Our laboratory’s work is focused on unraveling the molecular mechanisms, employed locally in the anatomic neural circuits, that control memory and behaviors related to fear. We chose to begin our investigation in the amygdala, the key region of the brain involved in learned and innate fear, for the following reasons:

Figure 1. The neural circuit of learned fear

       The neuroanatomy of the amygdala and its afferent pathways, which relay conditioned stimulus (CS) information to the amygdala, are well-defined (Pitkanen, 2000) (Figure 1). In our study, we focus primarily on the lateral nucleus of the amygdala (LA), because of its importance in the encoding and the possible storage of fear memory, and because it is the main nucleus of the amygdala for visual and auditory sensory inputs (McDonald, 1998)

       Learned fear can be assessed quantitatively by using Pavlovian fear conditioning, which is a quickly learned behavioral paradigm that leads to robust, long-lasting memory, measurable by freezing (Blanchard et al., 1970; Kapp et al., 1992; Kim and Fanselow, 1992; Pavlov, 1927; Phillips and LeDoux, 1992). In this task, an initially neutral CS acquires significance when paired with an aversive unconditioned stimulus (US). Fear learning produces a prolonged synaptic modification similar to long-term potentiation (LTP, a thoroughly studied form of synaptic plasticity) in both thalamic and cortical inputs to the LA (McKernan and Shinnick-Gallagher, 1997; Rogan et al., 1997; Tsvetkov et al., 2002). These and other findings provide strong evidence that at least some of the mechanisms for LTP, examined in brain slices, are recruited behaviorally and are involved in the storage of memory.

    The amygdala, a key component of the limbic system, is involved in a number of behaviors associated with learning and memory, emotions, drug addiction, and survival. Functional disturbances of the amygdala and the limbic system are generally believed to be at the root of a number of psychiatric disorders, including post-traumatic stress disorder, Alzheimer’s disease, autism, and schizophrenia.

	GRP-dependent negative feedback to principal neurons

       To study learned fear at the molecular level, we are focusing on the characterization of amygdala-enriched genes. We have identified some of the genetic components of amygdalar neural circuits that play an important role in how fear is regulated and experienced. We have identified the GRP gene, encoding gastrin-releasing peptide, as being highly expressed both in the lateral nucleus of the amygdala (the nucleus where associations for Pavlovian learned fear are formed) and in the regions that convey fearful auditory information to the lateral nucleus. Moreover, we found that the GRP receptor (GRPR) is expressed in GABAergic interneurons of the lateral nucleus. By binding to GRPR, GRP excites these interneurons and increases their inhibition of principal neurons through the release of the neurotransmitter GABA. GRPR-deficient mice showed decreased inhibition of principal neurons by the interneurons, enhanced long-term potentiation (LTP), and greater and more persistent long-term fear memory. In contrast, the knockout mice performed normally in hippocampus-dependent memory tests, such as the Morris water maze. These experiments provide genetic evidence that GRP and its neural circuitry operate as negative feedback regulating fear. They establish a causal relationship between GRPR gene expression, LTP, and amygdala-dependent memory of fear (Shumyatsky et. al. 2002).

       Little is known about the molecular mechanisms controlling both learned and innate fear. We have identified stathmin, an inhibitor of microtubule formation, as highly expressed in the lateral nucleus (LA) of the amygdala as well as in the thalamic and cortical structures that send information to the LA about the conditioned (learned fear) and unconditioned stimuli (innate fear). Whole-cell recordings from amygdala slices that are isolated from stathmin knockout mice show deficits in spike-timing-dependent long-term potentiation (LTP). The knockout mice also exhibit decreased memory in amygdala-dependent fear conditioning and fail to recognize danger in innately aversive environments. By contrast, these mice do not show deficits in the water maze, a spatial task dependent on the hippocampus, where stathmin is not normally expressed. We therefore conclude that stathmin is required for the induction of LTP in afferent inputs to the amygdala and is essential in regulating both innate and learned fear (Shumyatsky et. al 2005).

Innate parental behaviors and adult social interactions are essential for survival of the individual along with the species as a whole. Because these behaviors require risk assessment of the environment, it is plausible that they are regulated by the amygdala-associated neural circuitry of fear. However, the amygdala is not a single anatomic and functional unit, and nuclei of the amygdala have multiple inter- and intra-connections. This poses a question as to the exact role of different amygdala nuclei in these behaviors and the mechanisms involved. The basolateral complex of the amygdala nuclei (BLA) is particularly interesting in this regard: although BLA role in forming memories for learned fear is established, BLA role in innate behaviors is not well understood. We recently demonstrated that mice without an inhibitor of microtubules, stathmin, a gene enriched in BLA-associated circuitry, have deficiency in innate and learned fear. Here we show that the deficiency in fear processing in stathmin-/- females leads to improper risk assessment, which in turn affects innate parental care and adult social interactions. Profound deficiency is observed in maternal behavior of stathmin-/- females: they lack motivation for retrieving pups and are unable to choose a safe location for nest-building. Remarkably, stathmin-/- females have an enhancement in social interactions. BLA lesions in wildtype mice produce similar effects in maternal and social behaviors, confirming vital BLA participation. The findings implicate stathmin as the critical molecular component linking the BLA-associated neural circuitry with innate parental behaviors and adult social interactions (Martel et al. 2008).