Track-by-Day: A standardized approach to estrous cycle monitoring in biobehavioral research.

Despite known sex differences in brain function, female subjects are underrepresented in preclinical neuroscience research. This is driven in part by concerns about variability arising from estrous cycle-related hormone fluctuations, especially in fear- and anxiety-related research where there are conflicting reports as to whether and how the cycle influences behavior. The inconsistency may arise from a lack of common standards for tracking and reporting the cycle as opposed to inherent unpredictability in the cycle itself. The rat estrous cycle is conventionally tracked by assigning vaginal cytology smears to one of four qualitatively-defined stages. Although the cytology stages are of unequal length, the stage names are often, but not always, used to refer to the four cycle days. Subjective staging criteria and inconsistent use of terminology are not necessarily a problem in research on the cycle itself, but can lead to irreproducibility in neuroscience studies that treat the stages as independent grouping factors. We propose the explicit use of cycle days as independent variables, which we term Track-by-Day to differentiate it from traditional stage-based tracking, and that days be indexed to the only cytology feature that is a direct and rapid consequence of a hormonal event: a cornified cell layer formed in response to the pre-ovulatory 17ß-estradiol peak. Here we demonstrate that cycle length is robustly regular with this method, and that the method outperforms traditional staging in detecting estrous cycle effects on Pavlovian fear conditioning and on a separate proxy for hormonal changes, uterine histology.

Rehydration of Freeze Substituted Brain Tissue for Pre-embedding Immunoelectron Microscopy.

Electron microscopy (EM) volume reconstruction is a powerful tool for investigating the fundamental structure of brain circuits, but the full potential of this technique is limited by the difficulty of integrating molecular information. High quality ultrastructural preservation is necessary for EM reconstruction, and intact, highly contrasted cell membranes are essential for following small neuronal processes through serial sections. Unfortunately, the antibody labeling methods used to identify most endogenous molecules result in compromised morphology, especially of membranes. Cryofixation can produce superior morphological preservation and has the additional advantage of allowing indefinite storage of valuable samples. We have developed a method based on cryofixation that allows sensitive immunolabeling of endogenous molecules, preserves excellent ultrastructure, and is compatible with high-contrast staining for serial EM reconstruction.

The Track-by-Day Method for Monitoring the Rodent Estrous Cycle.

The exclusion of female subjects from preclinical neuroscience research has traditionally been justified in part by concerns about potential effects of cycling ovarian hormones on brain function. There is evidence that some behavioral and neurobiological measures do change over the estrous cycle and, as the use of female subjects becomes increasingly routine, there is a greater demand for accessible cycle-tracking methods. Conventional estrous cycle staging requires expert training in the qualitative interpretation of vaginal cytology smears, which serves as a barrier for novice researchers. In addition, definitions and reporting practices are not standardized across laboratories, which makes it difficult to compare results across studies and likely contributes to a false perception of the cycle as ephemeral and inconsistent. Here, we describe a streamlined method for monitoring the estrous cycle in rats, which we term Track-by-Day. It is simple to implement and inherently produces consistent reporting. Our protocol should serve to demystify and facilitate adoption of cycle tracking for those new to the practice. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Collection and staining of vaginal smears Basic Protocol 2: Track-by-Day classification of vaginal smears Support Protocol: Preparation of gelatin-subbed slides.

Automated classification of estrous stage in rodents using deep learning.

The rodent estrous cycle modulates a range of biological functions, from gene expression to behavior. The cycle is typically divided into four stages, each characterized by distinct hormone concentration profiles. Given the difficulty of repeatedly sampling plasma steroid hormones from rodents, the primary method for classifying estrous stage is by identifying vaginal epithelial cell types. However, manual classification of epithelial cell samples is time-intensive and variable, even amongst expert investigators. Here, we use a deep learning approach to achieve classification accuracy at expert level. Due to the heterogeneity and breadth of our input dataset, our deep learning approach ("EstrousNet") is highly generalizable across rodent species, stains, and subjects. The EstrousNet algorithm exploits the temporal dimension of the hormonal cycle by fitting classifications to an archetypal cycle, highlighting possible misclassifications and flagging anestrus phases (e.g., pseudopregnancy). EstrousNet allows for rapid estrous cycle staging, improving the ability of investigators to consider endocrine state in their rodent studies.

Persistent up-regulation of polyribosomes at synapses during long-term memory, reconsolidation, and extinction of associative memory.

Local protein synthesis at synapses can provide a rapid supply of proteins to support synaptic changes during consolidation of new memories, but its role in the maintenance or updating of established memories is unknown. Consolidation requires new protein synthesis in the period immediately following learning, whereas established memories are resistant to protein synthesis inhibitors. We have previously reported that polyribosomes are up-regulated in the lateral amygdala (LA) during consolidation of aversive-cued Pavlovian conditioning. In this study, we used serial section electron microscopy reconstructions to determine whether the distribution of dendritic polyribosomes returns to baseline during the long-term memory phase. Relative to control groups, long-term memory was associated with up-regulation of polyribosomes throughout dendrites, including in dendritic spines of all sizes. Retrieval of a consolidated memory by presentation of a small number of cues induces a new, transient requirement for protein synthesis to maintain the memory, while presentation of a large number of cues results in extinction learning, forming a new memory. One hour after retrieval or extinction training, the distribution of dendritic polyribosomes was similar except in the smallest spines, which had more polyribosomes in the extinction group. Our results demonstrate that the effects of learning on dendritic polyribosomes are not restricted to the transient translation-dependent phase of memory formation. Cued Pavlovian conditioning induces persistent synapse strengthening in the LA that is not reversed by retrieval or extinction, and dendritic polyribosomes may therefore correlate generally with synapse strength as opposed to recent activity or transient translational processes.

Axon TRAP reveals learning-associated alterations in cortical axonal mRNAs in the lateral amgydala.

Local translation can support memory consolidation by supplying new proteins to synapses undergoing plasticity. Translation in adult forebrain dendrites is an established mechanism of synaptic plasticity and is regulated by learning, yet there is no evidence for learning-regulated protein synthesis in adult forebrain axons, which have traditionally been believed to be incapable of translation. Here, we show that axons in the adult rat amygdala contain translation machinery, and use translating ribosome affinity purification (TRAP) with RNASeq to identify mRNAs in cortical axons projecting to the amygdala, over 1200 of which were regulated during consolidation of associative memory. Mitochondrial and translation-related genes were upregulated, whereas synaptic, cytoskeletal, and myelin-related genes were downregulated; the opposite effects were observed in the cortex. Our results demonstrate that axonal translation occurs in the adult forebrain and is altered after learning, supporting the likelihood that local translation is more a rule than an exception in neuronal processes.

Shifting patterns of polyribosome accumulation at synapses over the course of hippocampal long-term potentiation.

Hippocampal long-term potentiation (LTP) is a cellular memory mechanism. For LTP to endure, new protein synthesis is required immediately after induction and some of these proteins must be delivered to specific, presumably potentiated, synapses. Local synthesis in dendrites could rapidly provide new proteins to synapses, but the spatial distribution of translation following induction of LTP is not known. Here, we quantified polyribosomes, the sites of local protein synthesis, in CA1 stratum radiatum dendrites and spines from postnatal day 15 rats. Hippocampal slices were rapidly fixed at 5, 30, or 120 min after LTP induction by theta-burst stimulation (TBS). Dendrites were reconstructed through serial section electron microscopy from comparable regions near the TBS or control electrodes in the same slice, and in unstimulated hippocampus that was perfusion-fixed in vivo. At 5 min after induction of LTP, polyribosomes were elevated in dendritic shafts and spines, especially near spine bases and in spine heads. At 30 min, polyribosomes remained elevated only in spine bases. At 120 min, both spine bases and spine necks had elevated polyribosomes. Polyribosomes accumulated in spines with larger synapses at 5 and 30 min, but not at 120 min. Small spines, meanwhile, proliferated dramatically by 120 min, but these largely lacked polyribosomes. The number of ribosomes per polyribosome is variable and may reflect differences in translation regulation. In dendritic spines, but not shafts, there were fewer ribosomes per polyribosome in the slice conditions relative to in vivo, but this recovered transiently in the 5 min LTP condition. Overall, our data show that LTP induces a rapid, transient upregulation of large polyribosomes in larger spines, and a persistent upregulation of small polyribosomes in the bases and necks of small spines. This is consistent with local translation supporting enlargement of potentiated synapses within minutes of LTP induction.

Accumulation of Polyribosomes in Dendritic Spine Heads, But Not Bases and Necks, during Memory Consolidation Depends on Cap-Dependent Translation Initiation.

Translation in dendrites is believed to support synaptic changes during memory consolidation. Although translational control mechanisms are fundamental mediators of memory, little is known about their role in local translation. We previously found that polyribosomes accumulate in dendritic spines of the adult rat lateral amygdala (LA) during consolidation of aversive pavlovian conditioning and that this memory requires cap-dependent initiation, a primary point of translational control in eukaryotic cells. Here we used serial electron microscopy reconstructions to quantify polyribosomes in LA dendrites when consolidation was blocked by the cap-dependent initiation inhibitor 4EGI-1. We found that 4EGI-1 depleted polyribosomes in dendritic shafts and selectively prevented their upregulation in spine heads, but not bases and necks, during consolidation. Cap-independent upregulation was specific to spines with small, astrocyte-associated synapses. Our results reveal that cap-dependent initiation is involved in local translation during learning and that local translational control varies with synapse type.SIGNIFICANCE STATEMENT Translation initiation is a central regulator of long-term memory formation. Local translation in dendrites supports memory by providing necessary proteins at synaptic sites, but it is unknown whether this requires initiation or bypasses it. We used serial electron microscopy reconstructions to examine polyribosomes in dendrites when memory formation was blocked by an inhibitor of translation initiation. This revealed two major pools of polyribosomes that were upregulated during memory formation: one pool in dendritic spine heads that was initiation dependent and another pool in the bases and necks of small spines that was initiation independent. Thus, translation regulation differs between spine types and locations, and translation that occurs closest to individual synapses during memory formation is initiation dependent.

Mitochondrial support of persistent presynaptic vesicle mobilization with age-dependent synaptic growth after LTP.

Mitochondria support synaptic transmission through production of ATP, sequestration of calcium, synthesis of glutamate, and other vital functions. Surprisingly, less than 50% of hippocampal CA1 presynaptic boutons contain mitochondria, raising the question of whether synapses without mitochondria can sustain changes in efficacy. To address this question, we analyzed synapses from postnatal day 15 (P15) and adult rat hippocampus that had undergone theta-burst stimulation to produce long-term potentiation (TBS-LTP) and compared them to control or no stimulation. At 30 and 120 min after TBS-LTP, vesicles were decreased only in presynaptic boutons that contained mitochondria at P15, and vesicle decrement was greatest in adult boutons containing mitochondria. Presynaptic mitochondrial cristae were widened, suggesting a sustained energy demand. Thus, mitochondrial proximity reflected enhanced vesicle mobilization well after potentiation reached asymptote, in parallel with the apparently silent addition of new dendritic spines at P15 or the silent enlargement of synapses in adults.

Synapses lacking astrocyte appear in the amygdala during consolidation of Pavlovian threat conditioning.

There is growing evidence that astrocytes, long held to merely provide metabolic support in the adult brain, participate in both synaptic plasticity and learning and memory. Astrocytic processes are sometimes present at the synaptic cleft, suggesting that they might act directly at individual synapses. Associative learning induces synaptic plasticity and morphological changes at synapses in the lateral amygdala (LA). To determine whether astrocytic contacts are involved in these changes, we examined LA synapses after either threat conditioning (also called fear conditioning) or conditioned inhibition in adult rats by using serial section transmission electron microscopy (ssTEM) reconstructions. There was a transient increase in the density of synapses with no astrocytic contact after threat conditioning, especially on enlarged spines containing both polyribosomes and a spine apparatus. In contrast, synapses with astrocytic contacts were smaller after conditioned inhibition. This suggests that during memory consolidation astrocytic processes are absent if synapses are enlarging but present if they are shrinking. We measured the perimeter of each synapse and its degree of astrocyte coverage, and found that only about 20-30% of each synapse was ensheathed. The amount of synapse perimeter surrounded by astrocyte did not scale with synapse size, giving large synapses a disproportionately long astrocyte-free perimeter and resulting in a net increase in astrocyte-free perimeter after threat conditioning. Thus astrocytic processes do not mechanically isolate LA synapses, but may instead interact through local signaling, possibly via cell-surface receptors. Our results suggest that contact with astrocytic processes opposes synapse growth during memory consolidation.

CYFIP1 coordinates mRNA translation and cytoskeleton remodeling to ensure proper dendritic spine formation.

The CYFIP1/SRA1 gene is located in a chromosomal region linked to various neurological disorders, including intellectual disability, autism, and schizophrenia. CYFIP1 plays a dual role in two apparently unrelated processes, inhibiting local protein synthesis and favoring actin remodeling. Here, we show that brain-derived neurotrophic factor (BDNF)-driven synaptic signaling releases CYFIP1 from the translational inhibitory complex, triggering translation of target mRNAs and shifting CYFIP1 into the WAVE regulatory complex. Active Rac1 alters the CYFIP1 conformation, as demonstrated by intramolecular FRET, and is key in changing the equilibrium of the two complexes. CYFIP1 thus orchestrates the two molecular cascades, protein translation and actin polymerization, each of which is necessary for correct spine morphology in neurons. The CYFIP1 interactome reveals many interactors associated with brain disorders, opening new perspectives to define regulatory pathways shared by neurological disabilities characterized by spine dysmorphogenesis.

Inhibition of fear by learned safety signals: a mini-symposium review.

Safety signals are learned cues that predict the nonoccurrence of an aversive event. As such, safety signals are potent inhibitors of fear and stress responses. Investigations of safety signal learning have increased over the last few years due in part to the finding that traumatized persons are unable to use safety cues to inhibit fear, making it a clinically relevant phenotype. The goal of this review is to present recent advances relating to the neural and behavioral mechanisms of safety learning, and expression in rodents, nonhuman primates, and humans.

Stability of presynaptic vesicle pools and changes in synapse morphology in the amygdala following fear learning in adult rats.

Changes in synaptic strength in the lateral amygdala (LA) that occur with fear learning are believed to mediate memory storage, and both presynaptic and postsynaptic mechanisms have been proposed to contribute. In a previous study we used serial section transmission electron microscopy (ssTEM) to observe differences in dendritic spine morphology in the adult rat LA after fear conditioning, conditioned inhibition (safety conditioning), or naïve control handling (Ostroff et al. [2010] Proc Natl Acad Sci U S A 107:9418-9423). We have now reconstructed axons from the same dataset and compared their morphology and relationship to the postsynaptic spines between the three training groups. Relative to the naïve control and conditioned inhibition groups, the ratio of postsynaptic density (PSD) area to docked vesicles at synapses was greater in the fear-conditioned group, while the size of the synaptic vesicle pools was unchanged. There was significant coherence in synapse size between neighboring boutons on the same axon in the naïve control and conditioned inhibition groups, but not in the fear-conditioned group. Within multiple-synapse boutons, both synapse size and the PSD-to-docked vesicle ratio were variable between individual synapses. Our results confirm that synaptic connectivity increases in the LA with fear conditioning. In addition, we provide evidence that boutons along the same axon and even synapses on the same bouton are independent in their structure and learning-related morphological plasticity.

Molecular mechanisms of fear learning and memory.

Pavlovian fear conditioning is a particularly useful behavioral paradigm for exploring the molecular mechanisms of learning and memory because a well-defined response to a specific environmental stimulus is produced through associative learning processes. Synaptic plasticity in the lateral nucleus of the amygdala (LA) underlies this form of associative learning. Here, we summarize the molecular mechanisms that contribute to this synaptic plasticity in the context of auditory fear conditioning, the form of fear conditioning best understood at the molecular level. We discuss the neurotransmitter systems and signaling cascades that contribute to three phases of auditory fear conditioning: acquisition, consolidation, and reconsolidation. These studies suggest that multiple intracellular signaling pathways, including those triggered by activation of Hebbian processes and neuromodulatory receptors, interact to produce neural plasticity in the LA and behavioral fear conditioning. Collectively, this body of research illustrates the power of fear conditioning as a model system for characterizing the mechanisms of learning and memory in mammals and potentially for understanding fear-related disorders, such as PTSD and phobias.

Fear and safety learning differentially affect synapse size and dendritic translation in the lateral amygdala.

Fear learning is associated with changes in synapse strength in the lateral amygdala (LA). To examine changes in LA dendritic spine structure with learning, we used serial electron microscopy to re-construct dendrites after either fear or safety conditioning. The spine apparatus, a smooth endoplasmic reticulum (sER) specialization found in very large spines, appeared more frequently after fear conditioning. Fear conditioning was associated with larger synapses on spines that did not contain a spine apparatus, whereas safety conditioning resulted in smaller synapses on these spines. Synapses on spines with a spine apparatus were smaller after safety conditioning but unchanged with fear conditioning, suggesting a ceiling effect. There were more polyribosomes and multivesicular bodies throughout the dendrites from fear conditioned rats, indicating increases in both protein synthesis and degradation. Polyribosomes were associated with the spine apparatus under both training conditions. We conclude that LA synapse size changes bidirectionally with learning and that the spine apparatus has a central role in regulating synapse size and local translation.

Polyribosomes redistribute from dendritic shafts into spines with enlarged synapses during LTP in developing rat hippocampal slices.

The presence of polyribosomes in dendritic spines suggests a potential involvement of local protein synthesis in the modification of synapses. Dendritic spine and synapse ultrastructure were compared after low-frequency control or tetanic stimulation in hippocampal slices from postnatal day (P)15 rats. The percentage of spines containing polyribosomes increased from 12% +/- 4% after control stimulation to 39% +/- 4% after tetanic stimulation, with a commensurate loss of polyribosomes from dendritic shafts at 2 hr posttetanus. Postsynaptic densities on spines containing polyribosomes were larger after tetanic stimulation. Local protein synthesis might therefore serve to stabilize stimulation-induced growth of the postsynaptic density. Furthermore, coincident polyribosomes and synapse enlargement might indicate spines that are expressing long-term potentiation induced by tetanic stimulation.