Hypothalamic CRH neurons represent physiological memory of positive and negative experience.

Recalling a salient experience provokes specific behaviors and changes in the physiology or internal state. Relatively little is known about how physiological memories are encoded. We examined the neural substrates of physiological memory by probing CRHPVN neurons of mice, which control the endocrine response to stress. Here we show these cells exhibit contextual memory following exposure to a stimulus with negative or positive valence. Specifically, a negative stimulus invokes a two-factor learning rule that favors an increase in the activity of weak cells during recall. In contrast, the contextual memory of positive valence relies on a one-factor rule to decrease activity of CRHPVN neurons. Finally, the aversive memory in CRHPVN neurons outlasts the behavioral response. These observations provide information about how specific physiological memories of aversive and appetitive experience are represented and demonstrate that behavioral readouts may not accurately reflect physiological changes invoked by the memory of salient experiences.

MicroSweat: A Wearable Microfluidic Patch for Noninvasive and Reliable Sweat Collection Enables Human Stress Monitoring.

Stress affects cognition, behavior, and physiology, leading to lasting physical and mental illness. The ability to detect and measure stress, however, is poor. Increased circulating cortisol during stress is mirrored by cortisol release from sweat glands, providing an opportunity to use it as an external biomarker for monitoring internal emotional state. Despite the attempts at using wearable sensors for monitoring sweat cortisol, there is a lack of reliable wearable sweat collection devices that preserve the concentration and integrity of sweat biomolecules corresponding to stress levels. Here, a flexible, self-powered, evaporation-free, bubble-free, surfactant-free, and scalable capillary microfluidic device, MicroSweat, is fabricated to reliably collect human sweat from different body locations. Cortisol levels are detected corresponding to severe stress ranging from 25 to 125 ng mL-1 averaged across multiple body regions and 100-1000 ng mL-1 from the axilla. A positive nonlinear correlation exists between cortisol concentration and stress levels quantified using the perceived stress scale (PSS). Moreover, owing to the sweat variation in response to environmental effects and physiological differences, the longitudinal and personalized profile of sweat cortisol is acquired, for the first time, for various body locations. The obtained sweat cortisol data is crucial for analyzing human stress in personalized and clinical healthcare sectors.

Paraventricular nucleus CRH neurons encode stress controllability and regulate defensive behavior selection.

In humans and rodents, the perception of control during stressful events has lasting behavioral consequences. These consequences are apparent even in situations that are distinct from the stress context, but how the brain links prior stressful experience to subsequent behaviors remains poorly understood. By assessing innate defensive behavior in a looming-shadow task, we show that the initiation of an escape response is preceded by an increase in the activity of corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus (PVN) of the hypothalamus (CRHPVN neurons). This anticipatory increase is sensitive to stressful stimuli that have high or low levels of outcome control. Specifically, experimental stress with high outcome control increases CRHPVN neuron anticipatory activity, which increases escape behavior in an unrelated context. By contrast, stress with no outcome control prevents the emergence of this anticipatory activity and decreases subsequent escape behavior. These observations indicate that CRHPVN neurons encode stress controllability and contribute to shifts between active and passive innate defensive strategies.

Visual-looming Shadow Task with in-vivo Calcium Activity Monitoring to Assess Defensive Behaviors in Mice.

There has been a clear movement in recent years towards the adoption of more naturalistic experimental regimes for the study of behavior and its underlying neural architecture. Here we provide a protocol that allows experimenters working with mice, to mimic a looming and advancing predatory threat from the sky. This approach is easy to implement and can be combined with sophisticated neural recordings that allow access to real-time activity during behavior. This approach offers another option in a battery of tests that allow for a more comprehensive understanding of defensive behaviors.

Open-source, cost-effective system for low-light in vivo fiber photometry.

Fiber photometry uses genetically encoded optical reporters to link specific cellular activity in stereotaxically targeted brain structures to specific behaviors. There are still a number of barriers that have hindered the widespread adoption of this approach. This includes cost, but also the high-levels of light required to excite the fluorophore, limiting commercial systems to the investigation of short-term transients in neuronal activity to avoid damage of tissue by light. Here, we present a cost-effective optoelectronic system for in vivo fiber photometry that achieves high-sensitivity to changes in fluorescence intensity, enabling detection of optical transients of a popular calcium reporter with excitation powers as low as 100 nW. By realizing a coherent detection scheme and by using a photomultiplier tube as a detector, the system demonstrates reliable study of in vivo neuronal activity, positioning it for future use in the experiments inquiring into learning and memory processes. The system was applied to study stress-evoked calcium transients in corticotropin-releasing hormone neurons in the mouse hypothalamus.

Social transmission and buffering of synaptic changes after stress.

Stress can trigger enduring changes in neural circuits and synapses. The behavioral and hormonal consequences of stress can also be transmitted to others, but whether this transmitted stress has similar effects on synapses is not known. We found that authentic stress and transmitted stress in mice primed paraventricular nucleus of the hypothalamus (PVN) corticotropin-releasing hormone (CRH) neurons, enabling the induction of metaplasticity at glutamate synapses. In female mice that were subjected to authentic stress, this metaplasticity was diminished following interactions with a naive partner. Transmission from the stressed subject to the naive partner required the activation of PVN CRH neurons in both subject and partner to drive and detect the release of a putative alarm pheromone from the stressed mouse. Finally, metaplasticity could be transmitted sequentially from the stressed subject to multiple partners. Our findings demonstrate that transmitted stress has the same lasting effects on glutamate synapses as authentic stress and reveal an unexpected role for PVN CRH neurons in transmitting distress signals among individuals.

Tonic Local Brain Blood Flow Control by Astrocytes Independent of Phasic Neurovascular Coupling.

According to the current model of neurovascular coupling, blood flow is controlled regionally through phasic changes in the activity of neurons and astrocytes that signal to alter arteriole diameter. Absent in this model, however, is how brain blood flow is tonically regulated independent of regional changes in activity. This is important because a large fraction of brain blood flow is required to maintain basal metabolic needs. Using two-photon fluorescence imaging combined with patch-clamp in acute rat brain slices of sensory-motor cortex, we demonstrate that reducing resting Ca(2+) in astrocytes with intracellular BAPTA causes vasoconstriction in adjacent arterioles. BAPTA-induced vasoconstriction was eliminated by a general COX blocker and the effect is mimicked by a COX-1, but not COX-2, antagonist, suggesting that astrocytes provide tonic, steady-state vasodilation by releasing prostaglandin messengers. Tonic vasodilation was insensitive to TTX, as well as a variety of synaptic and extrasynaptic receptor antagonists, indicating that the phenomenon operates largely independent of neural activity. Using in vivo two-photon fluorescence imaging of the barrel cortex in fully awake mice, we reveal that acute COX-1 inhibition reduces resting arteriole diameter but fails to affect vasodilation in response to vibrissae stimulation. Our findings demonstrate that astrocytes provide tonic regulation of arterioles using resting intracellular Ca(2+) in a manner that is independent of phasic, neuronal-evoked vasodilation. Significance statement: The brain requires both phasic and tonic regulation of its blood supply to service energy needs over various temporal windows. While many mechanisms have been described for phasic blood flow regulation, how the brain accomplishes tonic control is largely unknown. Here we describe a way in which astrocytes contribute to the management of basal brain blood flow by providing steady-state vasodilation to arterioles via resting astrocyte Ca(2+) and the continuous release of prostaglandin messengers. This phenomenon may be important for understanding the declines in basal brain blood flow that occur in aging and dementia, as well as for the interpretation of fMRI data.

Arteriole dilation to synaptic activation that is sub-threshold to astrocyte endfoot Ca2+ transients.

Ca(2+)-dependent pathways in neurons and astrocyte endfeet initiate changes in arteriole diameter to regulate local brain blood flow. Whether there exists a threshold of synaptic activity in which arteriole diameter is controlled independent of astrocyte endfeet Ca(2+) remains unclear. We used two-photon fluorescence microscopy to examine synaptically evoked synthetic or genetic Ca(2+) indicator signals around penetrating arterioles in acute slices of the rat neocortex. We discovered a threshold below which vasodilation occurred in the absence of endfeet Ca(2+) signals but with consistent neuronal Ca(2+) transients, suggesting endfoot Ca(2+) is not necessary for activity-dependent vasodilation under subtle degrees of brain activation.

A slow or modulatory role of astrocytes in neurovascular coupling.

Astrocytes are thought to play an important role in NVC, a process that allows the brain to locally control blood flow in response to changes in activity. However, there is an ongoing debate as to when, and under what conditions astrocyte activity is required. In the following review we set forth the hypotheses that astrocytes: (i) act to modulate but not initiate functional hyperemia and (ii) help set the basal tone state of the brain microvasculature by the tonic release of vaso-active messengers. Through these actions astrocytes could help match metabolic demand with supply over a spectrum of activity timescales.

A high performance, cost-effective, open-source microscope for scanning two-photon microscopy that is modular and readily adaptable.

Two-photon laser scanning microscopy has revolutionized the ability to delineate cellular and physiological function in acutely isolated tissue and in vivo. However, there exist barriers for many laboratories to acquire two-photon microscopes. Additionally, if owned, typical systems are difficult to modify to rapidly evolving methodologies. A potential solution to these problems is to enable scientists to build their own high-performance and adaptable system by overcoming a resource insufficiency. Here we present a detailed hardware resource and protocol for building an upright, highly modular and adaptable two-photon laser scanning fluorescence microscope that can be used for in vitro or in vivo applications. The microscope is comprised of high-end componentry on a skeleton of off-the-shelf compatible opto-mechanical parts. The dedicated design enabled imaging depths close to 1 mm into mouse brain tissue and a signal-to-noise ratio that exceeded all commercial two-photon systems tested. In addition to a detailed parts list, instructions for assembly, testing and troubleshooting, our plan includes complete three dimensional computer models that greatly reduce the knowledge base required for the non-expert user. This open-source resource lowers barriers in order to equip more laboratories with high-performance two-photon imaging and to help progress our understanding of the cellular and physiological function of living systems.

The participation of NMDA receptors, PKC, and MAPK in Lymnaea memory extinction.

The aerial respiratory behavior of Lymnaea can be operantly conditioned to form a long-term memory (LTM) that will persist for >24h. LTM formation is dependent on altered gene activity and new protein synthesis, with the N-methyl-D-aspartate (NMDA) receptors, mitogen activated protein kinase (MAPK), and protein kinase C (PKC) pathways playing a critical role. LTM can also undergo extinction, whereby the original memory is temporarily masked by a new memory. Here we investigate if the formation of an extinction memory uses similar molecular pathways to those required for LTM formation. We find that the formation of the extinction memory can be blocked by inhibitors of NMDA receptors, PKC, and MAPK suggesting that extinction memory formation uses similar mechanisms to that of 'normal' memory formation.

The participation of NMDA receptors, PKC, and MAPK in the formation of memory following operant conditioning in Lymnaea.

BACKGROUND: Memory is the ability to store, retain, and later retrieve information that has been learned. Intermediate term memory (ITM) that persists for up to 3 h requires new protein synthesis. Long term memory (LTM) that persists for at least 24 h requires: DNA transcription, RNA translation, and the trafficking of newly synthesized proteins. It has been shown in a number of different model systems that NMDA receptors, protein kinase C (PKC) and mitogen activated protein kinase (MAPK) are all involved in the memory formation process. RESULTS: Here we show that snails trained in control conditions are capable of forming, depending on the training procedure used, either ITM or LTM. However, blockage of NMDA receptors (MK 801), inhibition of PKC (GF109203X hydrochloride) and MAPK activity (UO126) prevent the formation of both ITM and LTM. CONCLUSIONS: The injection of either U0126 or GF109203X, which inhibit MAPK and PKC activity respectively, 1 hour prior to training results in the inhibition of both ITM and LTM formation. We further found that NMDA receptor activity was necessary in order for both ITM and LTM formation.

Ecologically relevant stressors modify long-term memory formation in a model system.

Stress can alter adaptive behaviours, and as well either enhance or diminish learning, memory formation and/or memory recall. We focus attention on how environmentally relevant stressors (e.g. predator detection, crowding, and low concentrations of environmental Ca(++)) alter memory formation in the pond snail, Lymnaea stagnalis. We specifically look at operant conditioning of aerial respiration and whether or not long-term memory forms following the acquisition of the learned event, not performing aerial respiration. We will also examine the strain differences in Lymnaea which allow or cause isolated populations to possess different heritable cognitive capabilities, as manifested by differing abilities to form long-term memory.

A quantitative proteomic analysis of long-term memory.

BACKGROUND: Memory is the ability to store, retain, and later retrieve learned information. Long-term memory (LTM) formation requires: DNA transcription, RNA translation, and the trafficking of newly synthesized proteins. Several components of these processes have already been identified. However, due to the complexity of the memory formation process, there likely remain many yet to be identified proteins involved in memory formation and persistence. RESULTS: Here we use a quantitative proteomic method to identify novel memory-associated proteins in neural tissue taken from animals that were trained in vivo to form a long-term memory. We identified 8 proteins that were significantly up-regulated, and 13 that were significantly down-regulated in the LTM trained animals as compared to two different control groups. In addition we found 19 proteins unique to the trained animals, and 12 unique proteins found only in the control animals. CONCLUSIONS: These results both confirm the involvement of previously identified memory proteins such as: protein kinase C (PKC), adenylate cyclase (AC), and proteins in the mitogen-activated protein kinase (MAPK) pathway. In addition these results provide novel protein candidates (e.g. UHRF1 binding protein) on which to base future studies.

Enhancing memory formation by altering protein phosphorylation balance.

In Lymnaea, aerial respiration can be operantly conditioned and depending on the training procedure employed two forms of memory can result: intermediate-term (ITM) and long-term memory (LTM). ITM, which persists for 3h, is dependent on de novo protein synthesis whilst LTM, which persists for at least 24 h, is dependent on both de novo protein synthesis and altered gene activity. A single 0.5 h training session (i.e. ITM-training) leaves behind a residual molecular memory trace, which a second bout of ITM-training can activate and boost it to a LTM. Here we extend this finding to show that either inhibiting protein phosphatase activity with okadaic acid (1 microM), or increasing protein kinase C (PKC) activity and therefore protein phosphorylation with bryostatin (0.25 ng/mL) treatment prior to ITM-training, results in a LTM. However, following right pedal dorsal 1 (RPeD1) soma ablation neither of these treatments are effective in producing LTM following ITM-training, indicating transcription is a necessity. These findings suggest that the balance between phosphorylation and dephosphorylation in neurons is a key factor for LTM formation.

The perception of stress alters adaptive behaviours in Lymnaea stagnalis.

Stress can alter adaptive behaviours, and as well either enhance or diminish learning, memory formation and/or memory recall. We show here that two different stressors have the ability to alter such behaviours in our model system, Lymnaea stagnalis. One, a naturally occurring stressor - the scent of a predator (crayfish) - and the other an artificially controlled one - 25 mmol l(-1) KCl - significantly alter adaptive behaviours. Both the KCl stressor and predator detection enhance long-term memory (LTM) formation; additionally predator detection alters vigilance behaviours. The predator-induced changes in behaviour are also accompanied by specific and significant alterations in the electrophysiological properties of RPeD1 - a key neuron in mediating both vigilance behaviours and memory formation. Naive lab-bred snails exposed to crayfish effluent (CE; i.e. the scent of the predator) prior to recording from RPeD1 demonstrated both a significantly reduced spontaneous firing rate and fewer bouts of bursting activity compared with non-exposed snails. Importantly, in the CE experiments we used laboratory-reared snails that have not been exposed to a naturally occurring predator for over 250 generations. These data open a new avenue of research, which may allow a direct investigation from the behavioral to the neuronal level as to how relevant stressful stimuli alter adaptive behaviours, including memory formation and recall.

Canadian Association of Neurosciences Review: learning at a snail's pace.

While learning and memory are related, they are distinct processes each with different forms of expression and underlying molecular mechanisms. An invertebrate model system, Lymnaea stagnalis, is used to study memory formation of a non-declarative memory. We have done so because: (1) We have discovered the neural circuit that mediates an interesting and tractable behaviour; (2) This behaviour can be operantly conditioned and intermediate-term and long-term memory can be demonstrated; and (3) It is possible to demonstrate that a single neuron in the model system is a necessary site of memory formation. This article reviews how Lymnaea has been used in the study of behavioural and molecular mechanisms underlying consolidation, reconsolidation, extinction and forgetting.

Modulation of aerial respiratory behaviour in a pond snail.

Aerial respiratory in Lymnaea is driven by a three-neuron CPG whose sufficiency and necessity has been directly demonstrated. While this CPG is 'hard-wired' it displays a tremendous amount of plasticity. That is, it is possible by employing specific training procedures to alter how it functions in a specific hypoxic environment. Thus, it is possible to study directly the causal mechanisms of long-term memory formation, forgetting, and modulation of the memory at a single cell level. Thus, it is possible to use a relatively simple three-neuron CPG to study not only important questions concerning regulation of important homeostatic mechanisms but to also use it to study how learning and non-declarative memory are mediated at a cellular level.

Operant conditioning of an in vitro CNS-pneumostome preparation of Lymnaea.

Operant conditioning of aerial respiratory behaviour and its consolidation into long-term memory in Lymnaea has been previously studied in both intact, freely moving snails and in in vitro preparations made from previously trained snails. Here, we show in previously untrained semi-intact in vitro Lymnaea preparations that aerial respiratory behaviour can also be operantly conditioned. Neither yoked control nor 'run-down' control procedures in these in vitro preparations result in an alteration of aerial respiratory behaviour. Memory in the operantly trained semi-intact preparations persists for at least 1h after training. Intracellular recordings made from RPeD1, one of the 3-CPG neurons and the neuron that initiates CPG activity; show that there are specific changes in central excitatory input to this neuron concurrent with learning and its consolidation into memory. In addition following the acquisition of learning and its consolidation into memory the ability of RPeD1 and VI/J neurons when depolarized to cause a pneumostome opening is significantly decreased. Thus, previously untrained in vitro semi-intact preparations can be used to study changes in neuronal activity in a neuron known to be both necessary for the behaviour and for memory formation.

Memory, reconsolidation and extinction in Lymnaea require the soma of RPeD1.

The central pattern generator (CPG) that drives aerial respiratory behaviour in Lymnaea consists of 3 neurons. One of these, RPeD1--the cell that initiates activity in the circuit, plays an absolutely necessary role as a site for memory formation, memory reconsolidation, and extinction. Using an operant conditioning training procedure that results in a long-term non-declarative memory (LTM), we decrease the occurrence of aerial respiratory behaviour. Since snails can still breathe cutaneously learning this procedure is not harmful. Concomitant with behavioural memory are changes in the spiking activity of RPeD1. Going beyond neural correlates of memory we directly show that RPeD1 is a necessary site for LTM formation. Expanding on this finding we show that this neuron is also a necessary site for memory reconsolidation and 'Pavlovian' extinction. As far as we can determine, this is the first time a single neuron has been shown to be a necessary site for these different aspects memory. RPeD1 is thus a key neuron mediating different hierarchical aspects of memory. We are now in a position to determine the necessary neuronal, molecular and proteomic events in this neuron that are causal to memory formation, reconsolidation and extinction.