Gleb P. Shumyatsky

Identification of a signaling network in lateral nucleus of amygdala important for inhibiting memory specifically related to learned fear.

We 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 GRP receptor (GRPR) is expressed in GABAergic interneurons of the lateral nucleus. GRP excites these interneurons and increases their inhibition of principal neurons. 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. By contrast, these mice performed normally in hippocampus-dependent Morris maze. These experiments provide genetic evidence that GRP and its neural circuitry operate as a negative feedback regulating fear and establish a causal relationship between Grpr gene expression, LTP, and amygdala-dependent memory for fear.

stathmin, a gene enriched in the amygdala, controls both learned and innate fear.

Little is known about the molecular mechanisms of 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.

Bidirectional Regulation of Hippocampal Long-Term Synaptic Plasticity and Its Influence on Opposing Forms of Memory

Reference memory characterizes the long-term storage of information acquired through numerous trials. In contrast, working memory represents the short-term acquisition of trial-unique information. A number of studies in the rodent hippocampus have focused on the contribution of long-term synaptic potentiation (LTP) to long-term reference memory. In contrast, little is known about the synaptic plasticity correlates of hippocampal-based components of working memory. Here, we described a mouse with selective expression of a dominant-negative mutant of the regulatory subunit of protein kinase A (PKA) only in two regions of the hippocampus, the dentate gyrus and area CA1. This mouse showed a deficit in several forms of LTP in both hippocampal subregions and a lowered threshold for the consolidation of long-term synaptic depression (LTD). When trained with one trial per day in a water maze task, mutant mice displayed a deficit in consolidation of long-term memory. In contrast, these mice proved to be more flexible after a transfer test and also showed a delay-dependent increased performance in working memory, when repetitive information (proactive interference) was presented. We suggest that through its bidirectional control over synaptic plasticity PKA can regulate opposing forms of memory. The defect in L-LTP disrupts long-term memory consolidation. The persistence of LTD may allow acquisition of new information by restricting the body of previously stored information and suppressing interference.

Synaptically released zinc gates long-term potentiation in fear conditioning pathways

The functional role of releasable Zn2+ in the central nervous system remains unknown. Here we show that zinc transporter 3 (ZnT-3), which maintains a high concentration of Zn2+ in synaptic vesicles and serves as a marker for zinc-containing neurons, is enriched in the lateral nucleus of the amygdala and in the temporal area 3 of the auditory cortex, an area that conveys information about the auditory conditioned stimulus to the lateral nucleus of the amygdala, but not in other conditioned stimulus areas located in the auditory thalamus. Using whole-cell recordings from amygdala slices, we demonstrated that activity-dependent release of chelatable Zn2+ is required for the induction of spike timing-dependent long-term potentiation in cortical input to the amygdala implicated in fear learning. Our data indicate that synaptically released Zn2+ enables long-term potentiation at the cortico-amygdala synapses by depressing feed-forward GABAergic inhibition of principal neurons. This regulatory mechanism, implicating pathway-dependent release of Zn2+, may serve an essential control function in assuring spatial specificity of long-lasting synaptic modifications in the neural circuit of a learned behavior.

Zinc transporter 3 is involved in learned fear and extinction, but not in innate fear.

Synaptically released Zn²+ is a potential modulator of neurotransmission and synaptic plasticity in fear-conditioning pathways. Zinc transporter 3 (ZnT3) knock-out (KO) mice are well suited to test the role of zinc in learned fear, because ZnT3 is colocalized with synaptic zinc, responsible for its transport to synaptic vesicles, highly enriched in the amygdala-associated neural circuitry, and ZnT3 KO mice lack Zn²+ in synaptic vesicles. However, earlier work reported no deficiency in fear memory in ZnT3 KO mice, which is surprising based on the effects of Zn²+ on amygdala synaptic plasticity. We therefore reexamined ZnT3 KO mice in various tasks for learned and innate fear. The mutants were deficient in a weak fear-conditioning protocol using single tone-shock pairing but showed normal memory when a stronger, five-pairing protocol was used. ZnT3 KO mice were deficient in memory when a tone was presented as complex auditory information in a discontinuous fashion. Moreover, ZnT3 KO mice showed abnormality in trace fear conditioning and in fear extinction. By contrast, ZnT3 KO mice had normal anxiety. Thus, ZnT3 is involved in associative fear memory and extinction, but not in innate fear, consistent with the role of synaptic zinc in amygdala synaptic plasticity.

Stathmin reveals dissociable roles of the basolateral amygdala in parental and social behaviors

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 threat 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 the BLA role in forming memories for learned fear is established, the 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 threat 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 WT 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.

Zinc transporter ZnT3 is involved in memory dependent on the hippocampus and perirhinal cortex.

Since zinc transporter ZnT3 is localized to the hippocampus and perirhinal cortex, we used ZnT3 knockout mice (KO) to analyze the role of ZnT3 in memory and behavior dependent on these brain regions. ZnT3KO mice were normal in initial learning in the standard water maze but had difficulty finding a second platform location. The mutants showed increased social interaction but were deficient in social and object recognition memory. These data suggest that ZnT3 is involved in certain types of spatial memory and behavior dependent on the hippocampus and perirhinal cortex.

Pituitary adenylate cyclase-activating polypeptide induces postsynaptically expressed potentiation in the intra-amygdala circuit.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide expressed in the brain, where it may act as a neuromodulator or neurotransmitter contributing to different behavioral processes and stress responses. PACAP is highly expressed in the amygdala, a subcortical brain area involved in both innate and learned fear, suggesting a role for PACAP-mediated signaling in fear-related behaviors. It remains unknown, however, whether and how PACAP affects neuronal and synaptic functions in the amygdala. In this study, we focused on neurons in the lateral division of the central nucleus (CeL), where PACAP-positive presynaptic terminals were predominantly found within the amygdala. In our experiments on rat brain slices, exogenous application of PACAP did not affect either resting membrane potential or membrane excitability of CeL neurons. PACAP enhanced, however, excitatory synaptic transmission in projections from the basolateral nucleus (BLA) to the CeL, while inhibitory transmission in the same pathway was unaffected. PACAP-induced potentiation of glutamatergic synaptic responses persisted after the washout of PACAP and was blocked by the VPAC1 receptor antagonist, suggesting that VPAC1 receptors might mediate synaptic effects of PACAP in the CeL. Moreover, potentiation of synaptic transmission by PACAP was dependent on postsynaptic activation of protein kinase A and calcium/calmodulin-dependent protein kinase II, as well as synaptic targeting of GluR1 subunit-containing AMPA receptors. Thus, PACAP may upregulate excitatory neurotransmission in the BLA–CeL pathway postsynaptically, consistent with the known roles of PACAP in control of fear-related behaviors.

Learning-induced and stathmin-dependent changes in microtubule stability are critical for memory and disrupted in ageing

Changes in the stability of microtubules regulate many biological processes, but their role in memory remains unclear. Here we show that learning causes biphasic changes in the microtubule-associated network in the hippocampus. In the early phase, stathmin is dephosphorylated, enhancing its microtubule-destabilizing activity by promoting stathmin-tubulin binding, whereas in the late phase these processes are reversed leading to an increase in microtubule/KIF5-mediated localization of the GluA2 subunit of AMPA receptors at synaptic sites. A microtubule stabilizer paclitaxel decreases or increases memory when applied at the early or late phases, respectively. Stathmin mutations disrupt changes in microtubule stability, GluA2 localization, synaptic plasticity and memory. Aged wild-type mice show impairments in stathmin levels, changes in microtubule stability, and GluA2 localization. Blocking GluA2 endocytosis rescues memory deficits in stathmin mutant and aged wild-type mice. These findings demonstrate a role for microtubules in memory in young adult and aged individuals.

Murine GRPR and Stathmin Control in Opposite Directions both Cued Fear Extinction and Neural Activities of the Amygdala and Prefrontal Cortex

Extinction is an integral part of normal healthy fear responses, while it is compromised in several fear-related mental conditions in humans, such as post-traumatic stress disorder (PTSD). Although much research has recently been focused on fear extinction, its molecular and cellular underpinnings are still unclear. The development of animal models for extinction will greatly enhance our approaches to studying its neural circuits and the mechanisms involved. Here, we describe two gene-knockout mouse lines, one with impaired and another with enhanced extinction of learned fear. These mutant mice are based on fear memory-related genes, stathmin and gastrin-releasing peptide receptor (GRPR). Remarkably, both mutant lines showed changes in fear extinction to the cue but not to the context. We performed indirect imaging of neuronal activity on the second day of cued extinction, using immediate-early gene c-Fos. GRPR knockout mice extinguished slower (impaired extinction) than wildtype mice, which was accompanied by an increase in c-Fos activity in the basolateral amygdala and a decrease in the prefrontal cortex. By contrast, stathmin knockout mice extinguished faster (enhanced extinction) and showed a decrease in c-Fos activity in the basolateral amygdala and an increase in the prefrontal cortex. At the same time, c-Fos activity in the dentate gyrus was increased in both mutant lines. These experiments provide genetic evidence that the balance between neuronal activities of the amygdala and prefrontal cortex defines an impairment or facilitation of extinction to the cue while the hippocampus is involved in the context-specificity of extinction.