1,1,3 Tricyano-2-amino-1-propene (Triap) stimulates choline acetyltransferase activity in vitro and in vivo.

1,1,3 Tricyano-2-amino-1-propene (Triap) is a small molecule that has neurotrophic properties similar to nerve growth factor (NGF). Studies have shown that NGF increases choline acetyltransferase (ChAT) activity, the enzyme responsible for the synthesis of acetylcholine, in several cell lines and in the CNS of adult animals. To investigate whether Triap can cause similar increases in ChAT enzyme activity, we used the PC12 cell line and primary cultures of rat fetal brain tissue to examine Triap's effects. Nanomolar concentrations of Triap produced a 4.2- and 2.1-fold increase in the ChAT activity of PC12 cells and cultured rat fetal brain cells, respectively. This stimulation reached a plateau within 4 days of treatment in the primary fetal brain cultures with the first increases evident within 24 h. In the PC12 cell line, Triap's stimulation of ChAT activity was significantly greater than increases produced by optimal concentrations of NGF. Triap also matched NGF's stimulation of ChAT activity in primary neuronal culture. Triap also potentiated NGF's actions on ChAT activity in the PC12 cell line and in primary fetal neuronal cultures. These increases in enzyme activity correlated with increases in cellular enzyme levels as assessed using immunochemical identification of the ChAT enzyme. We also conducted experiments to determine if Triap also induced these same increases in ChAT activity in adult animals. Ten-day chronic injections of Triap in mice resulted in significant increases in specific ChAT enzyme activity in the cortex and septal-hippocampal area. Similar increases in ChAT enzyme levels were also detected using western blotting techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Effects of housing and muricidal behavior on serotonergic receptors and interactions with novel anxiolytic drugs.

Mouse killing by rats represents a predatory behavior that can be modified by drugs from several different therapeutic classes and by environmental conditions. Buspirone and gepirone, non-benzodiazepine anxiolytics that stimulate serotonergic receptors (5HT1a) and inhibit isolation-induced intraspecies aggression, were tested for inhibition of muricidal behavior by isolated rats. Neither buspirone (3.0 mg/kg s.c.) nor gepirone (from 5.0 to 40 mg/kg) inhibited muricide. Additional rats were housed, either aggregated or isolated, and tested for muricidal behavior 9 times over 5 weeks to establish which animals were muricidal: thus, there were 4 groups of rats: muricidal or non-muricidal under either isolated or aggregated housing condition. [3H]-Spiperone was used to determine striatal D2 receptor Bmax and Kd and prefrontal cortex D2 and 5HT2 receptor binding. There were no changes across the four groups. Binding of [3H]-5-hydroxytryptamine (5HT) to 5HT1a receptors decreased in septum of both groups of isolated rats and binding to 5HT1b receptors decreased 50% in hippocampus of isolated and aggregated muricidal rats. Binding of [3H]-5HT to either receptor was unchanged in amygdaloid area and hypothalamus across all groups. Thus, stimulating pre- and postsynaptic 5HT1a receptors does not alter muricidal behavior and changes in 5HT1 receptor binding occurs in limited areas. Whether this limited change in hippocampal 5HT1b binding is important for establishing muricidal behavior is unclear; however the direction of the change is consistent with reports that decreased serotonergic activity increases predatory behavior.

Influence of environment on GABA receptors in muricidal rats.

The influence of environment (isolation) on GABA receptor numbers ( [3H]-muscimol binding sites) and affinities was investigated in specific limbic areas known to be involved with the development of muricide. Olfactory bulbs of isolated rats were found to have identical numbers of [3H]-muscimol binding sites whether or not they were muricidal. However, in the olfactory bulbs of the aggregated animals a greater than two-fold increase was found in numbers of [3H]-muscimol binding sites in those rats that were muricidal. In the amygdala muricidal rats had a 32-34% increase in [3H]-muscimol binding sites over non-muricidal rats regardless of environment. In the septum non-muricidal rats had fewer [3H]-muscimol binding sites than muricidal rats and although the trend was evident, statistical vigor was seen only in the aggregated animals. Neither muricide nor isolation significantly influenced [3H]-muscimol binding numbers in the hypothalamus. GABA Ki values were examined in all brain regions and found to be the same in the isolated and aggregated animals whether or not they were muricidal. We concluded that environment appears to influence apparent GABA receptor numbers in the olfactory bulbs and septum whereas muricidal behavior correlates well with an increase in apparent number of GABA receptors in the amygdala. GABA receptors in the hypothalamus were not influenced by either environment or aggression.

Regional [3H]testosterone uptake in the brain of isolated non-aggressive mice.

Male mice which are housed individually develop a characteristic aggressive, 'fighting' behavior. A small percentage of mice so isolated fail to become aggressive and in fact, behaviorally are strikingly different in that they develop a 'timid' behavior. Isolation-induced aggression is an androgen-dependent behavior and androgen-sensitive neurons have been identified in mouse brain, mainly in the limbic system. When intact isolated aggressive mice and isolated and aggregated non-aggressive mice were injected with radioactive testosterone and sacrificed after 30 min no remarkable differences in uptake of radioactive testosterone occurred in various brain areas. However, in castrated animals the uptake of testosterone was greatly enhanced in the isolated non-fighters whereas testosterone uptake in the isolated fighters and aggregated non-fighters was in the range of the intact animals. This study represents, for the first time, a documented hormonal difference between isolated aggressive and non-aggressive animals.

Must antidepressants be anticholinergic to inhibit muricide?

Two classical tricyclic antidepressants possessing anticholinergic side effects, amitriptyline and desipramine, were compared to newer antidepressants lacking such activity, mianserin, trazodone, and bupropion, for their ability to inhibit muricidal behavior. As has been previously shown for the tricyclic antidepressants, the newer antidepressants without anticholinergic activity also depressed mouse-killing behavior. Scopolamine HBr, at a dose which lacked antimuricidal activity, was tested for its ability to potentiate the antimuricidal effect of these antidepressants. Although potentiation was not demonstrated, there was a trend for scopolamine to enhance the antimuricidal effect of all drugs tested, regardless of whether or not they had anticholinergic activity.

The effect of olfactory bulb lesions on agonistic, locomotory and social behavior in mice housed in aggregate.

Eleven Dublin ICR mice sustaining nearly complete bilateral aspiration lesions of the olfactory bulbs were compared to 16 sham-operated mice on the frequency of agonistic, locomotory, social, and maintenance behaviors. The animals were housed in aggregate with two cages of bulbectomized mice and two cages of shams. A time sampling technique was used to quantify the frequency of the various behaviors three times per week during one preoperative and four postoperative weeks. During the first two postoperative weeks the bulbectomized mice were observed to make frequent single bites of the head incision and tail of mice passing directly in front of them, with the bites resulting in mild to severe lesions. Following surgery, the bulbectomized mice also showed a temporary increase in locomotory responses and permanent suppression of social responses.

Histofluorescent identification of indoleamine-concentrating brain loci associated with intraspecies, reflexive biting and locomotor behavior in olfactory-bulbectomized mice.

The histofluorescent identification of indoleamine (serotonin)-concentrating neurons and the changes induced by olfactory bulbectomy (Obx) were correlated with reflexive biting and locomotor behavioral activities in grouped male mice. Between 2 and 35 days post-Obx, an increase in the number and fluorescent-intensity of indoleamine-containing neurons was identified in the olfactory tubercle, lateral olfactory tract (bed neurons) and piriform cortex. Neurons in the nucleus accumbens and anterior olfactory nucleus also demonstrated increased histofluorescence by Day 35 post-Obx as compared with controls, but to a lesser extent than observed in the other olfactory loci. The increase in serotonin concentration was inversely related to locomotor activity and directly correlated to biting behavior in Obx mice. These results demonstrate that the development of this type of intraspecies aggression is temporally correlated with an enhanced serotonin histofluorescence in specific forebrain, olfactory regions in mice.

Influence of isolation and training on fighting in mice with olfactory bulb lesions.

Isolation-induced aggressive mice receiving total bilateral bulbectomy failed to fight after chronic training. Animals receiving sub-total olfactory bulbectomy were capable of being trained to attack but the latency to attack was increased. When mice were bulbectomized before being isolated they were incapable of attack regardless of training or completeness of the lesion. These results indicated that isolation influences aggressive behavior in bilaterally bulbectomized mice.

Inhibition of isolation-induced aggressive behavior with GABA transaminase inhibitors.

Administration of aminooxyacetic acid (AOAA) and gamma-acetylenic GABA (GAG) resulted in a dose-dependent suppression of aggressive behavior and a concomitant increase in brain GABA. The increase in GABA levels persisted beyond the time when AOAA and GAG exerted an anti-aggressive effect. Brain GABA levels remained significantly elevated over controls for 120 h after a single injection of AOAA and 46 h after a single dose of GAG.

Resistance of androgen-mediated aggressive behavior in mice to flutamide, an antiandrogen.

Flutamide (FTA), an anti-androgenic compound, inhibited the effects of methyltestosterone (MT) on the weight of the ventral prostate, seminal vesicles and levator ani in male castrate mice. Castration prevented the development of aggressive behavior in mice isolated for 3 weeks. While chronic administration of MT to castrate isolated mice returned the incidence of fighting behavior to control values, chronic administration of FTA + MTdid not significantly reduce the incidence of fighting as compared to castrate + MT values. These results suggest that the mechanism for androgen stimulation of secondary sex organ weight may differ from that involved in the development and maintenance of aggression resulting from isolation.