The Precision of Place Fields Governs Their Fate across Epochs of Experience.

Spatial memories are represented by hippocampal place cells during navigation. This spatial code is dynamic, undergoing changes across time, known as representational drift, and across changes in internal state, even while navigating the same spatial environment with consistent behavior. A dynamic code may provide the hippocampus a means to track distinct epochs of experience that occur at different times or during different internal states and update spatial memories. Changes to the spatial code include place fields (PFs) that remap to new locations and place fields that vanish, while others are stable. However, what determines place field fate across epochs remains unclear. We measured the lap-by-lap properties of place cells in mice during navigation for a block of trials in a rewarded virtual environment. We then determined the position of the place fields in another block of trials in the same spatial environment either separated by a day (a distinct temporal epoch) or during the same session but with reward removed to change reward expectation (a distinct internal state epoch). We found that place cells with remapped place fields across epochs tended to have lower spatial precision during navigation in the initial epoch. Place cells with stable or vanished place fields tended to have higher spatial precision. We conclude that place cells with less precise place fields have greater spatial flexibility, allowing them to respond to, and track, distinct epochs of experience in the same spatial environment, while place cells with precise place fields generally preserve spatial information when their fields reappear.

A thalamic-hippocampal CA1 signal for contextual fear memory suppression, extinction, and discrimination.

The adaptive regulation of fear memories is a crucial neural function that prevents inappropriate fear expression. Fear memories can be acquired through contextual fear conditioning (CFC) which relies on the hippocampus. The thalamic nucleus reuniens (NR) is necessary to extinguish contextual fear and innervates hippocampal CA1. However, the role of the NR-CA1 pathway in contextual fear is unknown. We developed a head-restrained virtual reality CFC paradigm, and demonstrate that mice can acquire and extinguish context-dependent fear responses. We found that inhibiting the NR-CA1 pathway following CFC lengthens the duration of fearful freezing epochs, increases  fear generalization, and delays fear extinction. Using in vivo imaging, we recorded NR-axons innervating CA1 and found that NR-axons become tuned to fearful freezing following CFC. We conclude that the NR-CA1 pathway actively suppresses fear by disrupting contextual fear memory retrieval in CA1 during fearful freezing behavior, a process that also reduces fear generalization and accelerates extinction.

Direct Thalamic Inputs to Hippocampal CA1 Transmit a Signal That Suppresses Ongoing Contextual Fear Memory Retrieval.

Memory retrieval of fearful experiences is essential for survival but can be maladaptive if not appropriately suppressed. Fear memories can be acquired through contextual fear conditioning (CFC) which relies on the hippocampus. The thalamic subregion Nucleus Reuniens (NR) is necessary for contextual fear extinction and strongly projects to hippocampal subregion CA1. However, the NR-CA1 pathway has not been investigated during behavior, leaving unknown its role in contextual fear memory retrieval. We implement a novel head-restrained virtual reality CFC paradigm and show that inactivation of the NR-CA1 pathway prolongs fearful freezing epochs, induces fear generalization, and delays extinction. We use in vivo sub-cellular imaging to specifically record NR-axons innervating CA1 before and after CFC. We find NR-axons become selectively tuned to freezing only after CFC, and this activity is well-predicted by an encoding model. We conclude that the NR-CA1 pathway actively suppresses fear responses by disrupting ongoing hippocampal-dependent contextual fear memory retrieval.

Direct Thalamic Inputs to Hippocampal CA1 Transmit a Signal That Suppresses Ongoing Contextual Fear Memory Retrieval.

Memory retrieval of fearful experiences is essential for survival but can be maladaptive if not appropriately suppressed. Fear memories can be acquired through contextual fear conditioning (CFC) which relies on the hippocampus. The thalamic subregion Nucleus Reuniens (NR) is necessary for contextual fear extinction and strongly projects to hippocampal subregion CA1. However, the NR-CA1 pathway has not been investigated during behavior, leaving unknown its role in contextual fear memory retrieval. We implement a novel head-restrained virtual reality CFC paradigm and show that inactivation of the NR-CA1 pathway prolongs fearful freezing epochs, induces fear generalization, and delays extinction. We use in vivo sub-cellular imaging to specifically record NR-axons innervating CA1 before and after CFC. We find NR-axons become selectively tuned to freezing only after CFC, and this activity is well-predicted by an encoding model. We conclude that the NR-CA1 pathway actively suppresses fear responses by disrupting ongoing hippocampal-dependent contextual fear memory retrieval.

Place conditioning in humans: opportunities for translational research.

RATIONALE: Translational research, especially research that bridges studies with humans and nonhuman species, is critical to advancing our understanding of human disorders such as addiction. This advancement requires reliable and rigorous models to study the underlying constructs contributing to the maladaptive behavior. OBJECTIVE: In this commentary, we address some of the challenges of conducting translational research by examining a single procedure, place conditioning. Place conditioning is commonly used with laboratory animals to study the conditioned rewarding effects of drugs, and recent studies indicate that a similar procedure can be used in humans. RESULTS: We discuss the opportunities and challenges of making the procedure comparable across species, as well as discuss the benefits of more systematically applying the procedure to humans. CONCLUSION: We argue that the capacity of humans to report verbally on their internal experiences (perceptions, affective states, likes and dislikes) add an important dimension to the understanding of the procedures used in laboratory animals.

Reward Expectation Reduces Representational Drift in the Hippocampus.

Spatial memory in the hippocampus involves dynamic neural patterns that change over days, termed representational drift. While drift may aid memory updating, excessive drift could impede retrieval. Memory retrieval is influenced by reward expectation during encoding, so we hypothesized that diminished reward expectation would exacerbate representational drift. We found that high reward expectation limited drift, with CA1 representations on one day gradually re-emerging over successive trials the following day. Conversely, the absence of reward expectation resulted in increased drift, as the gradual re-emergence of the previous day's representation did not occur. At the single cell level, lowering reward expectation caused an immediate increase in the proportion of place-fields with low trial-to-trial reliability. These place fields were less likely to be reinstated the following day, underlying increased drift in this condition. In conclusion, heightened reward expectation improves memory encoding and retrieval by maintaining reliable place fields that are gradually reinstated across days, thereby minimizing representational drift.

Reward expectation extinction restructures and degrades CA1 spatial maps through loss of a dopaminergic reward proximity signal.

Hippocampal place cells support reward-related spatial memories by forming a cognitive map that over-represents reward locations. The strength of these memories is modulated by the extent of reward expectation during encoding. However, the circuit mechanisms underlying this modulation are unclear. Here we find that when reward expectation is extinguished in mice, they remain engaged with their environment, yet place cell over-representation of rewards vanishes, place field remapping throughout the environment increases, and place field trial-to-trial reliability decreases. Interestingly, Ventral Tegmental Area (VTA) dopaminergic axons in CA1 exhibit a ramping reward-proximity signal that depends on reward expectation and inhibiting VTA dopaminergic neurons largely replicates the effects of extinguishing reward expectation. We conclude that changing reward expectation restructures CA1 cognitive maps and determines map reliability by modulating the dopaminergic VTA-CA1 reward-proximity signal. Thus, internal states of high reward expectation enhance encoding of spatial memories by reinforcing hippocampal cognitive maps associated with reward.

Surveying the experience of postdocs in the United States before and during the COVID-19 pandemic.

In the interest of advocating for the postdoctoral community in the United States (US), we compared the results of surveys of postdocs carried out in 2019 and in late 2020. We found that respondents' mental health and wellness were significantly impacted by the pandemic irrespective of their gender, race, citizenship, or other identities. Career trajectories and progression were also affected, as respondents reported being less confident about achieving career goals, and having more negative perceptions of the job market compared to before the pandemic. Postdocs working in the US on temporary visas reported experiencing increased stress levels due to changes in immigration policy. Access to institutional Postdoctoral Offices or Associations positively impacted well-being and helped mitigate some of the personal and professional stresses caused by the pandemic.

Neural correlates of state transitions elicited by a chemosensory danger cue.

BACKGROUND: Detection of predator cues changes the brain state in prey species and helps them avoid danger. Dysfunctionality in changing the central state appropriately in stressful situations is proposed to be an underlying cause of multiple psychiatric disorders in humans. METHODS: Here, we investigate the dynamics of neural circuits mediating response to a threat, to characterize these states and to identify potential control networks. We use resonant scanning 2-photon microscopy for in vivo brain-wide imaging and custom designed behavioral assays for the study. RESULTS: We first show that 5-7 day old zebrafish larvae react to an alarm pheromone (Schreckstoff) with reduced mobility. They subsequently display heightened vigilance, as evidenced by increased dark avoidance. Calcium imaging indicates that exposure to Schreckstoff elicits stimulus-locked activity in olfactory sensory neurons innervating a lateral glomerulus and in telencephalic regions including the putative medial amygdala and entopeduncular nucleus. Sustained activity outlasting the stimulus delivery was detected in regions regulating neuromodulator release, including the lateral habenula, posterior tuberculum, superior raphe, and locus coeruleus. CONCLUSION: We propose that these latter regions contribute to the network that defines the "threatened" state, while neurons with transient activity serve as the trigger. Our study highlights the utility of the zebrafish larval alarm response system to examine neural circuits during stress dependent brain state transitions and to discover potential therapeutic agents when such transitions are disrupted.

A toxicogenomic approach for the risk assessment of the food contaminant acetamide.

Acetamide (CAS 60-35-5) is detected in common foods. Chronic rodent bioassays led to its classification as a group 2B possible human carcinogen due to the induction of liver tumors in rats. We used a toxicogenomics approach in Wistar rats gavaged daily for 7 or 28 days at doses of 300 to 1500 mg/kg/day (mkd) to determine a point of departure (POD) and investigate its mode of action (MoA). Ki67 labeling was increased at doses ≥750 mkd up to 3.3-fold representing the most sensitive apical endpoint. Differential gene expression analysis by RNA-Seq identified 1110 and 1814 differentially expressed genes in male and female rats, respectively, following 28 days of treatment. Down-regulated genes were associated with lipid metabolism while up-regulated genes included cell signaling, immune response, and cell cycle functions. Benchmark dose (BMD) modeling of the Ki67 labeling index determined the BMD10 lower confidence limit (BMDL10) as 190 mkd. Transcriptional BMD modeling revealed excellent concordance between transcriptional POD and apical endpoints. Collectively, these results indicate that acetamide is most likely acting through a mitogenic MoA, though specific key initiating molecular events could not be elucidated. A POD value of 190 mkd determined for cell proliferation is suggested for risk assessment purposes.

Expression and purification optimization of an N-terminal Pfs230 transmission-blocking vaccine candidate.

In an effort to control and eventually eliminate malaria, the development of transmission-blocking vaccines has long been sought. However, few antigens have been evaluated in clinical trials, often due to limitations in the expression and purification of the antigen in sufficient yield and quality. Pfs230, a surface antigen of gametocytes, has recently advanced to clinical evaluation as a conjugate vaccine using the Pseudomonas aeruginosa exoprotein A carrier protein. Here we continue to build upon prior work of developing a Pfs230 candidate in the baculovirus system, Pfs230C1 (aa 443-731), through systematic process development efforts to improve yield and purity. Various insect cells including High Five, Sf9 and Super Sf9 were first evaluated for quality and quantity of antigen, along with three insect cell media. In the selection of Sf9 cells, an intact Pfs230C1 was expressed and harvested at 48 h for downstream development. A downstream process, utilizing immobilized metal affinity column (IMAC), followed by ion exchange (IEX) membranes (Mustang S) and finally IEX chromatography (DEAE) yielded a pure Pfs230C1 protein. The complete process was repeated three times at the 20 L scale. To support the eventual chemistry manufacturing and controls (CMC) of Pfs230C1, analytical tools, including monoclonal antibodies, were developed to characterize the identity, integrity, and purity of Pfs230C1. These analytical tools, taken in combination with the optimized process, were implemented with Current Good Manufacturing Practices (cGMP) in mind with the ultimate objective of Phase I clinical trials.

Optical inhibition of larval zebrafish behaviour with anion channelrhodopsins.

BACKGROUND: Optical silencing of activity provides a way to test the necessity of neurons in behaviour. Two light-gated anion channels, GtACR1 and GtACR2, have recently been shown to potently inhibit activity in cultured mammalian neurons and in Drosophila. Here, we test the usefulness of these channels in larval zebrafish, using spontaneous coiling behaviour as the assay. RESULTS: When the GtACRs were expressed in spinal neurons of embryonic zebrafish and actuated with blue or green light, spontaneous movement was inhibited. In GtACR1-expressing fish, only 3 µW/mm2 of light was sufficient to have an effect; GtACR2, which is poorly trafficked, required slightly stronger illumination. No inhibition was seen in non-expressing siblings. After light offset, the movement of GtACR-expressing fish increased, which suggested that termination of light-induced neural inhibition may lead to activation. Consistent with this, two-photon imaging of spinal neurons showed that blue light inhibited spontaneous activity in spinal neurons of GtACR1-expressing fish, and that the level of intracellular calcium increased following light offset. CONCLUSIONS: These results show that GtACR1 and GtACR2 can be used to optically inhibit neurons in larval zebrafish with high efficiency. The activity elicited at light offset needs to be taken into consideration in experimental design, although this property can provide insight into the effects of transiently stimulating a circuit.

Characterization of a thalamic nucleus mediating habenula responses to changes in ambient illumination.

BACKGROUND: Neural activity in the vertebrate habenula is affected by ambient illumination. The nucleus that links photoreceptor activity with the habenula is not well characterized. Here, we describe the location, inputs and potential function of this nucleus in larval zebrafish. RESULTS: High-speed calcium imaging shows that light ON and OFF both evoke a rapid response in the dorsal left neuropil of the habenula, indicating preferential targeting of this neuropil by afferents conveying information about ambient illumination. Injection of a lipophilic dye into this neuropil led to bilateral labeling of a nucleus in the anterior thalamus that responds to light ON and OFF, and that receives innervation from the retina and pineal organ. Lesioning the neuropil of this thalamic nucleus reduced the habenula response to light ON and OFF. Optogenetic stimulation of the thalamus with channelrhodopsin-2 caused depolarization in the habenula, while manipulation with anion channelrhodopsins inhibited habenula response to light and disrupted climbing and diving evoked by illumination change. CONCLUSIONS: A nucleus in the anterior thalamus of larval zebrafish innervates the dorsal left habenula. This nucleus receives input from the retina and pineal, responds to increase and decrease in ambient illumination, enables habenula responses to change in irradiance, and may function in light-evoked vertical migration.

Imaging voltage in zebrafish as a route to characterizing a vertebrate functional connectome: promises and pitfalls of genetically encoded indicators.

Neural circuits are non-linear dynamical systems that transform information based on the pattern of input, current state and functional connectivity. To understand how a given stimulus is processed, one would ideally record neural activity across the entire brain of a behaving animal, at cellular or even subcellular resolution, in addition to characterizing anatomical connectivity. Given their transparency and relatively small size, larval zebrafish provide a powerful system for brain-wide monitoring of neural activity. Genetically encoded calcium indicators have been used for this purpose, but cannot directly report hyperpolarization or sub-threshold activity. Voltage indicators, in contrast, have this capability. Here, we test whether two different genetically encoded voltage reporters, ASAP1 and Bongwoori, can be expressed and report activity in the zebrafish brain, using widefield, two-photon and light sheet microscopy. We were unable to express ASAP1 in neurons. Bongwoori, in contrast expressed well, and because of its membrane localization, allowed visualization of axon trajectories in 3D. Bongwoori displayed stimulus-evoked changes in fluorescence, which could be detected in single trials. However, under high laser illumination, puncta on neural membranes underwent spontaneous fluctuations in intensity, suggesting that the probe is susceptible to blinking artefacts. These data indicate that larval zebrafish can be used to image electrical activity in the brain of an intact vertebrate at high resolution, although care is needed in imaging and analysis. Recording activity across the whole brain will benefit from further developments in imaging hardware and indicators.

Activation and inhibition of tph2 serotonergic neurons operate in tandem to influence larval zebrafish preference for light over darkness.

Serotonergic neurons have been implicated in a broad range of processes, but the principles underlying their effects remain a puzzle. Here, we ask how these neurons influence the tendency of larval zebrafish to swim in the light and avoid regions of darkness. Pharmacological inhibition of serotonin synthesis reduces dark avoidance, indicating an involvement of this neuromodulator. Calcium imaging of tph2-expressing cells demonstrates that a rostral subset of dorsal raphe serotonergic neurons fire continuously while the animal is in darkness, but are inhibited in the light. Optogenetic manipulation of tph2 neurons by channelrhodopsin or halorhodopsin expression modifies preference, confirming a role for these neurons. In particular, these results suggest that fish prefer swimming in conditions that elicits lower activity in tph2 serotonergic neurons in the rostral raphe.

The right dorsal habenula limits attraction to an odor in zebrafish.

BACKGROUND: The habenula consists of an evolutionarily conserved set of nuclei that control neuromodulator release. In lower vertebrates, the dorsal habenula receives innervation from sensory regions, but the significance of this is unclear. Here, we address the role of the habenula in olfaction by imaging neural activity in larval zebrafish expressing GCaMP3 throughout the habenula and by carrying out behavioral assays. RESULTS: Activity in several hundred neurons throughout the habenula was recorded using wide-field fluorescence microscopy, fast focusing, and deconvolution. This enabled the creation of 4D maps of odor-evoked activity. Odors activated the habenula in two broad spatiotemporal patterns. Increasing concentrations of a putative social cue (a bile salt) evoked a corresponding increase in neuronal activity in the right dorsal habenula. In behavioral assays, fish were attracted to intermediate concentration of this cue but avoided higher concentration. Increasing cholinergic activity through nicotine exposure rendered the intermediate concentration aversive in a habenula-dependent manner. Pharmacologically blocking nicotinic receptors or lesioning the right dorsal habenula attenuated avoidance. CONCLUSIONS: These data provide physiological and functional evidence that the habenula functions as a higher center in zebrafish olfaction and suggest that activity in the right dorsal subdomain gates innate attraction to specific odors.