TMT predator odor activated neural circuit in C57BL/6J mice indicates TMT-stress as a suitable model for uncontrollable intense stress.

Intense stressful events can result in chronic disorders such as posttraumatic stress disorder (PTSD). In vulnerable individuals, a single aversive experience can be sufficient to cause long-lasting behavioral changes. Candidate brain regions implicated in stress-related psychopathology are the amygdala, the bed nucleus of the stria terminalis (BNST), and the hypothalamic pituitary adrenal (HPA) axis. In rodents exposure to 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), an ethologically relevant stressor, has been shown to induce intense stress and innate anxiety responses. To study dispositions for the development of maladaptive stress responses, mice models are required. Therefore C57BL/6J mice were exposed to TMT and Fos expression was studied in key brain regions implicated in stress responses and anxiety-like behavior. Our results show TMT-induced activation of a distinct neural circuit involving the BNST, the lateral septum (LS), the paraventricular nucleus of the hypothalamus (PVN), the periaqueductal gray (PAG) and the locus coeruleus (LC). Anatomical interconnection of the BNST with all these regions could point to an important modulatory role of this nucleus. Since, the BNST gets direct input from the olfactory bulbs and projects to the PVN and PAG and is therefore well positioned to modulate behavioral and endocrine stress responses to TMT. Hence, we suggest that TMT exposure is suitable to investigate uncontrollable stress responses in mice which exhibit similarities to maladaptive stress responses underlying PTSD in humans.

Increased CRF mRNA expression in the sexually dimorphic BNST of male but not female GAD67 mice and TMT predator odor stress effects upon spatial memory retrieval.

The bed nucleus of the stria terminalis (BNST) is an important region for 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) predator odor-induced stress responses in mice. It is sexually dimorphic and a region for corticotropin-releasing factor (CRF)-enhanced stress responses. Dense GABAergic and CRF input from the amygdala to the BNST gives point to relevant interactions between CRF and GABA activity in these brain regions. Hence, to investigate sexual dimorphism of stress-induced neuronal changes, we studied effects of acute TMT exposure on CRF mRNA expression in stress-related brain regions in male and female GAD67 mice and their wild-type littermates. In GAD67 mice, heterozygous knock-in of GFP in GABAergic neurons caused a 50% decrease of GAD67 protein level in the brain [91,99]. Results show higher CRF mRNA levels in the BNST of male but not female GAD67 mice after TMT and control odor exposure. While CRF neurons in the BNST are predominantly GABAergic and CRF enhances GABAergic transmission in the BNST [20,51], the deficit in GABAergic transmission in GAD67 mice could induce a compensatory CRF increase. Sexual dimorphism of the BNST with greater density of GABA-ir neurons in females could explain the differences in CRF mRNA levels between male and female GAD67 mice. Effects of odor exposure were studied in a radial arm maze (RAM) task. Results show impaired retrieval of spatial memory after acute TMT exposure in both sexes and genotypes. However, only GAD67 mice show increased working memory errors after control odor exposure. Our work elicits GAD67 mice as a model to further study interactions of GABA and CRF in the BNST for a better understanding of how sex-specific characteristics of the brain may contribute to differences in anxiety- and stress-related psychological disorders.

Species-relevant inescapable stress differently influences memory consolidation and retrieval of mice in a spatial radial arm maze.

Stress affects learning and there are both facilitating and impairing actions of stressors on memory processes. Here we investigated the influence of acute exposure to 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), an ethological relevant stressor for rodents, on spatial memory formation and performance in a radial arm maze (RAM) task and studied TMT effects on corticosterone levels in GAD67-GFP knock-in mice and their wildtype littermates. Our results suggest that predator odor-exposure differently affects consolidation and retrieval of memory in a hippocampus-dependent spatial learning task in adult male mice, independently from their genotypes. Acute TMT-stress before retrieval facilitates performance, whereas repeated TMT-stress during consolidation exerts no influence. Additionally, we found genotype specific effects of TMT on corticosterone release. While TMT-stress tend to result in increased corticosterone release in wildtypes there was a significant decrease in transgenic mice. Taken together, these findings indicate that biologically significant predator odor-induced stress can have different actions on the strength of spatial memory formation depending on the timing with regard to memory phases. Furthermore, we suppose an impact of GABAergic mechanisms on HPA-stress axis activation to TMT resulting in absent peripheral corticosterone release of GAD67-GFP mice.

Behavioral effects and pattern of brain c-fos mRNA induced by 2,5-dihydro-2,4,5-trimethylthiazoline, a component of fox feces odor in GAD67-GFP knock-in C57BL/6 mice.

Predator odors, which are non-intrusive and naturalistic stressors of high ethological relevance, were used to study the neurobiology of innate fear in rodents. The present study investigates behavioral effects and the induction of c-fos mRNA in adult male predator naive mice caused by acute exposure to 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a component of the fox feces odor. On the behavioral level, TMT potently increased unconditioned freezing and decreased non-defensive grooming behavior. With quantitative real time PCR we established a strong TMT-induced activation in the bed nucleus of the stria terminalis (BNST) (eight-fold increase, p<0.016) and in the ventral olfactory bulb (two-fold increase, p<0.036). In contrast, no significant TMT-induced c-fos induction could be observed in the dorsal olfactory bulb or in the amygdala. Our results display robust fear responses of GAD67-GFP knock-in mice exposed to TMT and suggest that the ventral olfactory bulb and the BNST are strongly activated during the elicitation of fear through predator odor in these transgenic mice.

Disrupted visceral feedback reduces locomotor activity and influences background contextual fear conditioning in C57BL/6JOlaHsd mice.

The present experiments were designed to study fear conditioning as an emotional learning task with disrupted visceral feedback. For that purpose we used the peripherally acting beta1-adrenoceptor blocker atenolol and studied its effects on the behavior of male C57BL/6JOlaHsd mice in an exploration-related test and during fear-conditioning. In the first experiment, we treated mice with saline or different doses of the beta1-adrenergic blocker atenolol (5mg/kg and 20mg/kg body weight i.p.) 30 min before behavioral testing in a motility box. Only the high but not the low dose of atenolol led to a reduction of locomotor activity (p<0.02). Factors known to be related to emotionality (rearing, area preference) were unaffected. In a second experiment, saline- and atenolol-treated mice (same dosages and mode of application) were trained for auditory fear conditioning, and 24h later they were retested in the same environment. We found differences between the effects of atenolol upon contextual- and cue-fear conditioning. Animals treated with 20mg/kg BW doses of atenolol showed significantly decreased background contextual fear compared to saline-treated control animals. In contrast, no differences were found during CS presentation in the conditioning context between atenolol-treated animals and saline-treated controls, independent from a paired or an unpaired conditioning paradigm. Thus, the blockade of peripheral beta1-adrenoceptors by atenolol may have disrupted the positive feedback to the central nervous system via visceral afferents resulting in a decreased locomotor activity and background contextual fear.