Firing Frequency Of Neurons Research Articles

Glucocorticoids Selectively Inhibit Hippocampal CA1 Pyramidal Neurons Activity Through HCN Channels.

Glucocorticoids are known to influence hippocampal function, but their rapid non-genomic effects on specific neurons in the hippocampal trisynaptic circuit remain underexplored. This study investigated the immediate effects of glucocorticoids on CA1 and CA3 pyramidal neurons, and dentate gyrus (DG) granule neurons in rats using the patch-clamp technique. We found that a 5 min extracellular application of corticosterone significantly reduced action potential firing frequency in CA1 pyramidal neurons, while no effects were observed in CA3 or DG neurons. The corticosterone-induced inhibition in CA1 was blocked by the glucocorticoid receptor antagonist CORT125281, but remained unaffected by the mineralocorticoid receptor antagonist spironolactone. Notably, membrane-impermeable bovine serum albumin-conjugated dexamethasone mimicked corticosterone's effects on CA1 neurons, which exhibited prominent hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents. Pyramidal neurons in CA3 and granular neurons in the DG showed little HCN channel currents. Corticosterone enhanced HCN channel activity in CA1 neurons via glucocorticoid receptors, and the HCN channel inhibitor ZD7288 abolished corticosterone's suppressive effects on action potentials. These findings suggest that glucocorticoids selectively inhibit CA1 pyramidal neuron activity through HCN channels, providing new insight into the mechanisms of glucocorticoid action in hippocampal circuits.

A sexually dimorphic signature of activity-dependent BDNF signaling on the intrinsic excitability of pyramidal neurons in the prefrontal cortex.

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders with strong genetic heterogeneity and more prevalent in males than females. We and others hypothesize that diminished activity-dependent neural signaling is a common molecular pathway dysregulated in ASD caused by diverse genetic mutations. Brain-derived neurotrophic factor (BDNF) is a key growth factor mediating activity-dependent neural signaling in the brain. A common single nucleotide polymorphism (SNP) in the pro-domain of the human BDNF gene that leads to a methionine (Met) substitution for valine (Val) at codon 66 (Val66Met) significantly decreases activity-dependent BDNF release without affecting basal BDNF secretion. By using mice with genetic knock-in of this human BDNF methionine (Met) allele, our previous studies have shown differential severity of autism-like social deficits in male and female BDNF+/Met mice. Pyramidal neurons are the principal neurons in the prefrontal cortex (PFC), a key brain region for social behaviors. Here, we investigated the impact of diminished activity-dependent BDNF signaling on the intrinsic excitability of pyramidal neurons in the PFC. Surprisingly, diminished activity-dependent BDNF signaling significantly increased the intrinsic excitability of pyramidal neurons in male mice, but not in female mice. Notably, significantly decreased thresholds of action potentials were observed in male BDNF+/Met mice, but not in female BDNF+/Met mice. Voltage-clamp recordings revealed that the sodium current densities were significantly increased in the pyramidal neurons of male BDNF+/Met mice, which were mediated by increased transcriptional level of Scn2a encoding sodium channel NaV 1.2. Medium after hyperpolarization (mAHP), another important parameter to determine intrinsic neuronal excitability, is strongly associated with neuronal firing frequency. Further, the amplitudes of mAHP were significantly decreased in male BDNF+/Met mice only, which were mediated by the downregulation of Kcnn2 encoding small conductance calcium-activated potassium channel 2 (SK2). This study reveals a sexually dimorphic signature of diminished activity-dependent BDNF signaling on the intrinsic neuronal excitability of pyramidal neurons in the PFC, which provides possible cellular and molecular mechanisms underpinning the sex differences in idiopathic ASD patients and human autism victims who carry BDNF Val66Met SNP.

Frequency chimera state induced by time delays in FitzHugh-Nagumo neural networks

The delay of information transmission is an inherent factor of the nervous systems, which has great influences on their collective dynamic behaviors. In this paper, we constructed a ring neural network using FitzHugh-Nagumo (FHN) neuron model as the network node and memristive synapses as the connection mode. Our primary focus was on investigating the effects of time delay and coupling strength on the firing frequency of neurons. Simulation results revealed that the frequency chimera state could be induced in the neural network with appropriate time delay, which is a new type of chimera state characterized by firing frequency rather than traditional membrane potential. By adjusting the time delay properly, the neural network can also display multi-cluster frequency chimera states that coexisted with various incoherent regions and coherent regions. Meanwhile, we exhibit that initial value and coupling strength could have great influences on the effects of time delay on inducing frequency chimera state of the nervous systems.

Abstract 53: Chronic Angiotensin-(1-7) Treatment May Enhance GABAergic Neurotransmission onto Melanocortin-4 Receptor Neurons in The Hypothalamic Paraventricular Nucleus in a Sex-Specific Manner

Angiotensin (Ang)-(1-7) is a protective hormone of the renin-angiotensin system that lowers blood pressure in animal models of cardiovascular disease; however, the precise mechanisms remain incompletely understood. Recent data from our laboratory suggests that blood pressure lowering effects of Ang-(1-7) may involve the arcuate nucleus of the hypothalamus (ARC). We have shown that Ang-(1-7) activates proopiomelanocortin (POMC)-containing neurons in the ARC. While ARC POMC neuron activation canonically increases blood pressure by stimulating melanocortin-4 receptors (MC4R) in the hypothalamic paraventricular nucleus (PVN), these neurons also release the inhibitory neurotransmitter GABA. Selective activation of these GABAergic POMC neurons could serve as a novel mechanism to lower blood pressure. In this study, we hypothesized that Ang-(1-7) treatment inhibits PVN MC4R neuron activity via enhanced GABAergic function. To test this, male and female MC4R-GFP mice were implanted with osmotic mini pumps at 14 weeks of age and treated chronically with either Ang-(1-7) (400 ng/kg/min) or saline. After six weeks of drug administration, action potential firing of PVN MC4R-expressing neurons was examined using cell attached electrophysiology. To assess GABAergic function, changes in action potential firing frequency following bath application of the GABA A receptor antagonist bicuculline (10 μm) were compared between Ang-(1-7) and saline treated animals (n=4-6 mice/group). While Ang-(1-7) did not alter baseline firing frequency of MC4R PVN neurons in male mice (1.5±0.6 vs. 1.1±0.3 Hz saline; p=0.630, unpaired t-test), it enhanced bicuculline responsiveness as indicated by a greater peak increase in action potential firing frequency (156.9±18.0 vs. 106.2±6.8 % saline; p=0.013). In female mice, Ang-(1-7) increased baseline action potential firing frequency of PVN MC4R neurons (1.5±0.4 vs. 0.3±0.1 Hz saline; p=0.026) but did not alter bicuculline responsiveness (111.0±5.3 vs. 143.2±25.3 % saline; p=0.241). Overall, our data suggest that Ang-(1-7) enhances GABAergic neurotransmission onto PVN MC4R neurons in a sex-specific manner. These data provide new insight into the neural mechanisms of Ang-(1-7), which could have implications for understanding the beneficial cardiovascular effects of this hormone. Additional studies are needed to examine this inhibitory neurocircuit in the context of hypertension as well as precise mechanisms underlying the observed sex differences.

Electroacupuncture alleviates myocardial ischemia-reperfusion injury by inhibiting hypothalamic paraventricular nucleus neurons projecting to the rostral ventrolateral medulla.

Accumulating evidence suggests that electroacupuncture (EA) has obvious therapeutic effects and unique advantages in alleviating myocardial ischemia-reperfusion injury (MIRI), while the underlying neuromolecular mechanisms of EA intervention for MIRI have not been fully elucidated. The aim of the study is to investigate the role of the neural pathway of hypothalamic paraventricular nucleus (PVN) neurons projecting to the rostral ventrolateral medulla (RVLM) in the alleviation of MIRI rats by EA preconditioning. MIRI models were established by ligating the left anterior descending coronary artery for 30 min followed by reperfusion for 2h. Electrocardiogram recording, chemogenetics, enzyme-linked immunosorbent assay, multichannel physiology recording and haematoxylin-eosin and immunofluorescence staining methods were conducted to demonstrate that the firing frequencies of neurons in the PVN and the expression of c-Fos decreased by EA pretreatment. Meanwhile, EA preconditioning significantly reduced the levels of creatine kinase isoenzymes (CK-MB), cardiac troponin I (cTnI) and lactic dehydrogenase (LDH). Virus tracing showed a projection connection between PVN and RVLM. The inhibition of the PVN-RVLM neural pathway could replicate the protective effect of EA pretreatment on MIRI rats. However, the activation of the pathway weakened the effect of EA preconditioning. EA pretreatment alleviated MIRI by regulating PVN neurons projecting to RVLM. This work provides novel evidence of EA pretreatment for alleviating MIRI.

Potentiation of BKCa channels by cystic fibrosis transmembrane conductance regulator correctors VX-445 and VX-121.

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, ultimately leading to diminished transepithelial anion secretion and mucociliary clearance. CFTR correctors are therapeutics that restore the folding/trafficking of mutated CFTR to the plasma membrane. The large-conductance calcium-activated potassium channel (BKCa, KCa1.1) is also critical for maintaining lung airway surface liquid (ASL) volume. Here, we show that the class 2 (C2) CFTR corrector VX-445 (elexacaftor) induces K+ secretion across WT and F508del CFTR primary human bronchial epithelial cells (HBEs), which was entirely inhibited by the BKCa antagonist paxilline. Similar results were observed with VX-121, a corrector under clinical evaluation. Whole-cell patch-clamp recordings verified that CFTR correctors potentiated BKCa activity from both primary HBEs and HEK cells stably expressing the α subunit (HEK-BK cells). Furthermore, excised patch-clamp recordings from HEK-BK cells verified direct action on the channel and demonstrated a significant increase in open probability. In mouse mesenteric artery, VX-445 induced a paxilline-sensitive vasorelaxation of preconstricted arteries. VX-445 also reduced firing frequency in primary rat hippocampal and cortical neurons. We raise the possibilities that C2 CFTR correctors gain additional clinical benefit by activation of BKCa in the lung yet may lead to adverse events through BKCa activation elsewhere.

Direct Inhibition of BK Channels by Cannabidiol, One of the Principal Therapeutic Cannabinoids Derived from Cannabis sativa.

Cannabidiol (CBD), one of the main Cannabis sativa bioactive compounds, is utilized in the treatment of major epileptic syndromes. Its efficacy can be attributed to a multimodal mechanism of action that includes, as potential targets, several types of ion channels. In the brain, CBD reduces the firing frequency in rat hippocampal neurons, partly prolonging the duration of action potentials, suggesting a potential blockade of voltage-operated K+ channels. We postulate that this effect might involve the inhibition of the large-conductance voltage- and Ca2+-operated K+ channel (BK channel), which plays a role in the neuronal action potential's repolarization. Thus, we assessed the impact of CBD on the BK channel activity, heterologously expressed in HEK293 cells. Our findings, using the patch-clamp technique, revealed that CBD inhibits BK channel currents in a concentration-dependent manner with an IC50 of 280 nM. The inhibition is through a direct interaction, reducing both the unitary conductance and voltage-dependent activation of the channel. Additionally, the cannabinoid significantly delays channel activation kinetics, indicating stabilization of the closed state. These effects could explain the changes induced by CBD in action potential shape and duration, and they may contribute to the observed anticonvulsant activity of this cannabinoid.

SIGNIFICANCE OF THE NAV 1.8. VOLTAGE-GATED, TETRODOTOXIN-RESISTANT SODIUM CHANNEL FOR THE RENAL SENSORY INNERVATION IN MICE AND RATS

Objective: Our previous work repeatedly showed that the sensory innervation of the kidney in rats has a peculiarity containing predominantly (more than 50%) highly active tonic neurons to electrical stimulation. In a previous publication demonstrated an increased mRNA expression of the TTX-resistant sodium channel Nav1.8 in renal sensory neurons. Hence, we tested the hypothesis that tonic firing pattern is related to the specific expression of Nav1.8 on the cell surface of neurons with renal sensoric axons in the dorsal root ganglia (DRG Th12-L2). Design and method: Harvested dorsal root ganglion neurons (DRG Th11-L2) from Sprague Dawley (SD) with renal afferents were investigated in neuronal cell culture using current clamp mode to assess action potential generation during current injection and to characterize neurons as tonic highly active and phasic less active neurons using a Nav1.8 blocker (A-803467) before and after stimulation. Further, renal DRG neurons from a Nav1.8 knock out mouse (C57BL6J-Scn10atm1Jwo) were investigated in a current clamp mode. C57BL6 mice were used as controls. Results: At a concentration of 0.3μM the maximum AP firing frequency of tonic neurons was blocked from 13+/- 1.1 APs/600ms to 7.6+/-1.4 APs/600ms under superfusion with a Nav1.8 blocker. No blocker effects were seen at a concentration of 0.1 μM and due to superfusion with the solvent methanol alone. The firing pattern of renal neurons in the C57BL6 mouse was similar to that in the SD rat with a dominance of the tonic highly active neurons. In a Nav1.8 knock out mouse (C57BL6J-Scn10atm1Jwo) in the population of neurons with dendrites from the kidney only a single cell out of 70 showed tonic firing behavior (control vs Nav1.8 KO mouse, z-test, p<0.05). Conclusions: Under physiological conditions, renal sensory neurons exhibit predominantly a firing pattern associated with higher excitability. Our findings in this study support the significance of the TTX-resistant sodium channel Nav1.8 for the specific tonic firing pattern of neurons with renal projections. That might be of importance for pharmacological interventions to influence renal nerve activity, which likely plays a crucial role in the regulation of blood pressure and control of cardiovascular function.

WNK1 is a central osmolality sensor for arginine vasopressin release and acts through the OSR1/SPAK kinase cascade

Background: Maintaining internal osmolality constancy and water homeostasis is essential for life. The circumventricular organs (CVOs) of the brain, including the organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO), lack a blood-brain barrier. Neurons in OVLT and SFO detect increases in serum osmolality that stimulate the production of vasopressin (AVP) in paraventricular nuclei (PVN) to be released in the posterior pituitary. We have recently reported that WNK1 in sensory neurons in CVOs functions as an osmolality sensor for AVP release (Jin et al., JCI, 2023). WNK1 activates Kv3.1 to increase action potential, traveling down to PVN to increase AVP synthesis for release from the posterior pituitary. Here, we propose that OSR1/SPAK acts as the downstream kinase for WNK1 in regulating AVP release. We further investigated the regulation of vasopressin release by the WNK1-OSR1/SPAK cascade in response to extracellular hyperosmolality. Methods: Cre recombinase-carrying retrograde AAV virus was stereotaxically injected into PVN nuclei from normal mice and SPAK-null mice with a homozygous OSR1-floxed allele (SAPK−/−;OSR1flox/flox). Metabolic cage studies were performed on mice. Urine output, water intake, serum osmolality, serum AVP, and copeptin levels were measured. K+ currents were measured from the OVLT neurons by whole-cell patch clamp. Results: Increased osmolality, either by water restriction or mannitol injection, activated OSR1/SPAK in OVLT, as evident by increased serine-373 phospho-SPAK and serine-325 phospho-OSR. Like the WNK1 deletion, the double deletion of OSR/SPAK in OVLT caused polyuria with decreased urine osmolality that persisted in water restriction. Circulating levels of AVP and copeptin were increased by water restriction in control mice. In contrast, water restriction failed to increase the levels in OSR/SPAK-deletion mice. Knockdown of the Kv3.1 channel in OVLT by shRNA showed the central diabetes insipidus (DI) phenotype as well. Hypertonicity increased K+ currents and WNKs inhibitor prevented the increase in OVLT neurons. Conclusions: Mice with neuronal-specific OSR1/SPAK deletion have water homeostasis defects consistent with partial central DI; Evidence supporting that OSR1/SPAK acts downstream of WNK1 to regulate AVP release; WNK1-OSR1/SPAK activates Kv3.1 to increase firing frequency of neurons projecting from CVOs to PVN. Our findings reveal that an intracellular protein acts as a sensor for extracellular tonicity and provide fresh insights into the mechanisms by which the body maintains osmolality constancy. Future studies will investigate how OSR1/SPAK regulates Kv3.1 channels. NIDDK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Glutamatergic neurons in the paraventricular nucleus of the hypothalamus participate in the regulation of visceral pain induced by pancreatic cancer in mice.

Visceral pain induced by pancreatic cancer seriously affects patients' quality of life, and there is no effective treatment, because the mechanism of its neural circuit is unknown. Therefore, the aim of this study is to explore the main neural circuit mechanism regulating visceral pain induced by pancreatic cancer in mice. The mouse model of pancreatic cancer visceral pain was established on C57BL/6N mice by pancreatic injection of mPAKPC-luc cells. Abdominal mechanical hyperalgesia and hunch score were performed to assess visceral pain; the pseudorabies virus (PRV) was used to identify the brain regions innervating the pancreas; the c-fos co-labeling method was used to ascertain the types of activated neurons; in vitro electrophysiological patch-clamp technique was used to record the electrophysiological activity of specific neurons; the calcium imaging technique was used to determine the calcium activity of specific neurons; specific neuron destruction and chemogenetics methods were used to explore whether specific neurons were involved in visceral pain induced by pancreatic cancer. The PRV injected into the pancreas was detected in the paraventricular nucleus of the hypothalamus (PVN). Immunofluorescence staining showed that the majority of c-fos were co-labeled with glutamatergic neurons in the PVN. In vitro electrophysiological results showed that the firing frequency of glutamatergic neurons in the PVN was increased. The calcium imaging results showed that the calcium activity of glutamatergic neurons in the PVN was enhanced. Both specific destruction of glutamatergic neurons and chemogenetics inhibition of glutamatergic neurons in the PVN alleviated visceral pain induced by pancreatic cancer. Glutamatergic neurons in the PVN participate in the regulation of visceral pain induced by pancreatic cancer in mice, providing new insights for the discovery of effective targets for the treatment of pancreatic cancer visceral pain.

FAM81A is a postsynaptic protein that regulates the condensation of postsynaptic proteins via liquid-liquid phase separation.

Proteome analyses of the postsynaptic density (PSD), a proteinaceous specialization beneath the postsynaptic membrane of excitatory synapses, have identified several thousands of proteins. While proteins with predictable functions have been well studied, functionally uncharacterized proteins are mostly overlooked. In this study, we conducted a comprehensive meta-analysis of 35 PSD proteome datasets, encompassing a total of 5,869 proteins. Employing a ranking methodology, we identified 97 proteins that remain inadequately characterized. From this selection, we focused our detailed analysis on the highest-ranked protein, FAM81A. FAM81A interacts with PSD proteins, including PSD-95, SynGAP, and NMDA receptors, and promotes liquid-liquid phase separation of those proteins in cultured cells or in vitro. Down-regulation of FAM81A in cultured neurons causes a decrease in the size of PSD-95 puncta and the frequency of neuronal firing. Our findings suggest that FAM81A plays a crucial role in facilitating the interaction and assembly of proteins within the PSD, and its presence is important for maintaining normal synaptic function. Additionally, our methodology underscores the necessity for further characterization of numerous synaptic proteins that still lack comprehensive understanding.

Ultrasound: A new strategy for artificial synapses modulation

AbstractDue to its non‐invasive nature, ultrasound has been widely used for neuromodulation in biological systems, where its application influences the synaptic weights and the process of neurotransmitter delivery. However, such modulation has not been emulated in physical devices. Memristors are ideal electrical components for artificial synapses, but up till now they are hardly reported to respond to ultrasound signals. Here we design and fabricate a HfOx‐based memristor on 64°Y‐X LiNbO3 single crystal substrate, and successfully realize artificial synapses modulation by shear‐horizontal surface acoustic wave (SH‐SAW). It is a prominent short‐term resistance modulation, where ultrasound has been shown to cause resistance drop for various resistance states, which could fully recover after the ultrasound is shut off. The physical mechanism illustrates that ultrasound induced polarization potential in the HfOx dielectric layer acts on the Schottky barrier, leading to the resistance drop. The emulation of neuron firing frequency modulation through ultrasound signals is demonstrated. Moreover, the joint application of ultrasound and electric voltage yields fruitful functionalities, such as the enhancement of resistance window and synaptic plasticity through ultrasound application. All these promising results provide a new strategy for artificial synapses modulation, and also further advance neuromorphic devices toward system applications.image

Cognitive performance in aged rats is associated with differences in distinctive neuronal populations in the ventral tegmental area and altered synaptic plasticity in the hippocampus.

Deterioration of cognitive functions is commonly associated with aging, although there is wide variation in the onset and manifestation. Albeit heterogeneity in age-related cognitive decline has been studied at the cellular and molecular level, there is poor evidence for electrophysiological correlates. The aim of the current study was to address the electrophysiological basis of heterogeneity of cognitive functions in cognitively Inferior and Superior old (19-20 months) rats in the ventral tegmental area (VTA) and the hippocampus, having Young (12 weeks) rats as a control. The midbrain VTA operates as a hub amidst affective and cognitive facets, processing sensory inputs related to motivated behaviours and hippocampal memory. Increasing evidence shows direct dopaminergic and non-dopaminergic input from the VTA to the hippocampus. Aged Superior and Inferior male rats were selected from a cohort of 88 animals based on their performance in a spatial learning and memory task. Using in vivo single-cell recording in the VTA, we examined the electrical activity of different neuronal populations (putative dopaminergic, glutamatergic and GABAergic neurons). In the same animals, basal synaptic transmission and synaptic plasticity were examined in hippocampal slices. Electrophysiological recordings from the VTA and hippocampus showed alterations associated with aging per se, together with differences specifically linked to the cognitive status of aged animals. In particular, the bursting activity of dopamine neurons was lower, while the firing frequency of glutamatergic neurons was higher in VTA of Inferior old rats. The response to high-frequency stimulation in hippocampal slices also discriminated between Superior and Inferior aged animals. This study provides new insight into electrophysiological information underlying compromised cerebral ageing. Further understanding of brain senescence, possibly related to neurocognitive decline, will help develop new strategies towards the preservation of a high quality of life.

Neuroprotective effects of levothyroxine on cognition deficits and memory in an experimental model of Huntington's disease in rats: An electrophysiological study.

Huntington's disease (HD) is a neurodegenerative disorder characterized by cognitive deficits and motor function. Levothyroxine (L-T4) is a synthetic form of Thyroxine (T4), which can improve cognitive ability. The aim of the present study was to determine the neuroprotective effect of L-T4 administration in rats with 3-nitropropionic acid (3-NP)-induced Huntington's disease. Forty-eight Wistar male rats were divided into six groups (n = 8): Group 1 control group that received physiological saline, Group 2 and 3: which received L-T4 (30 and 100μg/kg), Group 4: HD group that received 3-NP and Groups 5 and 6: The treatment of the HD rats with L-T4 (30 and 100μg/kg). Spatial memory, locomotor activity, and frequency of neuronal firing were assessed. After decapitation, the Brain-Derived Neurotrophic Factor (BDNF) and Total antioxidant capacity (TAC) levels in the striatum was measured. The results showed that the indices of spatial memory (mean path length and latency time) and motor dysfunction (immobility time) significantly increased, while time spent in the goal quadrant, swimming speed, spike rate, and striatum levels of BDNF significantly decreased in the HD group compared to the control group. L-T4 treatment significantly enhanced time spent in the goal quadrant, swimming speed, motor activity (number of line crossing and rearing), spike rate and striatal BDNF level. This research showed that L-T4 prevented the disruption of motor activity and cognitive deficiencies induced by 3-NP. The beneficial effects of L-T4 may be due to an increase in the concentration of BDNF and enhancement of the spike rate in the striatum.

Inhibition of GABAergic neurons in the paraventricular nucleus of the hypothalamus precipitates visceral pain induced by pancreatic cancer in mice.

For patients with pancreatic cancer, visceral pain is a debilitating symptom that significantly compromises their quality of life. Unfortunately, the lack of effective treatment options can be attributed to our limited understanding of the neural circuitry underlying this phenomenon. The primary objective of this study is to elucidate the fundamental mechanisms governing visceral pain induced by pancreatic cancer in murine models. A mouse model of pancreatic cancer visceral pain was established in C57BL/6N mice through the intrapancreatic injection of mPAKPC-luc cells. Abdominal mechanical hyperalgesia and hunch score were employed to evaluate visceral pain, whereas the in vitro electrophysiological patch-clamp technique was utilized to record the electrophysiological activity of GABAergic neurons. Specific neuron ablation and chemogenetics methods were employed to investigate the involvement of GABAergic neurons in pancreatic cancer-induced visceral pain. In vitro electrophysiological results showed that the firing frequency of GABAergic neurons in the paraventricular nucleus of the hypothalamus (PVN) was decreased. Specific destruction of GABAergic neurons in the PVN exacerbated visceral pain induced by pancreatic cancer. Chemogenetics activation of GABAergic neurons in the PVN alleviated visceral pain induced by pancreatic cancer. GABAergic neurons located in PVN play a crucial role in precipitating visceral pain induced by pancreatic cancer in mice, thereby offering novel insights for identifying effective targets to treat pancreatic cancer-related visceral pain.

Elevated Heme Drives Evoked and Ongoing Pain Behaviors in WT Mice By Sensitization of Dorsal Root Ganglia Neurons

Individuals with sickle cell disease (SCD) suffer from complex stimulus-evoked and ongoing pain that is mediated in part by the hyperexcitability of their peripheral sensory neurons. However, it is unclear whether acute or chronic elevation of cell free heme, a key pathological feature of SCD, contributes to the aberrant activity of sensory neurons. Thus, determining whether heme drives pain behavior and enhanced excitability of dorsal root ganglia (DRG) primary sensory neurons involved in pain signal transduction is critical for the development of targeted pain therapies for SCD. In the current experiments, we use evoked and ongoing pain behavior assays and in vitro calcium imaging to evaluate the hypothesis that acute increases in heme causes direct sensitization of nociceptive DRG neurons in wildtype (WT) mice. Our preliminary data reveal that similar to pain behaviors in the mouse model of SCD, hind paw injection of heme into WT mice induces mechanical hypersensitivity (Figure 1). We also observed increased aversive responses to dynamic paintbrush and noxious needle stimulation as well as the onset of paw licking behavior suggesting that heme also drives ongoing, non-evoked pain. These data suggest that axon terminals in the skin become rapidly sensitized when exposed to elevated heme. We next administered heme to primary cultured DRG neurons in vitro and observed calcium flux across the neuronal cell membrane that was dependent on extracellular calcium. To determine if heme directly triggers action potentials or enhances spontaneous activity of sensory neurons, we conducted electrophysiological recordings of neuronal activity in neurons held at resting membrane potential at physiological temperature. Our preliminary data indicate that exposure to heme may directly enhance neuronal action potential firing frequency (Figure 2). Together, these data suggest that heme opens calcium channels on sensory neurons to initiate pain signal transduction and drive acute pain behaviors. We plan to conduct additional non-evoked pain behavior assays including facial grimace analysis and conditioned place aversion to determine whether heme injection is sufficient to drive ongoing pain. We will further determine whether exposure to heme enhances the intrinsic excitability of mechanical and cold sensitive neurons as heme-dependent hyperactivity of these neurons may lead to our observed acute onset mechanical and cold hypersensitivity in heme-injected mice. Completion of these experiments will shed light on the contributions of cell free heme to SCD pain and illuminate downstream effectors that may be targeted to provide analgesic relief for this historically understudied and debilitating disease.

Local Administration of the Phytochemical, Quercetin, Attenuates the Hyperexcitability of Rat Nociceptive Primary Sensory Neurons Following Inflammation Comparable to lidocaine

Although in vivo local injection of quercetin into the peripheral receptive field suppresses the excitability of rat nociceptive trigeminal ganglion (TG) neurons, under inflammatory conditions, the acute effects of quercetin in vivo, particularly on nociceptive TG neurons, remain to be determined. The aim of this study was to examine whether acute local administration of quercetin into inflamed tissue attenuates the excitability of nociceptive TG neurons in response to mechanical stimulation. The mechanical escape threshold was significantly lower in complete Freund’s adjuvant (CFA)-inflamed rats compared to before CFA injection. Extracellular single-unit recordings were made from TG neurons of CFA-induced inflammation in anesthetized rats in response to orofacial mechanical stimulation. The mean firing frequency of TG neurons in response to both non-noxious and noxious mechanical stimuli was reversibly inhibited by quercetin in a dose-dependent manner (1–10 mM). The mean firing frequency of inflamed TG neurons in response to mechanical stimuli was reversibly inhibited by the local anesthetic, 1% lidocaine (37 mM). The mean magnitude of inhibition on TG neuronal discharge frequency with 1 mM quercetin was significantly greater than that of 1% lidocaine. These results suggest that local injection of quercetin into inflamed tissue suppresses the excitability of nociceptive primary sensory TG neurons. PerspectiveLocal administration of the phytochemical, quercetin, into inflamed tissues is a more potent local analgesic than voltage-gated sodium channel blockers as it inhibits the generation of both generator potentials and action potentials in nociceptive primary nerve terminals. As such, it contributes to the area of complementary and alternative medicines.

Potentiated GABAergic neuronal activities in the basolateral amygdala alleviate stress-induced depressive behaviors.

Major depressive disorder is a severe psychiatric disorder that afflicts ~17% of the world population. Neuroimaging investigations of depressed patients have consistently reported the dysfunction of the basolateral amygdala in the pathophysiology of depression. However, how the BLA and related circuits are implicated in the pathogenesis of depression is poorly understood. Here, we combined fiber photometry, immediate early gene expression (c-fos), optogenetics, chemogenetics, behavioral analysis, and viral tracing techniques to provide multiple lines of evidence of how the BLA neurons mediate depressive-like behavior. We demonstrated that the aversive stimuli elevated the neuronal activity of the excitatory BLA neurons (BLACAMKII neurons). Optogenetic activation of CAMKII neurons facilitates the induction of depressive-like behavior while inhibition of these neurons alleviates the depressive-like behavior. Next, we found that the chemogenetic inhibition of GABAergic neurons in the BLA (BLAGABA ) increased the firing frequency of CAMKII neurons and mediates the depressive-like phenotypes. Finally, through fiber photometry recording and chemogenetic manipulation, we proved that the activation of BLAGABA neurons inhibits BLACAMKII neuronal activity and alleviates depressive-like behavior in the mice. Thus, through evaluating BLAGABA and BLACAMKII neurons by distinct interaction, the BLA regulates depressive-like behavior.

Discovery and Characterization of Novel CNS-Penetrant GPR55 Agonists.

From our NETSseq-derived human brain transcriptomics data, we identified GPR55 as a potential molecular target for the treatment of motor symptoms in patients with Parkinson's disease. From a high-throughput screen, we identified and optimized agonists with nanomolar potency against both human and rat GPR55. We discovered compounds with either strong or limited β-arrestin signaling and receptor desensitization, indicating biased signaling. A compound that showed minimal GPR55 desensitization demonstrated a reduction in firing frequency of medium spiny neurons cultured from rat striatum but did not reverse motor deficits in a rat hypolocomotion model. Further profiling of several desensitizing and non-desensitizing lead compounds showed that they are selective over related cannabinoid receptors CB1 and CB2 and that unbound brain concentrations well above the respective GPR55 EC50 can be readily achieved following oral administration. The novel brain-penetrant GPR55 agonists disclosed can be used to probe the role of this receptor in the brain.

Discovery of CVN417, a Novel Brain-Penetrant α6-Containing Nicotinic Receptor Antagonist for the Modulation of Motor Dysfunction.

Nicotinic acetylcholine receptor (nAChR) α6 subunit RNA expression is relatively restricted to midbrain regions and is located presynaptically on dopaminergic neurons projecting to the striatum. This subunit modulates dopamine neurotransmission and may have therapeutic potential in movement disorders. We aimed to develop potent and selective α6-containing nAChR antagonists to explore modulation of dopamine release and regulation of motor function in vivo. High-throughput screening (HTS) identified novel α6-containing nAChR antagonists and led to the development of CVN417. This molecule blocks α6-containing nAChR activity in recombinant cells and reduces firing frequency of noradrenergic neurons in the rodent locus coeruleus. CVN417 modulated phasic dopaminergic neurotransmission in an impulse-dependent manner. In a rodent model of resting tremor, CVN417 attenuated this behavioral phenotype. These data suggest that selective antagonism of α6-containing nAChR, with molecules such as CVN417, may have therapeutic utility in treating the movement dysfunctions observed in conditions such as Parkinson's disease.