Experience-Regulated Neuronal Signaling in Maternal Behavior.

Maternal behavior is shaped and challenged by the changing developmental needs of offspring and a broad range of environmental factors, with evidence indicating that the maternal brain exhibits a high degree of plasticity. This plasticity is displayed within cellular and molecular systems, including both intra- and intercellular signaling processes as well as transcriptional profiles. This experience-associated plasticity may have significant overlap with the mechanisms controlling memory processes, in particular those that are activity-dependent. While a significant body of work has identified various molecules and intracellular processes regulating maternal care, the role of activity- and experience-dependent processes remains unclear. We discuss recent progress in studying activity-dependent changes occurring at the synapse, in the nucleus, and during the transport between these two structures in relation to maternal behavior. Several pre- and postsynaptic molecules as well as transcription factors have been found to be critical in these processes. This role reflects the principal importance of the molecular and cellular mechanisms of memory formation to maternal and other behavioral adaptations.

Brain and blood biomarkers of tauopathy and neuronal injury in humans and rats with neurobehavioral syndromes following blast exposure.

Traumatic brain injury (TBI) is a risk factor for the later development of neurodegenerative diseases that may have various underlying pathologies. Chronic traumatic encephalopathy (CTE) in particular is associated with repetitive mild TBI (mTBI) and is characterized pathologically by aggregation of hyperphosphorylated tau into neurofibrillary tangles (NFTs). CTE may be suspected when behavior, cognition, and/or memory deteriorate following repetitive mTBI. Exposure to blast overpressure from improvised explosive devices (IEDs) has been implicated as a potential antecedent for CTE amongst Iraq and Afghanistan Warfighters. In this study, we identified biomarker signatures in rats exposed to repetitive low-level blast that develop chronic anxiety-related traits and in human veterans exposed to IED blasts in theater with behavioral, cognitive, and/or memory complaints. Rats exposed to repetitive low-level blasts accumulated abnormal hyperphosphorylated tau in neuronal perikarya and perivascular astroglial processes. Using positron emission tomography (PET) and the [18F]AV1451 (flortaucipir) tau ligand, we found that five of 10 veterans exhibited excessive retention of [18F]AV1451 at the white/gray matter junction in frontal, parietal, and temporal brain regions, a typical localization of CTE tauopathy. We also observed elevated levels of neurofilament light (NfL) chain protein in the plasma of veterans displaying excess [18F]AV1451 retention. These findings suggest an association linking blast injury, tauopathy, and neuronal injury. Further study is required to determine whether clinical, neuroimaging, and/or fluid biomarker signatures can improve the diagnosis of long-term neuropsychiatric sequelae of mTBI.

Internal Clocks, mGluR7 and Microtubules: A Primer for the Molecular Encoding of Target Durations in Cerebellar Purkinje Cells and Striatal Medium Spiny Neurons.

The majority of studies in the field of timing and time perception have generally focused on sub- and supra-second time scales, specific behavioral processes, and/or discrete neuronal circuits. In an attempt to find common elements of interval timing from a broader perspective, we review the literature and highlight the need for cell and molecular studies that can delineate the neural mechanisms underlying temporal processing. Moreover, given the recent attention to the function of microtubule proteins and their potential contributions to learning and memory consolidation/re-consolidation, we propose that these proteins play key roles in coding temporal information in cerebellar Purkinje cells (PCs) and striatal medium spiny neurons (MSNs). The presence of microtubules at relevant neuronal sites, as well as their adaptability, dynamic structure, and longevity, makes them a suitable candidate for neural plasticity at both intra- and inter-cellular levels. As a consequence, microtubules appear capable of maintaining a temporal code or engram and thereby regulate the firing patterns of PCs and MSNs known to be involved in interval timing. This proposed mechanism would control the storage of temporal information triggered by postsynaptic activation of mGluR7. This, in turn, leads to alterations in microtubule dynamics through a "read-write" memory process involving alterations in microtubule dynamics and their hexagonal lattice structures involved in the molecular basis of temporal memory.

Epigenetic regulation of Fgf1 transcription by CRTC1 and memory enhancement.

Recent evidence demonstrates that epigenetic regulation of gene transcription is critically involved in learning and memory. Here, we discuss the role of histone acetylation and DNA methylation, which are two best understood epigenetic processes in memory processes. More specifically, we focus on learning-strength-dependent changes in chromatin on the fibroblast growth factor 1 (Fgf1) gene and on the molecular events that modulate regulation of Fgf1 transcription, required for memory enhancement, with the specific focus on CREB-regulated transcription coactivator 1 (CRTC1).

Synaptically Localized Transcriptional Regulators in Memory Formation.

At the neuronal cell level, long-term memory formation emerges from interactions between initial activity-dependent molecular changes at the synapse and subsequent regulation of gene transcription in the nucleus. This in turn leads to strengthening of the connections back at the synapse that received the initial signal. However, the mechanisms through which this synapse-to-nucleus molecular exchange occurs remain poorly understood. Here we discuss recent studies that delineate nucleocytoplasmic transport of a special class of synaptically localized transcriptional regulators that upon receiving initial external signal by the synapse move to the nucleus to modulate gene transcription.

Susceptibility and Resilience to Posttraumatic Stress Disorder-like Behaviors in Inbred Mice.

BACKGROUND: The limited neurobiological understanding of posttraumatic stress disorder (PTSD) has been partially attributed to the need for improved animal models. Stress-enhanced fear learning (SEFL) in rodents recapitulates many PTSD-associated behaviors, including stress-susceptible and stress-resilient subgroups in outbred rats. Identification of subgroups requires additional behavioral phenotyping, a confound to mechanistic studies. METHODS: We employed a SEFL paradigm in inbred male and female C57BL/6 mice that combines acute stress with fear conditioning to precipitate traumatic-like memories. Extinction and long-term retention of extinction were examined after SEFL. Further characterization of SEFL effects on male mice was performed with additional behavioral tests, determination of regional activation by Fos immunofluorescence, and RNA sequencing of the basolateral amygdala. RESULTS: Stressed animals displayed persistently elevated freezing during extinction. While more uniform in females, SEFL produced male subgroups with differential susceptibility that were identified without posttraining phenotyping. Additional phenotyping of male mice revealed PTSD-associated behaviors, including extinction-resistant fear memory, hyperarousal, generalization, and dysregulated corticosterone in stress-susceptible male mice. Altered Fos activation was also seen in the infralimbic cortex and basolateral amygdala of stress-susceptible male mice after remote memory retrieval. Key behavioral outcomes, including susceptibility, were replicated by two independent laboratories. RNA sequencing of the basolateral amygdala revealed transcriptional divergence between the male subgroups, including genes with reported polymorphic association to patients with PTSD. CONCLUSIONS: This SEFL model provides a tool for development of PTSD therapeutics that is compatible with the growing number of mouse-specific resources. Furthermore, use of an inbred strain allows for investigation into epigenetic mechanisms that are expected to critically regulate susceptibility and resilience.

CRTC1 Nuclear Translocation Following Learning Modulates Memory Strength via Exchange of Chromatin Remodeling Complexes on the Fgf1 Gene.

Memory is formed by synapse-to-nucleus communication that leads to regulation of gene transcription, but the identity and organizational logic of signaling pathways involved in this communication remain unclear. Here we find that the transcription cofactor CRTC1 is a critical determinant of sustained gene transcription and memory strength in the hippocampus. Following associative learning, synaptically localized CRTC1 is translocated to the nucleus and regulates Fgf1b transcription in an activity-dependent manner. After both weak and strong training, the HDAC3-N-CoR corepressor complex leaves the Fgf1b promoter and a complex involving the translocated CRTC1, phosphorylated CREB, and histone acetyltransferase CBP induces transient transcription. Strong training later substitutes KAT5 for CBP, a process that is dependent on CRTC1, but not on CREB phosphorylation. This in turn leads to long-lasting Fgf1b transcription and memory enhancement. Thus, memory strength relies on activity-dependent changes in chromatin and temporal regulation of gene transcription on specific CREB/CRTC1 gene targets.

Genetic Demonstration of a Role for Stathmin in Adult Hippocampal Neurogenesis, Spinogenesis, and NMDA Receptor-Dependent Memory.

Neurogenesis and memory formation are essential features of the dentate gyrus (DG) area of the hippocampus, but to what extent the mechanisms responsible for both processes overlap remains poorly understood. Stathmin protein, whose tubulin-binding and microtubule-destabilizing activity is negatively regulated by its phosphorylation, is prominently expressed in the DG. We show here that stathmin is involved in neurogenesis, spinogenesis, and memory formation in the DG. tTA/tetO-regulated bitransgenic mice, expressing the unphosphorylatable constitutively active Stathmin4A mutant (Stat4A), exhibit impaired adult hippocampal neurogenesis and reduced spine density in the DG granule neurons. Although Stat4A mice display deficient NMDA receptor-dependent memory in contextual discrimination learning, which is dependent on hippocampal neurogenesis, their NMDA receptor-independent memory is normal. Confirming NMDA receptor involvement in the memory deficits, Stat4A mutant mice have a decrease in the level of synaptic NMDA receptors and a reduction in learning-dependent CREB-mediated gene transcription. The deficits in neurogenesis, spinogenesis, and memory in Stat4A mice are not present in mice in which tTA/tetO-dependent transgene transcription is blocked by doxycycline through their life. The memory deficits are also rescued within 3 d by intrahippocampal infusion of doxycycline, further indicating a role for stathmin expressed in the DG in contextual memory. Our findings therefore point to stathmin and microtubules as a mechanistic link between neurogenesis, spinogenesis, and NMDA receptor-dependent memory formation in the DG. SIGNIFICANCE STATEMENT: In the present study, we aimed to clarify the role of stathmin in neuronal and behavioral functions. We characterized the neurogenic, behavioral, and molecular consequences of the gain-of-function stathmin mutation using a bitransgenic mouse expressing a constitutively active form of stathmin. We found that stathmin plays an important role in adult hippocampal neurogenesis and spinogenesis. In addition, stathmin mutation led to impaired NMDA receptor-dependent and neurogenesis-associated memory and did not affect NMDA receptor-independent memory. Moreover, biochemical analysis suggested that stathmin regulates the synaptic transport of NMDA receptors, which in turn influence CREB-mediated gene transcription machinery. Overall, these data suggest that stathmin is an important molecule for neurogenesis, spinogenesis, and NMDA receptor-dependent learning and memory.

Deceivingly dynamic: Learning-dependent changes in stathmin and microtubules.

Microtubules, one of the major cytoskeletal structures, were previously considered stable and only indirectly involved in synaptic structure and function in mature neurons. However, recent evidence demonstrates that microtubules are dynamic and have an important role in synaptic structure, synaptic plasticity, and memory. In particular, learning induces changes in microtubule turnover and stability, and pharmacological manipulation of microtubule dynamics alters synaptic plasticity and long-term memory. These learning-induced changes in microtubules are controlled by the phosphoprotein stathmin, whose only known cellular activity is to negatively regulate microtubule formation. During the first eight hours following learning, changes in the phosphorylation of stathmin go through two phases causing biphasic shifts in microtubules stability/instability. These shifts, in turn, regulate memory formation by controlling in the second phase synaptic transport of the GluA2 subunit of AMPA receptors. Improper regulation of stathmin and microtubule dynamics has been observed in aged animals and in patients with Alzheimer's disease and depression. Thus, recent work on stathmin and microtubules has identified new molecular players in the early stages of memory encoding.

Quantifying resilience: Theoretical or pragmatic for translational research?

Quantifying resilience allows for several testable hypotheses, such as that resilience is equal to the number of mental health problems given a known quantity of stressor load. The proposed model lends itself well to prospective studies with data collection pre- and post-adversity; however, prestressor assessments are not always available. Challenges remain for adapting quantifying resilience to animal research, even if the idea of its translation value is significant.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.