Bruce K. Schrier

Neurons from fetal rat brain in a new cell culture system: A multidisciplinary analysis

A new culture system for cells from the mammalian brain was developed by a modification of a previously established technique. This modification involved the use of fluorodeoxyuridine and adult horse serum. The cultures contained large, easily visualized neurons both isolated from other neurons and in networks of varying complexity. These cells were large enough to permit reliable intracellular electrophysiologic recording and were often sufficiently dispersed to allow examination of membrane responses to iontophoretically applied neurotransmitter candidates. Many responses characteristic of central neurons in situ were seen, including evoked and spontaneous action potentials, complex patterns of inhibitory and excitatory post-synaptic potentials, and neurotransmitter-induced membrane responses. These preparations were examined by phase contrast microscopy, by light microscopy after silver impregnation and by Nomarski interference optics. Total choline acetyltransferase (CAT) activity was little changed and specific activity was increased in the new culture system as compared with the earilier system. Conditions which gave the highest specific activity of CAT also provided the best cultures from the standpoint of electrophysiologic and morphologic analysis. This new approach will allow, in culture, detailed multidisciplinary analyses of individual neurons and small networks of neurons from the mammalian brain.

Choline Acetyltransferase Activity Is Increased in Combined Cultures of Spinal Cord and Muscle Cells from Mice

The activity of choline acetyltransferase was more than tenfold greater in combined cultures of spinal cord and muscle cells than in cultures of spinal cord cells alone. This increase was associated with the formation of functional neuromuscular junctions in culture. Counts of silver-stained cells and determinations of other enzyme activities indicated that the increased choline acetyltransferase activity was not due to nonspecific neuronal survival but reflected greater activity in the surviving neurons. Hence, muscle had a marked, highly specific trophic effect on the cholinergic neurons that innervated it.

SEQUENCE DIVERSITY STUDIES OF RAT BRAIN RNA: EFFECTS OF ENVIRONMENTAL COMPLEXITY ON RAT BRAIN RNA DIVERSITY

The sequence complexities of rat brain RNAs were measured by RNA‐driven hybridization reactions with nonrepetitive rat DNA. The total sequence complexity of rat brain HnRNA was estimated to be 6.61 x 108 nucleotides while rat brain poly(A)‐mRNA sequence complexity was 1.32 x 108 nucleotides. Up to 33.7% of the total transcribable nonrepetitive DNA was expressed in the nuclear RNA. The nuclear RNAs reacted with complex kinetics over at least 4.5 decades of equivalent Rot (product of RNA concentration and time), with an apparent division into three major RNA abundance classes. The abundances of average nuclear RNA species in these classes ranged from 2.9 x 109 copies per brain (18 copies per cell) to 2.4 x 105 copies per brain (1.5 x 10−3 copies per cell). Poly(A)‐mRNA diversity was sufficient to code for 8.8 x 104 polypeptides of 50,000 daltons. There were also three distinguishable abundance classes of poly(A)‐mRNA with frequencies which ranged from 8.9 x 108 copies per brain (5.5 copies per cell) to 3.2 x 105 copies per brain (2 x 10−3 copies per cell). Evidence for compartmentalization of expressed RNA sequences supports the concept that the extensive morphological and physiological specialization evident in brain parallels extensive transcriptional specialization at the cellular level.

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.

Source and target cell specificities of a conditioned medium factor that increases choline acetyltransferase activity in cultured spinal cord cells

Abstract A macromolecular factor(s) in muscle conditioned medium (CM), when applied to spinal cord (SC) cells in culture, causes large increases in the activity of choline acetyltransferase (CAT), the enzyme which synthesizes the neurotransmitter acetylcholine. We have found apparent specificity of both species and cell type for the production, release, or action of this CAT stimulation component (CSC). Rat and mouse muscle CMs contained CSC which was active in mouse SC cells; chick muscle CM did not. In addition to muscle CM, the CM from cell cultures of mouse heart, liver, and kidney contained CSC. However, CM from secondary cultures of liver cells contained little if any CSC. These apparent specificities were not due to differences in the protein content of either the cells providing CM or of the CM itself. There was also apparent specificity of response to CSC among cholinergic cells in culture. Cultures of cells from only two of four regions of the mouse central nervous system, and from one of five neuronal cell lines tested, had increased CAT activity after treatment with muscle CM. The response in NG108-15 neuroblastoma-glioma hybrid cells was further characterized, and was used to develop a more convenient and rapid assay for CSC.

[77] Cultured cell systems and methods for neurobiology

Publisher Summary This chapter describes methods for the culture of cells from the nervous system of the mouse or rat, and for determining the activities of enzymes required for communication between neurons, such as choline acetyltransferase (EC 2.3.1.6); tyrosine hydroxylase (EC 1.14.3a); glutamate decarboxylase (EC 4.1.1.15); acetylcholinesterase (EC 3.1.1.7), and catechol O-methyltransferase (EC 2.1.1.1). The methods are sufficiently sensitive so that specific activities of most of the above enzymes usually can be determined with protein harvested from one petri dish. 50 to 100 assays are performed routinely in one day.

Development of choline acetyltransferase activity in chick cranial neural crest cells in culture.

Abstract Cultures of chick cranial neural crest cells, when grown in medium containing horse serum (HS), underwent morphological changes leading to the formation, after 6 days in culture, of dense aggregates of cells, some of which produced processes resembling neurite fascicles. In contrast, cranial neural crest cells grown under identical conditions, but with fetal calf serum (FCS) instead of HS, did not form aggregates; after 7–8 days, most of these cells were heavily pigmented. Choline acetyltransferase (CAT) activity was absent or low in freshly dissected neural tubes from embryos of stage 9 and in neural tubes in culture for 2 days in medium containing either HS or FCS. CAT activity was also absent in neural crest cells which had grown out in the presence of FCS for 6 days or in the presence of HS for 2 or 4 days. Cells grown in medium containing HS for 6 days developed significant levels of CAT activity. These results demonstrate that cranial neural crest cells in culture have the capacity to differentiate into cholinergic cells under specific conditions. When neural crest cells which had migrated from neural tubes in medium containing HS were shifted to medium containing FCS for 4 days, no CAT activity was detected. However, when neural crest cells which had migrated in medium containing FCS were shifted to medium containing HS for 4 days, the cells formed aggregates and CAT activity was present. This demonstrates that the kind of serum present in the medium did not select for the outgrowth of a specific predetermined population of neural crest cells from the neural tube, but rather that some factor associated with subsequent culture conditions influenced the cells to become cholinergic cells or pigmented cells.

On GABA function and physiology in the pineal gland

Pineal gamma-aminobutyric acid (GABA) content and glutamic acid decarboxylase (GAD) activity were found not to be influenced by environmental light, catecholamines, sympathetic innervation, or input via the pineal stalk. The observation that GAD activity did not disappear after pineal stalk section, ganglionectomy, or 48 h of organ culture leads us to suggest that GAD activity is not located in nerve processes entering the pineal gland. Treatment in organ culture with an inhibitor of protein synthesis did not greatly influence the slow rate of decrease of GAD activity. This finding is consistent with the conclusion that GAD turnover is slow. Treatment of denervated glands or glands containing functional sympathetic nerve structures with GABA, amino-oxyacetic acid (AOAA) or bicuculline in organ culture did not alter unstimulated levels, or significantly block the adrenergic stimulation of the activity of pineal serotonin N-acetyl transferase (NAT). It is clear from our studies that GABA does not influence or modulate the adrenergic regulation of.pineal NAT activity, and that GABA content and synthesis are not regulated by an adrenergic mechanism. The role of GABA in the pineal gland remains to be discovered.

An in situ Assay for Determination of Benzodiazepine Binding

Benzodiazepine (BDZ) receptors have been demonstrated recently in a variety of mammalian tissues. However, assay methods for such receptors have required that disrupted tissues be used. We have developed an in situ assay for this receptor utilizing intact cells cultured from the cerebral cortices of fetal mice which is more sensitive and physiologic than those used previously. Results obtained with this assay differ in the following ways from those in which disrupted tissues are used: (1) total and specific BDZ binding was as much as 10-fold higher in the in situ assays; (2) Scatchard analysis of the binding data is consistently nonlinear, revealing at least two binding sites with KD values of 5.5 and 303 nM, and (3) presumed nonneuronal receptors were found in abundance.

Interpeduncular nucleus: differential effects of habenula lesions on choline acetyltransferase and glutamic acid decarboxylase.

The effects of ablation of the habenula on choline acetyltransferase and glutamic acid decarboxylase activities in the interpeduncular nucleus were studied at 3-, 8-, 15-, and 30-day postoperative survival periods. Choline acetyltransferase in the interpeduncular nucleus decreased markedly (P ≤ 0.001) after 3 days of habenular lesion. This is in accord with the time course of degeneration of the habenulointerpeduncular tract, which provides the major cholinergic innervation to that nucleus. In contrast, glutamic acid decarboxylase activity increased slowly to almost twice control values (P ≤ 0.001) at the 30-day postoperative period. These data suggest collateral sprouting of γ-aminobutyric acid-containing terminals within the interpeduncular nucleus in response to degeneration of the habenulointerpeduncular tract terminals by habenular ablation.