The general finding that long-term plasticity and long-term memory recruit transcription in the nucleus, an organelle shared by all synapses of a neuron, has raised a question that we have begun to explore in Aplysia and mice: Are long-term changes cell-wide, or can induced gene products be spatially compartmentalized so that they selectively alter the function of some synapses and not others? In mice, we have also explored the molecular mechanisms whereby attention modifies and stabilizes internal representation of space.
Persistence of Synaptic Facilitation for Learned Fear in Aplysia
In both Aplysia and mice, we have found that long-term synapse-specific plasticity can occur and that this requires a local marking signal. In Aplysia we found that one component of the synapse-specific marking signals requires local protein synthesis at the activated synapse. Local protein synthesis serves two functions in Aplysia: (1) it marks the activated synapse that confers synapse specificity, and (2) it stabilizes the synaptic growth associated with long-term facilitation. We have found that a neuron-specific isoform of cytoplasmic polyadenylation element–binding protein (CPEB) regulates this synaptic protein synthesis in an activity-dependent manner. Aplysia CPEB protein is up-regulated locally in activated synapses; it is needed not for the initiation but for the stable maintenance of long-term facilitation. Recent work suggests that Aplysia CPEB is the stabilizing component of the synaptic mark.
We have found that CPEB may serve as a stabilizer because it has prion-like properties. Prion proteins have the unusual ability to fold into functionally distinct conformations, one of which is self-perpetuating. When prion proteins convert to the self-perpetuating state, they can cause disease (in mammals) or a nonfunctioning protein (in yeast). Compared to other CPEBs, the neuronal form in Aplysia has an amino-terminal extension which shares characteristics of yeast prion determinants: a high glutamine content and predicted conformational flexibility. When fused to a reporter protein in yeast, this region is sufficient to confer upon it the prototypical epigenetic changes in state that characterize yeast prions. Full-length CPEB undergoes similar changes in yeast but, surprisingly, the dominant, self-perpetuating prion-like form has the greatest capacity to stimulate translation of CPEB-regulated mRNA. Our preliminary studies suggest that conversion of CPEB to a prion-like state in stimulated synapses helps to maintain long-term synaptic changes associated with memory storage.
Explicit Memory Storage in Mammals also requires a Prion-like Protein
Is a functional prion also important for the persistence of memory in mammals? In mouse and in human there are four distinct CPEB genes; CPEB-1 to CPEB-4. Only the contribution of the N-terminal domain of CPEB3 has so far been functionally interrogated at the molecular and behavioral level. As a first step, we expressed CPEB3 in yeast and found that it displayed the two essential features of a prion-like protein: 1) it forms amyloidogenic aggregates and 2) aggregates are heritable across cell division.
Does a prion-like conversion of CPEB3 also occur in the brain? We found that in the basal state CPEB3 binds to and represses the translation of its target mRNAs in the brain such as the AMPA receptor subunits GluA1 and GluA2. In turn, CPEB3 promotes the translation of the AMPAr following mono-ubiquitination by the ubiquitin ligase Neuralized. Together these data suggest that CPEB3 can act as a repressor in the basal state and can be converted to an activator in aggregation by post-translational modification.
Similar to Aplysia, mouse CPEB3 forms aggregates upon synaptic activation in culture as well as performing a behavioral task in vivo. Moreover the dual role in translation, the switch from repression to activation, is correlated with change of CPEB3 from a soluble to an aggregated form. The propensity of CPEB3 to form aggregates derive from its N-terminus domain which comprises two regions rich in glutamine and a low complexity sequence which is predicted to be poorly structured and to form aggregates.
To determine the role of CPEB3 in the persistence of synaptic plasticity and memory we generated a conditional knockout strain of CPEB3. We found that CPEB3-mediated protein synthesis is required for maintenance, but not for memory acquisition. We also found that CPEB3 loses its ability to maintain long-term synaptic plasticity and long-term memory if its prion-like N-terminus domain is deleted. Therefore, like the Aplysia CPEB, CPEB3 can sustain the persistence of memory through a stimulus-induced conformation change, which causes protein aggregation and a change in function that allows enhanced translation of CPEB3 target mRNAs. These results provide the first evidence for a prion-like mechanisms to sustain memory in the mouse brain, during consolidation and maintenance.
The dominant nature of the CPEB3 aggregates characteristic of other CPEB related functional prions led us to next search for inhibitory constraints that might be important in regulating aggregate formation. We found that Small-ubiquitin-like modifier or SUMOylation of CPEB3 acts on an inhibitory constraint. In its basal state CPEB3 is SUMOylated in hippocampal neurons and in its SUMOylated form CPEB3 is monomeric and acts as a repressor of translation. Following neuronal stimulation, CPEB3 is converted into an active form, which is associated with a decrease in SUMOylation and an increase of aggregation.
Since SUMOylation keeps it in an inactive state what activates CPEB3? We found that CPEB3 is activated by Neuralized1, an E3 ubiquitin ligase. Overexpressing Neuralized1 specifically in the forebrain increases the levels of monomeric CPEB3 in the hippocampus, without affecting CPEB-1 and CPEB-4. We found that CPEB3 interacts with Neuralized1 via its N-terminal, prion-like domain, and that this interaction leads to the monoubiquitination and consequent activation of CPEB3. Strikingly, overexpression of Neuralized1 activates CPEB3 in cultured hippocampal neurons.
These results suggest a model whereby Neuralized1-mediated ubiquination facilitates hippocampal plasticity and hippocampal-dependent memory storage by modulating the activity of CPEB3 and CPEB3-dependent protein synthesis. In response to synaptic activity, the protein levels of Neuralized1 are increased, leading to the ubiquination and activation of CPEB3, and consequent production of synaptic components critical for the formation of new functional synaptic connections. Since CPEB3 can be SUMOylated as well as ubiquitinated the relationship between these two post-translational modification is of interest.
Learned Fear and Learned Safety in the Mouse
Fear in mice, monkeys, and people is mediated by the amygdala, a structure that lies deep within the cerebral cortex. To develop a molecular approach to learned fear in the mouse, we identified two genes 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. One of these, the Grp gene, encodes gastrin-releasing peptide. We next found that the GRP receptor (GRPR) is expressed in GABAergic interneurons of the lateral nucleus. GRP excites these interneurons and increases their inhibition of the principal neurons of the nucleus. 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 the 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 learned fear.
We also have identified a second gene, stathmin, an inhibitor of microtubule formation, as highly expressed in the lateral nucleus of the amygdala as well as in the thalamic and cortical structures that send information to the lateral nucleus about the conditioned (learned fear) and unconditioned (innate) fear. Mice deficient in stathmin show a deficit in LTP. The knockout mice are bold—they exhibit decreased memory in amygdala-dependent fear conditioning and fail to recognize danger in innately aversive environments. These mice also 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 essential in regulating both innate and learned fear.
We have explored the opposite of fear—safety and security. The ability to identify, develop, and exploit conditions of safety and security is central to survival and mental health, but little is known of the neurobiology of these processes or associated positive modulations of affective state. We have studied electrophysiological and affective correlates of learned safety by negatively correlating an auditory conditioned stimulus (CS). This CS came to signify a period of protection, reducing fear responses to predictors of the US and increasing adventurous exploration of a novel environment. In nonaversive conditions, mice turn on the CS when given the opportunity. Thus, conditioned safety involves a reduction of learned and instinctive fear, as well as positive affective responses. In concurrent electrophysiological measurements, we have identified a safety learning-induced long-lasting depression of CS-evoked activity in the lateral nucleus of the amygdala, consistent with fear reduction, and an increase of CS-evoked activity in a region of the striatum involved in positive affect, euphoric responses, and reward.
A Reductionist Approach to Attention
The hippocampal formation plays a critical role in the acquisition and consolidation of memories. When recorded in freely moving animals, hippocampal pyramidal neurons fire in a location-specific manner; they are "place" cells, and are thought to generate an internal representation of space. To explore the relationship between place cells and spatial memory, we recorded from the hippocampal pyramidal cells of mice under various degrees of task demands. We found that long-term stability of place cells correlates with the degree of task demands and that successful performance of a spatial task is associated with stable place fields. This suggests that the storage and retrieval of place cells is modulated by a top-down cognitive process resembling attention. Consistent with the idea of an attention-like process, conditions that maximize place field stability greatly increase orientation to novel cues. These results suggest that place cells are neural correlates of spatial memory and that the rodent analog of selective attention modulates place field stability. We implicate dopamine in this process and suggest a learning model wherein attention recruits a neuromodulatory input which switches short-term homosynaptic plasticity to long-term heterosynaptic plasticity.
As of March 10, 2016