Thomas Endres

TMT-induced autonomic and behavioral changes and the neural basis of its processing.

One of the main interests in the field of neuroscience is the investigation of the neural basis of fear. During recent years, an increasing number of studies have used trimethylthiazoline (TMT), a component of red fox feces, as a stimulus to induce fear in predator naive rats, mice, and voles. The aim of the present review is to summarize these studies. We present an overview to the autonomic and behavioral changes that are induced by TMT exposure. Then, we summarize the small number of studies that have examined the neural processing of the TMT stimulus. Finally, we compare these studies with those using a natural predator or predator odor to induce fear and discuss the possible use of TMT exposure in rodents as an animal model of unconditioned fear in humans.

2,3,5-Trimethyl-3-thiazoline (TMT), a component of fox odor : Just repugnant or really fear-inducing?

During recent years, an increasing number of studies have used 2,3,5-trimethyl-3-thiazoline (TMT), a component of fox feces, as a stimulus to induce fear in predator naive rodents. The use of TMT is controversially discussed: There are some clear advantages of TMT against natural predator odors (e.g. stimulus intensity can be better controlled) but also still some open questions and objections regarding TMT. The aim of the present article is to discuss four often mentioned objections against TMT: (1) In some cases, TMT failed to produce fear behavior, (2) TMT is rather a noxious than a fear-inducing stimulus, (3) TMT does not support fear conditioning, and (4) there are different neural pathways processing natural predator odors and TMT. We summarize data showing different sensitivity to TMT in different rat strains. Then, new data are presented showing that TMT concentrations which are not avoided by rats induce fear behavior, and that concentrations of TMT and of the control odor butyric acid, which are similarly avoided, are totally different in their ability to induce fear behavior. Furthermore, we summarize and discuss data showing that fear conditioning to a TMT-paired context is possible and that there is an overlap between the neural basis for TMT- and cat odor-induced fear behavior. In conclusion, the recent data do not support the idea that TMT is simply a noxious stimulus which non-specifically induces fear behavior. Therefore, TMT is still a viable alternative stimulus to natural predator odors to investigate effects of predator odors on behavior.

Behavioral Changes Induced in Rats by Exposure to Trimethylthiazoline, a Component of Fox Odor

Trimethylthiazoline (TMT), a component of fox feces, has been used in various studies as a natural predator stimulus to induce autonomic and behavioral signs of fear (e.g., higher levels of stress hormones, freezing, and risk assessment). The present study investigated whether 2 further behavioral signs of fear are induced in rats by TMT exposure: potentiation of the acoustic startle response and inhibition of appetitive behavior. In addition, the authors tested the rats for dose dependency of TMT-induced freezing behavior. The study confirmed that behavioral changes observed during TMT exposure are caused by TMT-induced fear and are dose dependent.

Are rats predisposed to learn 22 kHz calls as danger-predicting signals?

Alarm calls are widely used in mammals. Their biological function is to deter predators and warn relatives of danger. Despite this important function of alarm calls, the development of alarm call recognition is poorly understood. Using laboratory rats, the present study investigated in a first experiment whether alarm calls are recognized innately. In experimentally naive animals, we found significantly increased freezing if stimuli in the 22 kHz range were presented but this response was not specific to conspecific 22 kHz calls. Therefore, a second experiment addressed the hypothesis whether recognition of conspecific 22 kHz calls can be learned and whether this learning is facilitated by a preparedness to acquire defensive responses to alarm calls. Our data show that rats learned quickly to associate the 22 kHz calls with aversive stimuli. Interestingly, the animals were more reluctant to extinguish this memory, and this information retained longer in memory than in the case of other types of calls and ultrasonic stimuli. We, therefore, conclude that rats are predisposed to acquire adaptive defensive behaviour in response to alarm calls. In particular, our data indicate that better encoding of such learning in rats leads to a stable memory which better resists extinction.

Postsynaptic BDNF signalling regulates long-term potentiation at thalamo-amygdala afferents

Non‐technical summary  Long‐term potentiation (LTP) in the lateral amygdala (LA) is a widely accepted cellular correlate of fear learning. In the present study we analysed the involvement of the neurotrophin brain‐derived neurotrophic factor (BDNF) in amygdala LTP by stimulating selectively thalamic or cortical sensory inputs into the LA. In heterozygous BDNF knockout mice we observed that LTP in the cortico‐amygdala pathway remained intact, whereas LTP in the thalamo‐amygdala pathway was abolished. Likewise, acute interference with BDNF/TrkB signalling in wild‐type mice by application of a BDNF scavenger (TrkB‐IgG) led to inhibition of LTP in this pathway. In addition, postsynaptic inhibition of TrkB receptors, which mediate BDNF effects, blocked LTP in the thalamic pathway. Thus, our data demonstrate for the first time a crucial role for postsynaptic BDNF signalling in mediating LTP selectively in the thalamo‐amygdala pathway in the LA.

Impaired fear extinction learning in adult heterozygous BDNF knock-out mice

Brain-derived neurotrophic factor (BDNF) is a crucial regulator of neuroplasticity, which underlies learning and memory processes in different brain areas. To investigate the role of BDNF in the extinction of amygdala-dependent cued fear memories, we analyzed fear extinction learning in heterozygous BDNF knock-out mice, which possess a reduction of endogenous BDNF protein levels to ~50% of wild-type animals. Since BDNF expression has been shown to decline with aging of animals, we tested the performance in extinction learning of these mice at 2 months (young adults) and 7 months (older adults) of age. The present study shows that older adult heterozygous BDNF knock-out mice, which have a chronic 50% lack of BDNF, also possess a deficit in the acquisition of extinction memory, while extinction learning remains unaffected in young adult heterozygous BDNF knock-out mice. This deficit in extinction learning is accompanied by a reduction of BDNF protein in the hippocampus, amygdala and the prefrontal cortex.

Aversion- vs fear-inducing properties of 2,4,5-trimethyl-3-thiazoline, a component of fox odor, in comparison with those of butyric acid.

SUMMARY 2,4,5-trimethyl-3-thiazoline (TMT), a component of fox feces, is a widely used odorant to induce innate fear behavior in rats and mice. However, based on the slight acrid smell it was argued that the observed behavioral effects are a result of the aversive and not of the fear-inducing properties of TMT. In the present study, we tried to directly compare the aversive and fear-inducing properties of TMT with those of the aversive control odor butyric acid. We first identified concentrations of butyric acid and TMT that induce similar amounts of avoidance behavior in rats, indicating that these concentrations have similar aversive properties. In a second experiment, these two concentrations were then tested for their ability to induce freezing, a species-specific defensive response. Only TMT but not butyric acid induced freezing in the rats. This supports the hypothesis that TMT indeed has specific fear-inducing properties and that the observed behavioral effects could not simply be reduced to the aversive properties of TMT.

Age-dependent deficits in fear learning in heterozygous BDNF knock-out mice

Beyond its trophic function, the neurotrophin BDNF (brain-derived neurotrophic factor) is well known to crucially mediate synaptic plasticity and memory formation. Whereas recent studies suggested that acute BDNF/TrkB signaling regulates amygdala-dependent fear learning, no impairments of cued fear learning were reported in heterozygous BDNF knock-out mice (BDNF(+/-)). Since brain BDNF levels are known to decline with aging, we hypothesized that BDNF(+/-) mice might show reduced fear learning at older ages. Indeed, BDNF(+/-) animals revealed an age-dependent deficit in fear learning 3 mo after birth and beyond. Since there were no alterations between the two genotypes during the conditioning training and when testing short-term memory, this learning deficit most likely reflects a deficit in memory consolidation. Importantly, there were no differences in spontaneous motor behavior and baseline anxiety in BDNF(+/-) animals at any age tested. Following behavioral testing quantification of BDNF levels in the basolateral amygdala with a sensitive BDNF ELISA revealed a positive correlation between the levels of BDNF in the amygdala and the individual learning performance. However, the age-dependent decline in the efficiency of fear conditioning in BDNF(+/-) mice was not accompanied by reduced BDNF expression in the amygdala. Thus, while reduced BDNF levels in general correlate with less efficient fear learning, this lack of BDNF can be compensated in young but not in older animals, suggesting that the cellular mechanisms responsible for fear learning consolidation become BDNF-dependent 3 mo after birth.

Conditioned behavioral responses to a context paired with the predator odor trimethylthiazoline.

Contextual fear conditioning is the learning of a fear response in a specific context in response to repeated application of aversive stimuli (e.g., foot shocks) or danger-related stimuli (predator odors) within that context. Cat odor, a danger-related stimulus common in laboratory studies of fear in the past, has often been replaced recently with trimethylthiazoline (TMT), a component of fox feces. No contextual fear conditioning in response to TMT has been reported so far, whereas cat odor has often been shown to induce such fear conditioning in rats. Using TMT in both a 1-compartment and a 2-compartment setup, the authors found conditioned fear behavior (expressed as avoidance behavior) in the 2-compartment setup but not--as reported by others--in the 1-compartment setup. Detailed analysis revealed 2 different coping strategies in the 2-compartment setup: Half of the rats showed pronounced avoidance behavior, whereas the other half showed intense risk assessment behavior. These results indicate that expression of conditioned fear behavior in response to a TMT-paired context is dependent on the experimental setup used, as well as the strategy of the individual rat.

Acute and chronic interference with BDNF/TrkB-signaling impair LTP selectively at mossy fiber synapses in the CA3 region of mouse hippocampus

Brain-derived neurotrophic factor (BDNF) signaling via TrkB crucially regulates synaptic plasticity in the brain. Although BDNF is abundant at hippocampal mossy fiber (MF) synapses, which critically contribute to hippocampus dependent memory, its role in MF synaptic plasticity (long-term potentiation, LTP) remained largely unclear. Using field potential recordings in CA3 of adult heterozygous BDNF knockout (ko, BDNF+/-) mice we observed impaired (∼50%) NMDAR-independent MF-LTP. In contrast to MF synapses, LTP at neighboring associative/commissural (A/C) fiber synapses remained unaffected. To exclude that impaired MF-LTP in BDNF+/- mice was due to developmental changes in response to chronically reduced BDNF levels, and to prove the importance of acute availability of BDNF in MF-LTP, we also tested effects of acute interference with BDNF/TrkB signaling. Inhibition of TrkB tyrosine kinase signaling with k252a, or with the selective BDNF scavenger TrkB-Fc, both inhibited MF-LTP to the same extent as observed in BDNF+/- mice. Basal synaptic transmission, short-term plasticity, and synaptic fatigue during LTP induction were not significantly altered by treatment with k252a or TrkB-Fc, or by chronic BDNF reduction in BDNF+/- mice. Since the acute interference with BDNF-signaling did not completely block MF-LTP, our results provide evidence that an additional mechanism besides BDNF induced TrkB signaling contributes to this type of LTP. Our results prove for the first time a mechanistic action of acute BDNF/TrkB signaling in presynaptic expression of MF-LTP in adult hippocampus.