's) and very high membrane input resistances (Rin's). Many were not able to produce action potentials (AP's). In contrast, the majority of cells recorded from 2- and 3-week-old grafts had stable RMP's, moderate Rin's, and were able to produce regenerative AP's. In grafts over 4 weeks of age, the majority of P19-derived neurons had mature neuronal electrophysiological characteristics including RMP's of −60 mV, Rin's of 100–300 MΩ, and overshooting AP's. Morphologically, P19 derived neurons increase in soma size from 12–15 μ in diameter in 7–14-day-old grafts, to 25–35 μ in diameter in grafts 50–120 days old. Developing neurons exhibited a variety of morphotypes with increasingly complex processes and lengths of process extension. Our results demonstrate a developmental progression of the electrophysiology of P19-derived neurons, culminating in mature characteristics closely resembling those of adult rodent hippocampal or cortical pyramidal neurons. The ability to easily alter these cells genetically provides a powerful model for addressing issues specific to neuronal development.     

Differentiation and maturation of zebrafish dorsal root and sympathetic ganglion neurons

The trunk neural crest of vertebrate embryos gives rise to dorsal root ganglion (DRG) sensory neurons and autonomic sympathetic neurons, among other derivatives. We have examined the development of DRG and sympathetic neurons during development in the zebrafish. We found that sensory neurons differentiate rapidly and that their overt neuronal differentiation significantly precedes that of sympathetic neurons in the trunk. Sympathetic neurons in different regions differentiate at different times. The most rostral population, which we call the cervical ganglion, differentiates several days before trunk sympathetic neurons. After undergoing overt neuronal differentiation, sympathetic neurons subsequently express the adrenergic differentiation markers dopamine beta-hydroxylase and tyrosine hydroxylase. A second population of adrenergic nonneuronal cells initially localized with cervical sympathetic neurons appears to represent adrenal chromaffin cells. In more mature fish, these cells were present in clusters within the kidneys. Individual DRG and sympathetic ganglia initially contain few neurons. However, the number of neurons in DRG and sympathetic ganglia increases continuously at least up to 4 weeks of age. Analysis of phosphohistone H3 expression and bromodeoxyuridine incorporation studies suggests that the increases in DRG and sympathetic ganglion neuronal cell number are due wholly or in part to the division of neuronal cells within the ganglia.     

Hypothalamic neurons in dissociated cell culture

We have developed a mechanical-enzymatic technique for the isolation and culture of dissociated hypothalamic neurons from both newborn and mature rats. The disaggregated cells were suspended in defined growth medium and plated onto collagen-coated coverslips. The cells derived from newborn animals attached themselves to the substrate more quickly and the cultures ultimately possessed significantly more neurons. Cultures were maintained for periods up to 5 weeks.We suggest that these preparations represent a useful model system for the investigation of a variety of functional studies of hypothalamic tissue. In particular, the possible effects hormonal steroids on monoamine metabolism may be approached using fluorescence microscopy.     

Characterization of West Nile viral replication and maturation in peripheral neurons in culture

The North American West Nile virus (WNV), New York 1999 strain, appears to be highly neurotropic, and its neuroinvasiveness is an important aspect of human disease. The authors have developed an in vitro model to study WNV replication and protein processing in neurons. They compared WNV infection of the dorsal root ganglion (DRG) neurons (sensory neurons) and PC-12 cells (sympathetic neurons) to WNV infection of the mosquito cell line, C6/36, and Vero cells. WNV infection of both neuronal cell types and C6/36 cells was not cytopathic up to 30 days post infection, and continual viral shedding was observed during this period. However, WNV infection of Vero cells was lytic. Interestingly, WNV infection of neurons was not efficient, requiring a high multiplicity of infection of > or = 10. Indirect immunofluorescence assays using normal and confocal microscopy with flavivirus-reactive antibodies and WNV-infected neurons demonstrated viral antigen mostly associated with the plasma membrane and in the neurite processes. Treatment of WNV-infected C6/36, PC-12, or DRG cells with brefeldin A (BFA; a trans-Golgi inhibitor) or nocadazole (a beta-tubulin inhibitor) had little effect on viral maturation and secretion. Treatment of WNV-infected Vero cells with BFA resulted in a 1000-fold decrease in viral titer, but nocodazole had no effect. Our studies suggest that even though PC-12 and DRG neurons are mammalian cells, viral protein processing and maturation in these cells more closely resembles replication in C6/36 insect cells than in mammalian Vero cells.     

Expression of necdin, an embryonal carcinoma-derived nuclear protein, in developing mouse brain.

Necdin is a polypeptide sequence encoded by neural differentiation-specific mRNA derived from embryonal carcinoma cells. We have examined the expression of necdin and its mRNA in cultured cells and mouse brain by Northern blot analysis and immunohistochemistry. Among various established cell lines including neuroblastoma and glioma cells, only differentiated embryonal carcinoma cells (P19 and F9) expressed necdin mRNA. Necdin immunoreactivity was localized in the nuclei of differentiated neurons derived from P19 cells. Necdin mRNA was detected throughout brain regions of adult mouse; the relative abundances in the hypothalamus and midbrain were the highest, whereas those in the olfactory bulb and cerebellum were the lowest. In developing mouse brain, necdin mRNA was expressed during early periods of neuronal generation and differentiation, and the peak levels were attained during postnatal days 1-4. Necdin immunoreactivity was not detected in the neural stem cells on embryonic day 10, but was concentrated in the nuclei of brain cells, mostly neurons, at advanced stages of differentiation. The majority of differentiated neurons in the brain had necdin-immunoreactive nuclei on postnatal day 33. Thus, necdin may represent a valuable molecular marker for differentiated neurons both in vitro and in vivo.     

Rat hippocampal neurons in dispersed cell culture

An in vitro system has been developed for the study of isolated hippocampal neurons from 18- or 19-day rat featuses. Following trypsinization the cells are plated out at low density on polylysine-treated coverslips in an enriched medium. The isolated neurons rapidly attach to the substrate and initiate process extension. Little reaggregation occurs and the number of non-neuronal cells present is minimal. Unless co-cultured with tissue explants the neurons survive for only a few days; in the presence of hippocampal explants the initial growth of the isolated cells is improved and their survival in culture is extended to about two weeks. Some of the cells in such cultures develop a characteristic branching pattern closely resembling that of maturing hippocampal pyramidal cells in vivo. There is a clear relationship between the stage of the cells' development and their growth in culture. Cells which had completed DNA synthesis about 48 h before dissociation, and which were in the process of migration to the cortical plate, survived best in our cultures. Early post-mitotic cells which were still within the ventricular zone and cells which had already reached the cortical plate grew poorly. This system should permit the study not only of process formation by these cells, but also of their capacity to form specific synapses in vitro and of the biochemical constituents of their surfaces.     

Morphological maturation and survival of chicken and rat embryonic neurons on different culture substrata

Neuronal cells from chicken and rat embryonic cerebral hemispheres were plated at a low cell concentration and cultured either on collagen or on a supporting continuous glial layer for periods of up to 21 days. The glial layer was either homologous or heterologous with regard to the animal species; the survival and maturation of the neuronal cells in these different conditions were investigated by light and electron microscopy.Neuronal cells cultured on collagen formed aggregates similar to those formed by neuronal cells plated at high cell density as described in a previous paper; a few aggregated neurons formed processes after 24 h and, only after 48 h of culture, more fibres had developed; the majority of the cells progressively degenerated between days 7 and 21 of culture. In contrast to this, neuronal cells cultured on a supporting glial layer, whether homologous or heterologous, progressively differentiated: neuronal perikarya remained well separated from each other and many processes were already formed after 24 h; later on, networks of fibres developed. At the electron microscopic level, microtubules and neurofilaments were present at a high density in the cells and fibres; immature synapses could be found, but infrequently. Differentiated cells were represented mostly by neurons; oligodendroglial cells were absent, and myelinated fibres could not be detected.The highest positive effect on the maturation and survival of neuronal cells was observed in the presence of a layer of glial cells from the same species. These results emphasize the essential role of glial cells for the neuronal maturation in the absence of contact between neuroblasts.     

Anticonvulsant drug actions on neurons in cell culture

Summary Two actions of clinically used antiepileptic drugs have been studied using mouse neurons in primary dissociated cell culture. The antiepileptic drugs phenytoin, carbamazepine and valproic acid were demonstrated to limit sustained high frequency repetitive firing of action potentials at free serum concentratons that are achieved in patients being treated for epilepsy. Furthermore, an active metabolite of carbamazepine also limited sustained high frequency repetitive firing while inactive metabolites of phenytoin and carbamazepine did not limit sustained high frequency repetitive firing. Phenobarbital and clinically used benzodiazepines limited sustained high frequency repetitive firing of action potentials, but only at concentrations achieved during the treatment of generalized tonic-clonic status epilepticus. Ethosuximide did not limit sustained high frequency repetitive firing even at concentrations four times those achieved in the serum of patients treated for generalized absence seizures. Phenobarbital and clinically used benzodiazepines enhanced postsynaptic GABA responses at concentrations achieved free in the serum during treatment of generalized tonic-clonic or generalized absence seizures. However, phenytoin, carbamazepine, valproic acid and ethosuximide did not modify postsynaptic GABA responses at therapeutic free serum concentrations. These results suggest that the ability of antiepileptic drugs to block generalized tonicclonic seizures and generalized tonic-clonic status epilepticus may be related to their ability to block high frequency repetitive firing of neurons. The mechanism underlying blockade of myoclonic seizures may be related to the ability of antiepileptic drugs to enhance GABAergic synaptic transmission. The mechanism underlying management of generalized absence seizures remains unclear.     

Survival of chick embryo sympathetic neurons in cell culture

Changes in neuronal numbers during the development of the chick embryo paravertebral sympathetic nervous system have been examined using cell culture techniques. Early sympathetic ganglia contain predominantly cells having neuronal phenotypes and these increase in number until embryonic day 9. Subsequently there is a large decrease in the number of neurons and an increase in the population of non-neuronal cells. This in vivo pattern is maintained when the neurons are grown in vitro, where Nerve Growth Factor more readily prevents the death of neurons cultured from 12-day or older embryos than those from earlier stages of development.     

Physiology and pharmacology of olfactory bulb neurons in dissociated cell culture

Cells from olfactory bulbs of embryonic rats were grown in dissociated cell culture for up to 5 weeks. Both neurons and non-neuronal cells grew in these cultures, with a variety of neuronal populations appearing. A population of 20–25% of the neurons were GABAergic by the criterion of [³H]GABA uptake. Electrophysiologic measurements were made of the baseline activity of the cultured neurons. Cells showed a mean resting potential of60.1 ± 1.2mV and a mean input resistance of87.6 ± 9.5MΩ. All cells were sensitive to microperfusion of GABA with half-maximal effect occurring at about 20 μM. Glutamate was universally excitatory but with variations in degree. Carnosine (β-Ala-l-His), tested over the concentration range of 10 nM to 100 μM, had no effect on input resistance, resting potential, action potential shape, on-going synaptic activity or the responsiveness to either GABA or glutamate. These results are further evidence against a role for carnosine as the excitatory transmitter of the primary olfactory afferents.     

Glutamate-immunoreactive sensory neurons in quail neural crest cell culture

This study addresses the question of whether embryonic sensory neuroblasts of the quail are glutamate (Glu)-immunoreactive as a neurotransmitter in vivo and when grown in neural crest cell culture. Using a monoclonal antibody against carbodiimide-treated glutamate, we found a subset of neural crest-derived cells in culture. They were large, round and contained long pseudouni- or bipolar processes. There was Glu-immunoreactivity in the cytoplasm of the soma and more intense fluorescence within processes. Double-labeling experiments using a monoclonal antibody to Glu in conjunction with polyclonal antibodies to either neurofilament (NF), substance P (SP), or tyrosine hydroxylase (TH) revealed that: (1) the Glu-immunoreactive cells are a subpopulation of NF-positive sensory neuroblasts; (2) intensely Glu-immunoreactive cells were usually SP-negative. However, cells less intensely staining with both antibodies were observed as well; (3) the Glu-immunoreactive cells were TH-negative, whereas TH-positive adrenergic neuroblasts were Glu-negative; and (4) frozen sections of the spinal ganglia of 9- and 15-day-old quail embryos contained Glu-immunoreactive cells. These data combined indicate that the Glu-immunoreactive cells are a subpopulation of the sensory neuroblasts that were previously identified in neural crest cell cultures with antibodies against NF proteins and the stage-specific embryonic antigen 1 (SSEA-1). The results should prove valuable in future studies on the mechanisms that govern neural crest cell differentiation into autonomic and sensory neurons.     

Differentiation of NTERA-2 clonal human embryonal carcinoma cells into neurons involves the induction of all three neurofilament proteins

Monoclonal antibodies were used in indirect immunofluorescence and immunoblot studies to examine the expression of four different classes of intermediate filaments, namely, neurofilaments, glial filaments, cytokeratin, and vimentin, in NTERA-2 cl.D1 (NT2/D1) pluripotent human embryonal carcinoma (EC) cells, and in the neurons derived from these cells by differentiation induced with retinoic acid. In the EC cell cultures, grown in the absence of retinoic acid, cytokeratin was the predominant intermediate filament detected by immunofluorescence; only a few cells expressed vimentin, and none expressed glial filament protein or any of the three neurofilament proteins (NF195, NF170, and NF70). Immunoblot analyses of cytoskeletal extracts of these cells supported these data. Two days after exposure to retinoic acid, all three neurofilament subunits were detected in a few cells with a non-neuronal morphology and, by double indirect immunofluorescence, were observed to colocalize with cytokeratin. The number of neurofilament-positive cells increased with time after initial exposure to retinoic acid, and although 95% of these cells contained cytokeratin initially, less than 5% of the neurofilament-positive cells retained cytokeratin 2 weeks later. By this time, many of the cells expressing all three neurofilaments but no cytokeratin exhibited a neuronal morphology. Vimentin was evident in a large number of cells in the cultures, but it was not detected in the neurofilament-positive cells. Also, many of the neurofilament-negative cells continued to express cytokeratin. No cells expressing glial filament proteins were found. Immunoblot analysis of the differentiated cultures also revealed all three neurofilament subunits, and vimentin and cytokeratin, but no glial filament protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocompetent cell markers in human fetal astrocytes and neurons in culture.

During the past few years, evidence has accumulated that interaction with peripheral immune cells as well as immunoregulatory functions in the central nervous system (CNS) can be played by several types of brain resident cells. Since very little information is available in man, however, we investigated the presence of markers so far considered typical of immunocompetent cells in in vitro cultures of human fetal brain. Immunocytochemistry at the light, scanning, and transmission electron microscopic levels revealed positivity for a very restricted range of macrophage antigens in astrocytes, which, however, were incapable of phagocytosis. In particular, expression of the major histocompatibility complex-class II antigen HLA-DR was observed in the cytoplasm and on the cell surface of GFA-P+ astrocytes and increased with time in culture and cell passages. Among the T-lymphocyte markers tested, Thy.1 and CD4 were positive. Both neurons and astrocytes carried Thy.1 from early cell passages. Noteworthy was the presence of CD4, which serves as the receptor for AIDS virus, in neurons from the first 2 weeks, whereas astrocytes became positive after only 4-6 weeks. Even if most staining was in the cytoplasm, some was exposed on cell surface. Astrocytes were found positive for the B-lymphocyte marker CD21, the cellular receptor for Epstein-Barr virus, whereas CD24 was detected in both neurons and astrocytes. Both antigens are related to B-cell proliferation. Results are in favour of the hypothesis of human brain cells being actively involved in CNS immunological events.     

Further observations on hippocampal neurons in dispersed cell culture.

The growth of the processes of dissociated hippocampal neurons from 18- to 20-day-old rat fetuses has been studied in vitro using a method that permits cells to be maintained at relatively low plating densities for periods up to three weeks (Banker and Cowan, '77). Most of the cells are spherical or ovoid when they first attach to the substratum (either polylysine or collagen) but within 24 to 48 hours they begin to put out processes, and by the end of the first week in culture a significant proportion (approximately 45%) come to resemble normal pyramidal cells with a more-or-less triangular shaped soma, a single dominant dendrite-like process emerging from the apex of the soma, and several "basal dendrites" arising from the opposite pole of the cell. Comparisons of the lengths of these dendrite-like processes with those of hippocampal cells in the brains of animals sacrificed on the fourth post-natal day and impregnated by a variant of the Golgi-Cox method, indicate that in some cases the rate of process formation in vitro approximates that in vivo and that the general form of the neurons is remarkably like that of immature pyramidal cells. After a week in culture a second type of process can be recognized. These tend to be finer than those we have identified as dendrites; they are relatively uniform in diameter, frequently give off branches at right angles, and in the electron microscope can be seen to form synapses upon the larger processes and cell somata. These axon-like processes differ from the axons of normal pyramidal cells in two important respects: (i) most commonly they arise from one of the dendritic processes, including, on occasion, the putative "apical" dendrites, and only rarely from the base of the perikaryon; (ii) there may be two or more such processes from a single cell. The thicker, tapering processes can be shown (after incubation in 3H-uridine) to contain large amounts of RNA; newly-synthesized RNA does not extend into the finer, axon-like processes.     

Quinolinate is a weak excitant of cortical neurons in cell culture

Defined concentrations of quinolinate (QUIN) were administered to murine cortical neurons in culture impaled for intracellular recording. In physiological recording medium containing 1 mM Mg, concentrations of QUIN up to 2 mM had minimal effect on membrane potential and input resistance; only at higher concentrations did QUIN produce consistent depolarizations, which were accompanied by apparent increases in membrane resistance. In the absence of Mg, responses to QUIN were larger and were accompanied by decreases in membrane resistance, but QUIN was still a weak neuroexcitant, exhibiting an ED50 of greater than 1 mM. Phthalic, dipicolinic and nicotinic acids, structural analogues of QUIN, were even less potent neuroexcitants. The relationship between QUIN depolarization amplitude and membrane potential was linear in the absence of Mg, but in the presence of 1 mM Mg showed a non-linearity consistent with the voltage-dependent Mg block of N-methyl-D-aspartate (NMDA) receptor-mediated responses described by others. QUIN responses had a marked dependence on the presence of extracellular Na, and an extrapolated reversal potential of +12 mV, consistent with the large involvement of an Na influx. The responses were attenuated by the selective NMDA receptor antagonists, DL-2-amino-5-phosphonovalerate and ketamine, as well as by the broad spectrum antagonist kynurenate, but not by L-glutamate diethylester or gamma-D-glutamylaminomethyl sulfonate, compounds reported to block quisqualate or kainate receptors. The present study is consistent with the suggestion of other workers that QUIN neuroexcitation is mediated in large part by an Na influx through cation permeable NMDA-activated channels, but provides new quantitative data suggesting that the potency of QUIN as a cortical neuroexcitant is low. This low potency may argue against a role for QUIN as a traditional fast excitatory neurotransmitter.     

Neuronal maturation in mammalian cell culture is dependent on spontaneous electrical activity

Fetal mouse spinal cord (SC) and dorsal root ganglion (DRG) neurons undergo a process of maturation in cell culture lasting a month or more. We have investigated the role of electrical activity in this maturational process with the use of tetrodotoxin (TTX), the specific blocker of the voltage-sensitive sodium channel responsible for action potential generation. This agent completely eliminates the spikes and related synaptic activity which occur abundantly in untreated cultures. Such blockade of electrical activity in the cultures, when begun early (day 1 or day 8 in vitro), results in a 85-95% reduction in the number of large SC neurons, without affecting DRG neuron numbers. TTX treatment initiated when cultures are mature (day 70) has no significant effect on either DRG or SC neurons. Intermediate effects are obtained when treatment is initiated at day 35 in vitro. The activity of the nerve-specific enzyme choline acetyltransferase, is significantly decreased by early TTX treatment, while DNA and protein content of the cultures (primarily contributed by glial and fibroblastic cells) is not affected.     

Developmental changes in calcium conductances contribute to the physiological maturation of cerebellar Purkinje neurons in culture.

The relationship between calcium conductances and developmental changes in the active and passive membrane properties of cerebellar Purkinje neurons from rats was studied in a culture model system by using current-clamp and voltage-clamp techniques. These cultures, at 6-21 d of age, represented the main period of morphological and physiological development of the Purkinje neuron. In the current-clamp studies, input resistance decreased and the current-voltage curve became more S-shaped as the neurons matured in culture. Spike-generating properties also changed. Immature Purkinje neurons without dendritic structure produced repetitive, fast TTX-sensitive simple spikes when stimulated electrically. The simple spike frequency increased with maturation. In older neurons (greater than or equal to 12 d in vitro) with well-developed dendritic structure, a burst event, the complex spike, preceded the repetitive simple spike firing. Magnesium (10 mM) and cadmium (50-100 microM), calcium channel blockers, antagonized the repetitive simple spike firing in both young and old neurons. The complex spike of the older neurons was also antagonized by magnesium (10 mM) but was resistant to cadmium (50-100 microM), suggesting that a pharmacologically distinct calcium conductance mediated this spike event. Whole-cell voltage-clamp recordings showed that the older Purkinje neurons expressed two calcium currents, a low-threshold rapidly inactivating calcium current resistant to cadmium (50-100 microM) and a high-threshold slowly inactivating calcium current antagonized by cadmium (50-100 microM). In young Purkinje neurons without dendritic structure (6-9 d in vitro), only the high-threshold calcium current was evident. The amplitude of this current increased approximately 50% during development. These results indicate that the developmental expression of calcium conductances plays a prominent role in the physiological maturation of the cultured Purkinje neurons, which closely simulate the physiologic cells they model. The high-threshold calcium conductance is expressed early in development and contributes to repetitive simple spike firing of both the young and old neurons. The low-threshold calcium conductance appears later in development, coincident with dendritic expression, and plays a major role in the generation of the complex spike.     

Electrophysiological chracteristics of chick embryo sympathetic neurons in dissociated cell culture

Intracellular recordings were obtained from 400 neurons in low-density dissociated cell cultures of 11-day chick embryo paravertebral sympathetic ganglia. Both the passive and active electrical membrane properties of the cultured neurons resembled those previously found with in vivo preparations of sympathetic neurons. Active responses (spikes) were elicited by depolarizing stimuli in about 60% of the cells tested. Spontaneous electrical activity was not observed. The generation of spikes was blocked in the presence of 3 × 10^?7M tetrodotoxin. Active responses could be evoked from the cultured neurons within 3 days after plating. Neither the proportion of electrically excitable cells nor their membrane properties appeared to change as a function of the period of culture (3 days to 3 weeks). Depolarizing responses were elicited from the cultured neurons by the iontophoretic application of acetylcholine.These findings indicate that the cultured sympathetic neurons display a number of differentiated electrical properties. This behavior is expressed in the absence of normal excitatory synaptic inputs or postsynaptic target tissues and with little interaction with non-neuronal cells.     

Effects of norepinephrine on rat neocortical neurons in dissociated cell culture

Intracellular recordings were made from neurons in dissociated cell culture of neocortex during application of norepinephrine (NE) or other adrenergic agonists. In the population of neurons generally studied, greater than 18 μm in diameter, adrenergic agonists from 1 nM to 50 μM produced no change in membrane potential or input resistance (120 cells). Adrenergic agonists increased synaptic activity impinging on the impaled cell in 25/120 neurons (21%). In neurons in cocultures of locus coeruleus and cerebral cortex, again the same synaptic response to perfusion with NE was noted in 13/93 neurons (14%). In addition, direct effects of NE were noted on 6/93 neurons recorded from in cocultures, all close to the explant. In these cells, NE hyperpolarized the membrane in association with a small decrease in input resistance (11%). These reponsive cells may have originated within the explant. A paradigm was used for testing the possibility of a responsive element in the cultures distinct from the impaled soma. ‘Hot spots’ were found using concentrations of isoproterenol as low as 10 nM.