Biofilm exopolysaccharides alter sensory-neuron-mediated sickness during lung infection.

Infections of the lung cause observable sickness thought to be secondary to inflammation. Signs of sickness are crucial to alert others via behavioral-immune responses to limit contact with contagious individuals. Gram-negative bacteria produce exopolysaccharide (EPS) that provides microbial protection; however, the impact of EPS on sickness remains uncertain. Using genome-engineered Pseudomonas aeruginosa (P. aeruginosa) strains, we compared EPS-producers versus non-producers and a virulent Escherichia coli (E. coli) lung infection model in male and female mice. EPS-negative P. aeruginosa and virulent E. coli infection caused severe sickness, behavioral alterations, inflammation, and hypothermia mediated by TLR4 detection of the exposed lipopolysaccharide (LPS) in lung TRPV1+ sensory neurons. However, inflammation did not account for sickness. Stimulation of lung nociceptors induced acute stress responses in the paraventricular hypothalamic nuclei by activating corticotropin-releasing hormone neurons responsible for sickness behavior and hypothermia. Thus, EPS-producing biofilm pathogens evade initiating a lung-brain sensory neuronal response that results in sickness.

Optogenetic recruitment of hypothalamic corticotrophin-releasing-hormone (CRH) neurons reduces motivational drive.

Impaired motivational drive is a key feature of depression. Chronic stress is a known antecedent to the development of depression in humans and depressive-like states in animals. Whilst there is a clear relationship between stress and motivational drive, the mechanisms underpinning this association remain unclear. One hypothesis is that the endocrine system, via corticotropin-releasing hormone (CRH) in the paraventricular nucleus of the hypothalamus (PVN; PVNCRH), initiates a hormonal cascade resulting in glucocorticoid release, and that excessive glucocorticoids change brain circuit function to produce depression-related symptoms. Another mostly unexplored hypothesis is that the direct activity of PVNCRH neurons and their input to other stress- and reward-related brain regions drives these behaviors. To further understand the direct involvement of PVNCRH neurons in motivation, we used optogenetic stimulation to activate these neurons 1 h/day for 5 consecutive days and showed increased acute stress-related behaviors and long-lasting deficits in the motivational drive for sucrose. This was associated with increased Fos-protein expression in the lateral hypothalamus (LH). Direct stimulation of the PVNCRH inputs in the LH produced a similar pattern of effects on sucrose motivation. Together, these data suggest that PVNCRH neuronal activity may be directly responsible for changes in motivational drive and that these behavioral changes may, in part, be driven by PVNCRH synaptic projections to the LH.

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.

Disruption of tonic endocannabinoid signalling triggers cellular, behavioural and neuroendocrine responses consistent with a stress response.

BACKGROUND AND PURPOSE: Endocannabinoid (eCB) signalling gates many aspects of the stress response, including the hypothalamic-pituitary-adrenal (HPA) axis. The HPA axis is controlled by corticotropin releasing hormone (CRH) producing neurons in the paraventricular nucleus of the hypothalamus (PVN). Disruption of eCB signalling increases drive to the HPA axis, but the mechanisms subserving this process are poorly understood. EXPERIMENTAL APPROACH: Using an array of cellular, endocrine and behavioural readouts associated with activation of CRH neurons in the PVN, we evaluated the contributions of tonic eCB signalling to the generation of a stress response. KEY RESULTS: The CB1 receptor antagonist/inverse agonist AM251, neutral antagonist NESS243 and NAPE PLD inhibitor LEI401 all uniformly increased Fos in the PVN, unmasked stress-linked behaviours, such as grooming, and increased circulating CORT, recapitulating the effects of stress. Similar effects were also seen after direct administration of AM251 into the PVN, while optogenetic inhibition of PVN CRH neurons ameliorated stress-like behavioural changes produced by disruption of eCB signalling. CONCLUSIONS AND IMPLICATIONS: These data indicate that under resting conditions, constitutive eCB signalling restricts activation of the HPA axis through local regulation of CRH neurons in the PVN.

α/ß-Peptides as Nanomolar Triggers of Lipid Raft-Mediated Endocytosis through GM1 Ganglioside Recognition.

Cell delivery of therapeutic macromolecules and nanoparticles is a critical drug development challenge. Translocation through lipid raft-mediated endocytic mechanisms is being sought, as it can avoid rapid lysosomal degradation. Here, we present a set of short α/ß-peptide tags with high affinity to the lipid raft-associated ganglioside GM1. These sequences induce effective internalization of the attached immunoglobulin cargo. The structural requirements of the GM1-peptide interaction are presented, and the importance of the membrane components are shown. The results contribute to the development of a receptor-based cell delivery platform.

A versatile computational algorithm for time-series data analysis and machine-learning models.

Here we introduce Local Topological Recurrence Analysis (LoTRA), a simple computational approach for analyzing time-series data. Its versatility is elucidated using simulated data, Parkinsonian gait, and in vivo brain dynamics. We also show that this algorithm can be used to build a remarkably simple machine-learning model capable of outperforming deep-learning models in detecting Parkinson's disease from a single digital handwriting test.

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.

Optogenetic Activation of A11 Region Increases Motor Activity.

Limbic brain regions drive goal-directed behaviors. These behaviors often require dynamic motor responses, but the functional connectome of limbic structures in the diencephalon that control locomotion is not well known. The A11 region, within the posterior diencephalon has been postulated to contribute to motor function and control of pain. Here we show that the A11 region initiates movement. Photostimulation of channelrhodopsin 2 (ChR2) transfected neurons in A11 slice preparations showed that neurons could follow stimulation at frequencies of 20 Hz. Our data show that photostimulation of ChR2 transfected neurons in the A11 region enhances motor activity often leading to locomotion. Using vGluT2-reporter and vGAT-reporter mice we show that the A11 tyrosine hydroxylase positive (TH) dopaminergic neurons are vGluT2 and vGAT negative. We find that in addition to dopaminergic neurons within the A11 region, there is another neuronal subtype which expresses the monoenzymatic aromatic L-amino acid decarboxylase (AADC), but not TH, a key enzyme involved in the synthesis of catecholamines including dopamine. This monoaminergic-based motor circuit may be involved in the control of motor behavior as part of a broader diencephalic motor region.

Endocannabinoid and nitric oxide systems of the hypothalamic paraventricular nucleus mediate effects of NPY on energy expenditure.

OBJECTIVE: Neuropeptide Y (NPY) is one of the most potent orexigenic peptides. The hypothalamic paraventricular nucleus (PVN) is a major locus where NPY exerts its effects on energy homeostasis. We investigated how NPY exerts its effect within the PVN. METHODS: Patch clamp electrophysiology and Ca2+ imaging were used to understand the involvement of Ca2+ signaling and retrograde transmitter systems in the mediation of NPY induced effects in the PVN. Immuno-electron microscopy were performed to elucidate the subcellular localization of the elements of nitric oxide (NO) system in the parvocellular PVN. In vivo metabolic profiling was performed to understand the role of the endocannabinoid and NO systems of the PVN in the mediation of NPY induced changes of energy homeostasis. RESULTS: We demonstrated that NPY inhibits synaptic inputs of parvocellular neurons in the PVN by activating endocannabinoid and NO retrograde transmitter systems via mobilization of Ca2+ from the endoplasmic reticulum, suggesting that NPY gates the synaptic inputs of parvocellular neurons in the PVN to prevent the influence of non-feeding-related inputs. While intraPVN administered NPY regulates food intake and locomotor activity via NO signaling, the endocannabinoid system of the PVN selectively mediates NPY-induced decrease in energy expenditure. CONCLUSION: Thus, within the PVN, NPY stimulates the release of endocannabinoids and NO via Ca2+-influx from the endoplasmic reticulum. Both transmitter systems appear to have unique roles in the mediation of the NPY-induced regulation of energy homeostasis, suggesting that NPY regulates food intake, energy expenditure, and locomotor activity through different neuronal networks of this nucleus.

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.

Role of TRH/UCN3 neurons of the perifornical area/bed nucleus of stria terminalis region in the regulation of the anorexigenic POMC neurons of the arcuate nucleus in male mice and rats.

Two anorexigenic peptides, thyrotropin-releasing hormone (TRH) and urocortin 3 (UCN3), are co-expressed in a continuous neuronal group that extends from the perifornical area to the bed nucleus of stria terminalis, raising the possibility that this cell group may be involved in the regulation of energy homeostasis. In this study, therefore, we tested the hypothesis that the TRH/UCN3 neurons regulate food intake by influencing feeding-related neuropeptide Y (NPY) and/or proopiomelanocortin (POMC) neurons in the arcuate nucleus (ARC). Triple-labeled immunofluorescent preparations demonstrated that only very few NPY neurons (4.3 ± 1.3%) were contacted by double-labeled TRH/UCN3 axons in the ARC. In contrast, more than half of the POMC neurons (52.4 ± 8.5%) were contacted by double-labeled axons. Immuno-electron microscopy demonstrated that the UCN3 axons established asymmetric synapses with POMC neurons, indicating the excitatory nature of these synaptic specializations. Patch clamp electrophysiology revealed that TRH and UCN3 have antagonistic effects on the POMC neurons. While UCN3 depolarizes and increases the firing rate of POMC neurons, TRH prevents these effects of UCN3. These data demonstrate that TRH/UCN3 neurons in the perifornical/BNST region establish abundant synaptic associations with the POMC neurons in the ARC and suggest a potentially important role for these neurons in the regulation of food intake through an antagonistic interaction between TRH and UCN3 on the electrophysiological properties of POMC neurons.

Hypothalamic CRH neurons orchestrate complex behaviours after stress.

All organisms possess innate behavioural and physiological programmes that ensure survival. In order to have maximum adaptive benefit, these programmes must be sufficiently flexible to account for changes in the environment. Here we show that hypothalamic CRH neurons orchestrate an environmentally flexible repertoire of behaviours that emerge after acute stress in mice. Optical silencing of CRH neurons disrupts the organization of individual behaviours after acute stress. These behavioural patterns shift according to the environment after stress, but this environmental sensitivity is blunted by activation of PVN CRH neurons. These findings provide evidence that PVN CRH cells are part of a previously unexplored circuit that matches precise behavioural patterns to environmental context following stress. Overactivity in this network in the absence of stress may contribute to environmental ambivalence, resulting in context-inappropriate behavioural strategies.

Characterization of A11 neurons projecting to the spinal cord of mice.

The hypothalamic A11 region has been identified in several species including rats, mice, cats, monkeys, zebrafish, and humans as the primary source of descending dopamine (DA) to the spinal cord. It has been implicated in the control of pain, modulation of the spinal locomotor network, restless leg syndrome, and cataplexy, yet the A11 cell group remains an understudied dopaminergic (DAergic) nucleus within the brain. It is unclear whether A11 neurons in the mouse contain the full complement of enzymes consistent with traditional DA neuronal phenotypes. Given the abundance of mouse genetic models and tools available to interrogate specific neural circuits and behavior, it is critical first to fully understand the phenotype of A11 cells. We provide evidence that, in addition to tyrosine hydroxylase (TH) that synthesizes L-DOPA, neurons within the A11 region of the mouse contain aromatic L-amino acid decarboxylase (AADC), the enzyme that converts L-DOPA to dopamine. Furthermore, we show that the A11 neurons contain vesicular monoamine transporter 2 (VMAT2), which is necessary for packaging DA into vesicles. On the contrary, A11 neurons in the mouse lack the dopamine transporter (DAT). In conclusion, our data suggest that A11 neurons are DAergic. The lack of DAT, and therefore the lack of a DA reuptake mechanism, points to a longer time of action compared to typical DA neurons.

The CB1 receptor mediates the peripheral effects of ghrelin on AMPK activity but not on growth hormone release.

This study aimed to investigate whether the growth hormone release and metabolic effects of ghrelin on AMPK activity of peripheral tissues are mediated by cannabinoid receptor type 1 (CB1) and the central nervous system. CB1-knockout (KO) and/or wild-type mice were injected peripherally or intracerebroventricularly with ghrelin and CB1 antagonist rimonabant to study tissue AMPK activity and gene expression (transcription factors SREBP1c, transmembrane protein FAS, enzyme PEPCK, and protein HSL). Growth hormone levels were studied both in vivo and in vitro. Peripherally administered ghrelin in liver, heart, and adipose tissue AMPK activity cannot be observed in CB1-KO or CB1 antagonist-treated mice. Intracerebroventricular ghrelin treatment can influence peripheral AMPK activity. This effect is abolished in CB1-KO mice and by intracerebroventricular rimonabant treatment, suggesting that central CB1 receptors also participate in the signaling pathway that mediates the effects of ghrelin on peripheral tissues. Interestingly, in vivo or in vitro growth hormone release is intact in response to ghrelin in CB1-KO animals. Our data suggest that the metabolic effects of ghrelin on AMPK in peripheral tissues are abolished by the lack of functional CB1 receptor via direct peripheral effect and partially through the central nervous system, thus supporting the existence of a possible ghrelin-cannabinoid-CB1-AMPK pathway.

CB1 receptor mediates the effects of glucocorticoids on AMPK activity in the hypothalamus.

AMP-activated protein kinase (AMPK), a regulator of cellular and systemic energy homeostasis, can be influenced by several hormones. Tissue-specific alteration of AMPK activity by glucocorticoids may explain the increase in appetite, the accumulation of lipids in adipose tissues, and the detrimental cardiac effects of Cushing's syndrome. Endocannabinoids are known to mediate the effects of various hormones and to influence AMPK activity. Cannabinoids have central orexigenic and direct peripheral metabolic effects via the cannabinoid receptor type 1 (CB1). In our preliminary experiments, WT mice received implants of a corticosterone-containing pellet to establish a mouse model of Cushing's syndrome. Subsequently, WT and Cb1 (Cnr1)-knockout (CB1-KO) littermates were treated with corticosterone and AMPK activity in the hypothalamus, various adipose tissues, liver and cardiac tissue was measured. Corticosterone-treated CB1-KO mice showed a lack of weight gain and of increase in hypothalamic and hepatic AMPK activity. In adipose tissues, baseline AMPK activity was higher in CB1-KO mice, but a glucocorticoid-induced drop was observed, similar to that observed in WT mice. Cardiac AMPK levels were reduced in CB1-KO mice, but while WT mice showed significantly reduced AMPK activity following glucocorticoid treatment, CB1-KO mice showed a paradoxical increase. Our findings indicate the importance of the CB1 receptor in the central orexigenic effect of glucocorticoid-induced activation of hypothalamic AMPK activity. In the periphery adipose tissues, changes may occur independently of the CB1 receptor, but the receptor appears to alter the responsiveness of the liver and myocardial tissues to glucocorticoids. In conclusion, our data suggest that an intact cannabinoid pathway is required for the full metabolic effects of chronic glucocorticoid excess.

Characterization of corticotropin-releasing hormone neurons in the paraventricular nucleus of the hypothalamus of Crh-IRES-Cre mutant mice.

Corticotropin-releasing hormone (CRH)-containing neurons in the paraventricular nucleus of the hypothalamus (PVN) initiate and control neuroendocrine responses to psychogenic and physical stress. Investigations into the physiology of CRH neurons, however, have been hampered by the lack of tools for adequately targeting or visualizing this cell population. Here we characterize CRH neurons in the PVN of mice that express tdTomato fluorophore, generated by crosses of recently developed Crh-IRES-Cre driver and Ai14 Cre-reporter mouse strains. tdTomato containing PVN neurons in Crh-IRES-Cre;Ai14 mice are readily visualized without secondary-detection methods. These neurons are predominantly neuroendocrine and abundantly express CRH protein, but not other PVN phenotypic neuropeptides. After an acute stress, a large majority of tdTomato cells express neuronal activation marker c-Fos. Finally, tdTomato PVN neurons exhibit homogenous intrinsic biophysical and synaptic properties, and can be optogenetically manipulated by viral Cre-driven expression of channelrhodopsin. These observations highlight basic cell-type characteristics of CRH neurons in a mutant mouse, providing validation for its future use in probing neurophysiology of endocrine stress responses.