Hydra head activator peptide has trophic activity for eukaryotic neurons.

The synthetic undecameric peptide, pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe, known as the hydra head activator peptide, present in high concentrations in mammalian hypothalamus and intestine, was tested for neurotrophic activity in a survival assay using cultured chick embryonic sympathetic and dorsal root ganglion cells, and for morphological differentiation activity on neuroblastoma cells. Hydra head activator peptide supported neuron survival. The optimal active concentration, 1 pM, was very similar to the concentration that causes bud and head formation in hydra. Maximal neuron survival obtained with hydra head activator peptide was close to that obtained with nerve growth factor: both substances enhanced survival up to 3 times that of control cultures. Bradykinin, which has some amino acid sequence homology with hydra head activator, was inactive as a neurotrophic factor. Hydra head activator induced rapid morphological differentiation of the mouse neuroblastoma cell line Neuro-2A. Neuro-2A responded to the peptide by process extension, 4 h after its addition to the culture medium. Neurotrophic factors isolated to date have been characterized by their ability to maintain cell viability and enhance neurite outgrowth. Hydra head activator peptide met these two criteria when tested in 3 different neuron culture systems. Our results suggest that the head activator peptide may act as a neurotrophic factor for neurons in other species, including mammals.

1,1,3 tricyano-2-amino-1-propene (Triap): a small molecule which mimics or potentiates nerve growth factor.

1,1,3 Tricyano-2-amino-1-propene (Triap) is a small molecular weight compound which increases the rate of nerve and tissue regeneration in several experimental systems. Early experiments with this compound showed that, like nerve growth factor (NGF), Triap induced neurite formation in chick spinal ganglia. To assess the similarity between NGF and Triap, we compared the effects of Triap and NGF on a rat pheochromocytoma cell line (PC12) and on cell survival in a primary chick neuronal culture. In the latter, Triap at less than 0.01 nM preserved neurons and caused them to extend neurites as did 1 nM NGF. Triap induced neurite outgrowth in the PC12 cell line giving a maximal response (40-50% of the maximal response of NGF) at a concentration of 20 micrograms/ml (151 microM). Triap's morphological effects were not inhibited by antibodies directed against NGF or the NGF receptor. Low concentrations of Triap also potentiated the morphological effects of NGF. Triap induced an increase in cell-substratum adhesion and cellular hypertrophy in PC12 cells and also potentiated the adhesive actions of NGF. Triap had no effect on ornithine decarboxylase activity even though it potentiated NGF's effects on this enzyme. These data indicate that Triap induces neurotrophic effects and does not seem to act through the same mechanisms as NGF but can potentiate many of NGF's morphological and biochemical actions.

Expression of neurotrophic activity in Xenopus oocytes injected with mRNA from wounded rat cerebral cortex.

Injury to the cerebral cortex of the rat brain has been shown to induce the expression of neurotrophic factors for dissociated peripheral and central neurons in culture. We confirm this phenomenon and report that Xenopus laevis oocytes injected with mRNA extracted from wounded rat cortex expressed similar neurotrophic activity. To detect the low amounts of neurotrophic factors that could be expected from the oocyte translation system, a miniaturization of the assay for neurotrophic and cell-surviving activity was developed using Terasaki microtiter plates for culture of chicken embryo sympathetic ganglion cells. Messenger RNA (mRNA) was size-fractionated on a sucrose gradient and RNAs from each fraction were injected into oocytes. Neurotrophic activity was recovered from the homogenates and from the incubation media of oocytes injected with mRNA from 7 day post-lesion cortex. Messenger RNAs in the active fractions ranged in size from 0.8 to 1.8 kb. As much as 20% of the activity was secreted by the oocytes. No significant neurotrophic activity was detected from oocytes injected with mRNA fractions extracted from the cortex of control rats or from other gradient fractions from post-lesion cortex.

Toxic effect of phenytoin on developing cortical neurons in culture.

Studies were undertaken to determine the effect of chronic phenytoin exposure on developing neurons. Cerebral cortex from 16-day fetal mice was utilized to prepare primary dissociated cell cultures. Phenytoin was added to the cultures 10 days after plating and the cultures were harvested on day 17. Cortical cultures were assayed for neuronal cell number by phase microscopy and for high-affinity uptake of 3H-labeled gamma-aminobutyric acid (GABA) by both radioautography and scintillation spectrometry. Neuronal cell counts demonstrated a highly significant decrement in the number of neurons in cultures exposed to phenytoin at 15, 25, and 50 micrograms/ml. 3H-GABA-labeled neurons constituted 13% of the neurons present in both control and phenytoin-exposed cultures. These data indicate that phenytoin is toxic to cortical neurons in culture and that GABAergic neurons are affected to the same extent as the total neuronal population.

Benzodiazepine receptor development in cultures of fetal mouse cerebral cortex mimics its development in vivo.

Using a recently developed assay method which maintains cell integrity, we have performed a developmental study of the benzodiazepine (BDZ) receptor on cells of fetal mouse cortex in cultures which were either 'neuron-poor' or 'neuron-rich'. The developmental profile of the BDZ receptor in culture, when assayed on the intact cell, closely mimicked its development in vivo, reaching maximum values at 35-42 days after conception. Further, the time course of development paralleled that reported for GABA neurons both in vivo and in vitro. We have also shown that the relative delay in development of the clonazepam-displaceable portion of BDZ binding approximated a similar delay in the reported development of glutamic acid decarboxylase activity, both parameters presumably reflecting specific neuronal development. Thus, the ontogenesis of specific BDZ receptor binding in culture bears marked similarity to the time course of development of the GABAergic system.

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.

Adverse effects of phenobarbital on morphological and biochemical development of fetal mouse spinal cord neurons in culture.

The effects of chronic phenobarbital treatment on neuronal development were assessed in cell cultures of fetal rodent spinal cord. The activity of the principal enzyme involved in acetylcholine synthesis, choline acetyltransferase (CAT), and counts of large spinal cord neurons were used as indicators of neuronal development Phenobarbital (30 to 120 micrograms/ml) added to cultures for two- or six-week periods produced dose-dependent decreases in both CAT activity and neurons counts. Barbituric acid at doses equimolar to those at which phenobarbital produced these decreases had no effect.

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.

Neuroblastoma differentiation involves both the disappearance of old and the appearance of new poly(A)+ messenger RNA sequences in polyribosomes.

The cholinergic mouse neuroblastoma cell line NS20Y was adapted to undifferentiated growth in suspension culture. When suspension cells were transferred to surface culture and treated with dibutyryl cyclic AMP, the cells underwent differentiation as assessed by biochemical, morphological, and physiological criteria. Differentiated NS20Y cells in co-culture with mouse muscle cells had the capacity to form functional neuromuscular junctions with the muscle cells. The sequence complexities of the poly(A)-containing messenger RNA (poly(A)+ mRNA) of the differentiated, process-forming cells (P-cells) and undifferentiated cells in suspension culture (S-cells) were measured by analysis of the kinetics of hybridization of the mRNAs with their complementary DNAs (cDNAs). There were less than 100 high abundance and approximately 8000 low abundance poly(A)+ mRNAs in both differentiation states. Heterologous hybridization reactions and recycling of the cDNA probes revealed that 9.7% and 6.8% of the messages in P- and S-cells, respectively, were specific to those differentiation states. The P-cell-specific sequences included approximately 3 high abundance and 320 low abundance poly(A)+ mRNAs. The S-cell-specific sequences included approximately 3 high abundance and 250 low abundance poly(A)+ mRNAs. We conclude that the increment in NS20Y differentiation results in both the disappearance of old, and the appearance of new mRNAs in polyribosomes.