vlPAG Neurons Research Articles

Alamandine injection in the periaqueductal gray and rostral ventromedial medulla attenuates allodynia induced by sciatic nerve ligation in rats

Alamandine, a peptide known to interact with Mas-related G protein-coupled receptor subtype D (MrgD), has been implicated in moderating inflammatory signals. MrgD receptors are abundantly found in pain transmission pathways, but the role of alamandine/MrgD in pain modulation has not been thoroughly explored. This study aimed to investigate the effects of alamandine (10, 40, and 100 pmol) in a rat model of allodynia induced by sciatic nerve ligation, with a specific focus on examining the involvement of MrgD receptors, NMDAR1, and serotonin transporter (SERT) in the ventrolateral periaqueductal gray (vlPAG) and rostral ventromedial medulla (RVM).Microinjection of alamandine into the vlPAG at a dose of 100 pmol and into the RVM at doses of 40 and 100 pmol resulted in a significant increase in paw withdrawal threshold (PWT). Additionally, co-administration of D-Pro7-Ang-(1–7) at 50 pmol, an MrgD receptor antagonist, effectively blocked the analgesic effects of alamandine.Immunofluorescence analysis confirmed the presence of MrgD receptors in both the vlPAG and RVM regions. Importantly, an upregulation of MrgD receptor expression was observed following allodynia induction, suggesting a potential compensatory mechanism in response to pain. Our findings support the co-localization of MrgD receptors with NMDAR1 in vlPAG neurons, suggesting their ability to initiate analgesic pathways similar to those activated by NMDA receptors in the vlPAG. Furthermore, our results underscore a significant co-localization of MrgD receptors with the SERT in the RVM, underscoring their potential impact on serotonergic neurons involved in promoting analgesic effects.

Bed Nucleus of the Stria Terminalis (BNST) neurons containing the serotonin 5HT2c receptor modulate operant alcohol self-administration behavior in mice.

The serotonin 5HT2c receptor has been widely implicated in the pathophysiology of alcohol use disorder (AUD), particularly alcohol seeking and the affective consequences of chronic alcohol consumption. However, little is known about the brain sites in which 5HT2c exerts its effects on specific alcohol-related behaviors, especially in females. Here, we investigated the effects of site-specific manipulation of the 5HT2c receptor system in the BNST on operant alcohol self-administration behaviors in adult mice of both sexes, including the acquisition and maintenance of fixed-ratio responding, motivation for alcohol (progressive ratio), and quinine-adulterated responding for alcohol on a fixed-ratio schedule (punished alcohol seeking). Knockdown of 5HT2c in the BNST did not affect the acquisition or maintenance of operant alcohol self-administration, nor did it affect progressive ratio responding for alcohol. This manipulation had only a subtle effect on responding for quinine alcohol selectively in females. On the other hand, chemogenetic inhibition of BNST 5HT2c-containing neurons (BNST5HT2c) increased operant alcohol self-administration behavior in both sexes on day 2, but not day 9, of testing. It also increased operant responding for 1000 μM quinine-adulterated alcohol selectively in males. Importantly, chemogenetic inhibition of BNST5HT2c did not alter operant sucrose responding or motivation for sucrose in either sex. We then performed cell-type specific anterograde tracing, which revealed that BNST5HT2c project to similar regions in males and females, many of which have been previously implicated in AUD. We next used chemogenetics and quantification of the immediate early gene cFos to characterize the functional influence of BNST5HT2c inhibition on vlPAG activity. We show that chemogenetic inhibition of BNST5HT2c reduces vlPAG cFos in both sexes, but that this reduction is more robust in males. Together these findings suggest that BNST5HT2c neurons, and to a small extent the BNST 5HT2c receptor, serve to promote aversive responses to alcohol consumption, potentially through sex-dependent disinhibition of vlPAG neurons.

BTRX-246040 Acts Through the Ventrolateral Periaqueductal Gray to Exert Antidepressant-Relevant Actions in Mice.

BTRX-246040, a nociceptin/orphanin FQ peptide receptor antagonist, is being developed for the treatment of depressive patients. However, the underlying mechanism of this potential antidepressant is still largely unclear. Here, we studied the antidepressant-related actions of BTRX-246040 in the ventrolateral periaqueductal gray (vlPAG). The tail suspension test, forced swim test, female urine sniffing test, sucrose preference test, and learned helplessness (LH) combined with pharmacological approaches were employed to examine the antidepressant-like effects and drug effects on LH-induced depressive-like behavior in C57BL/6J mice. Electrophysiological recordings in vlPAG neurons were used to study synaptic activity. Intraperitoneal administration of BTRX-246040 produced antidepressant-like behavioral effects in a dose-dependent manner. Systemic BTRX-246040 (10 mg/kg) resulted in an increased frequency and amplitude of miniature excitatory postsynaptic currents (EPSCs) in the vlPAG. Moreover, slice perfusion of BTRX-246040 directly elevated the frequency and amplitude of miniature EPSCs and enhanced the evoked EPSCs in the vlPAG, which were blocked by pretreatment with the nociceptin/orphanin FQ peptide receptor agonist Ro 64-6198. In addition, intra-vlPAG application of BTRX-246040 produced antidepressant-like behavioral effects in a dose-dependent manner. Moreover, intra-vlPAG pretreatment with 6-cyano-7-nitroquinoxaline-2,3-dione reversed both systemic and local BTRX-246040-mediated antidepressant-like behavioral effects. Furthermore, both systemic and local BTRX-246040 decreased the LH phenotype and reduced LH-induced depressive-like behavior. The results suggested that BTRX-246040 may act through the vlPAG to exert antidepressant-relevant actions. The present study provides new insight into a vlPAG-dependent mechanism underlying the antidepressant-like actions of BTRX-246040.

Persistent inflammation selectively activates opioid-sensitive phasic-firing neurons within the vlPAG.

The ventrolateral periaqueductal gray (vlPAG) is a key brain area within the descending pain modulatory pathway and an important target for opioid-induced analgesia. The vlPAG contains heterogeneous neurons with respect to neurotransmitter content, receptor and channel expression, and in vivo response to noxious stimuli. This study characterizes intrinsic membrane properties of vlPAG neurons to identify neuron types that respond to inflammation and determine whether the pain-responsive neurons are inhibited by opioids. Surveying 382 neurons identified four neuron types with distinct intrinsic firing patterns: Phasic (48%), Tonic (33%), Onset (10%), and Random (9%). Mu-opioid receptor (MOR) expression was determined by the ability of a selective MOR agonist (DAMGO) to activate G protein-coupled inwardly rectifying potassium channel (GIRK) currents. Opioid-sensitive neurons were observed within each neuron type. Opioid sensitivity did not correlate with other intrinsic firing features, including low-threshold spiking that has been previously proposed to identify opioid-sensitive GABAergic neurons in the vlPAG of mice. Complete Freund's adjuvant (CFA)-induced acute inflammation (2 h) had no effect on vlPAG neuron firing patterns. However, persistent inflammation (5-7 days) selectively activated Phasic neurons through a significant reduction in their firing threshold. Opioid-sensitive neurons were strongly activated compared with the opioid-insensitive Phasic neurons. Overall, this study provides a framework to further identify neurons activated by persistent inflammation so that they may be targeted for future pain therapies.NEW & NOTEWORTHY Intrinsic firing properties define four distinct vlPAG neuron populations, and a subset of each population expresses MORs coupled to GIRK channels. Persistent, but not acute, inflammation selectively activates opioid-sensitive Phasic vlPAG neurons. Although the vlPAG is known to contribute to the descending inhibition of pain, the activation of a single physiologically defined neuron type in the presence of persistent inflammation represents a mechanism by which the vlPAG participates in descending facilitation of pain.

An analgesic pathway from parvocellular oxytocin neurons to the periaqueductal gray in rats

The hypothalamic neuropeptide oxytocin (OT) exerts prominent analgesic effects via central and peripheral action. However, the precise analgesic pathways recruited by OT are largely elusive. Here we discovered a subset of OT neurons whose projections preferentially terminate on OT receptor (OTR)-expressing neurons in the ventrolateral periaqueductal gray (vlPAG). Using a newly generated line of transgenic rats (OTR-IRES-Cre), we determined that most of the vlPAG OTR expressing cells targeted by OT projections are GABAergic. Ex vivo stimulation of parvocellular OT axons in the vlPAG induced local OT release, as measured with OT sensor GRAB. In vivo, optogenetically-evoked axonal OT release in the vlPAG of as well as chemogenetic activation of OTR vlPAG neurons resulted in a long-lasting increase of vlPAG neuronal activity. This lead to an indirect suppression of sensory neuron activity in the spinal cord and strong analgesia in both female and male rats. Altogether, we describe an OT-vlPAG-spinal cord circuit that is critical for analgesia in both inflammatory and neuropathic pain models.

Activation of HCN channels caused by elevated cAMP levels in periaqueductal gray promotes bone cancer pain

The periaqueductal gray (PAG) is an important relay center for the descending pathways that regulate nociceptive information transduction. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play critical roles in the nerve injury-induced pain hypersensitivity. Previous studies have identified that HCN1 and HCN2 channel protein located in the ventral-lateral periaqueductal gray (vlPAG), a region important for pain regulation. However, it is not clear whether the HCN channel in vlPAG is involved in bone cancer pain (BCP). In this study, we assessed the role of HCN channels in BCP by measuring changes of HCN channel expression and activity in vlPAG neurons in bone cancer rats. In the present study, the BCP model was established by injecting SHZ-88 breast cancer cells into the right tibia bone marrow in rats. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured to evaluate pain behavior in rats. HCN1 and HCN2 channels expression in vlPAG were detected by using Western Blot and immunohistochemistry. In addition, the cAMP level in vlPAG neurons was detected by ELISA, and HCN channel current (Ih) of vlPAG neurons was recorded by whole cell patch-clamp to evaluate HCN channel activity. As a result, decreased MWT and TWL were observed in rats on 7d after SHZ-88 cell inoculation, and the allodynia was sustained until 21d after inoculation. At the same time, HCN1 and HCN2 channels expression and neuronal Ih in vlPAG were significantly increased in BCP rats. In addition, the level of cAMP in vlPAG also increased after SHZ-88 cell inoculation. Furthermore, intravlPAG injection of ZD7288 (HCN channels antagonist) could significantly reduce hyperalgesia and the elevation of cAMP in vlPAG in BCP rats. Our observations suggest that the elevation of cAMP may promote the activation of HCN channels in vlPAG in bone cancer rats, thereby promoting the development of bone cancer pain.

Supraspinal melatonin MT2 receptor agonism alleviates pain via a neural circuit that recruits mu opioid receptors.

Melatonin, through its G protein-coupled receptor (GPCR) (MTNR1B gene)MT2 , is implicated in analgesia, but the relationship between MT2 receptors and the opioid system remains elusive. In a model of rodent neuropathic pain (spared nerve injured [SNI]), the selective melatonin MT2 agonist UCM924 reversed the allodynia (a pain response to a non-noxious stimulus), and this effect was nullified by the pharmacological blockade or genetic inactivation of the mu opioid receptor (MOR), but not the delta opioid receptor (DOR). Indeed, SNI MOR, but not DOR knockout mice, did not respond to the antiallodynic effects of the UCM924. Similarly, the nonselective opioid antagonist naloxone and the selective MOR antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) blocked the effects of UCM924 in SNI rats, but not the DOR antagonist naltrindole (NTI). Electrophysiological recordings in the rostral-ventromedial medulla (RVM) revealed that the typical reduction of the firing activity of pronociceptive ON-cells, and the enhancement of the firing of the antinociceptive OFF-cells, induced by the microinjection of the MT2 agonist UCM924 into the ventrolateral periaqueductal gray (vlPAG) were blocked by MOR, but not DOR, antagonism. Immunohistochemistry studies showed that MT2 receptors are expressed in both excitatory (CaMKIIα+ ) and inhibitory (GAD65+ ) neuronal cell bodies in the vlPAG (~2.16% total), but not RVM. Only 0.20% of vlPAG neurons coexpressed MOR and MT2 receptors. Finally, UCM924 treatment induced an increase in the enkephalin precursor gene (PENK) in the PAG of SNI mice. Collectively, the melatonin MT2 receptor agonism requires MORs to exert its antiallodynic effects, mostly through an interneuronal circuit involving MOR and MT2 receptors.

Cellular and circuit diversity determines the impact of endogenous opioids in the descending pain modulatory pathway.

The descending pain modulatory pathway exerts important bidirectional control of nociceptive inputs to dampen and/or facilitate the perception of pain. The ventrolateral periaqueductal gray (vlPAG) integrates inputs from many regions associated with the processing of nociceptive, cognitive, and affective components of pain perception, and is a key brain area for opioid action. Opioid receptors are expressed on a subset of vlPAG neurons, as well as on both GABAergic and glutamatergic presynaptic terminals that impinge on vlPAG neurons. Microinjection of opioids into the vlPAG produces analgesia and microinjection of the opioid receptor antagonist naloxone blocks stimulation-mediated analgesia, highlighting the role of endogenous opioid release within this region in the modulation of nociception. Endogenous opioid effects within the vlPAG are complex and likely dependent on specific neuronal circuits activated by acute and chronic pain stimuli. This review is focused on the cellular heterogeneity within vlPAG circuits and highlights gaps in our understanding of endogenous opioid regulation of the descending pain modulatory circuits.

Examination of the periaqueductal gray as a site for controlling arterial pressure in the conscious spontaneously hypertensive rat.

Our understanding of central nervous system regulation of the set-point of arterial pressure remains incomplete, especially in conditions of hypertension. The ventrolateral periaqueductal gray (vlPAG) is of particular interest given that its acute activation induces hypotension and sympatho-inhibition in anaesthetised, normotensive animals, and recent preliminary studies have shown that vlPAG stimulation can reduce blood pressure in refractory hypertensive patients. To assist our mechanistic understanding, we investigated whether electrical stimulation of the vlPAG had depressor actions in a model of neurogenic hypertension, the spontaneously hypertensive (SH) rat. We found that electrical stimulation of the lateral and vlPAG (2-6V, 20-40Hz, 0.18-0.2ms pulse width) decreased arterial pressure (-19±4mmHg, n=8) and heart rate (median-18bpm) in anaesthetised SH rats. In contrast, in conscious freely-moving SH rats fitted with blood pressure telemetry, stimulation of this same region produced failed to evoked a hypotensive response (n=13; either no change, n=9; or an increase in arterial pressure of 23±4mmHg, n=4). The hypotensive action of the vlPAG observed in anaesthetised animals has been attributed to inhibition of pre-sympathetic neurones originating in the rostral ventrolateral medulla. We therefore used an un-anaesthetised, decerebrate SH rat preparation to investigate whether activation of vlPAG neurons produced sympatho-inhibition that might be below the threshold at which a peripheral vascular response could be observed. Only sympatho-excitatory responses to electrical and excitatory amino acid microinjections were observed, and these were evoked from both the dorsal and ventral PAG; no responses were evoked from the vlPAG. We conclude that the vlPAG is not a reliable antihypertensive locus in the awake SH rat. We discuss the potential importance of the state-dependency of the hypotensive response that can be evoked from the vlPAG, which has important implications for translating to humans.

Periaqueductal gray is required for controlling chronic stress-induced depression-like behavior

BackgroundEnduring exposure to psychological stress is associated with an elevated risk of major depressive disorder (MDD). There is an enormous need to investigate the unexplored mechanisms of MDD. We examined whether pain-free stress alters synaptic transmission, causing depression-like behaviors in the ventrolateral periaqueductal gray (vlPAG), a brain stem nucleus that controls stress-related depression-like behavior. MethodsIn the current study, we studied neuronal changes in the vlPAG and behavioral transforms using electrophysiological recordings, behavioral tests, and pharmacological approaches. ResultsWe found that chronic restraint stress (CRS) diminished glutamatergic transmission in the vlPAG, leading to maladaptive behavioral despair and anhedonia in mice demonstrated by the forced swimming test (FST), tail suspension test (TST) and female urine sniffing test (FUST). Moreover, CRS increased behavioral hypersensitivity shown by the von Frey test. Bath perfusion with the rapid-acting antidepressant (2R,6R)-hydroxynorketamine (HNK) increased both the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) in vlPAG neurons in the CRS and control groups. Functionally, (2R,6R)-HNK directly enhanced the action potential firing rate in vlPAG neurons. Behaviorally, intravlPAG microinjection of (2R,6R)-HNK alleviated chronic restraint stress-induced depression-like behaviors and behavioral hypersensitivity. ConclusionsThese results demonstrate that psychological stress-elicited depression-like behavior is related to a remarkable decrease in glutamatergic transmission in the vlPAG. The maladaptive behaviors are attributed to hypoactivity of glutamatergic neurons in the vlPAG, and direct enhancement of glutamatergic neuronal activity in the vlPAG rescues depression-like behaviors. The present results prove that vlPAG is critical for controlling stress-induced depression-like behaviors through alteration of glutamatergic transmission.

Activation of CamKII\u03b1 expressing neurons on ventrolateral periaqueductal gray improves behavioral hypersensitivity and thalamic discharge in a trigeminal neuralgia rat model

BackgroundPreceding studies have reported the association of chronic neuropathic orofacial pain with altered ongoing function in the ventrolateral periaqueductal gray (vlPAG). However, its role in trigeminal neuralgia (TN) lacks attention. We here reported the aspect that vlPAG neurons play in TN nociceptive processing by employing excitatory neuron-specific optogenetic approaches.MethodsTN was generated via unilateral infraorbital nerve chronic constriction in Sprague Dawley rats which induced mechanical and thermal pain sensitivity in air puff and acetone test, respectively. Channelrhodopsin conjugated virus with CamKIIα promoter was used to specifically activate the excitatory vlPAG neuronal population by optogenetic stimulation and in vivo microdialysis was done to determine its effect on the excitatory-inhibitory balance. In vivo extracellular recordings from ventral posteromedial (VPM) thalamus were assessed in response to vlPAG optogenetic stimulation. Depending on the experimental terms, unpaired student’s t test and two-way analysis of variance (ANOVA) were used for statistical analysis.ResultsWe observed that optogenetic activation of vlPAG subgroup neurons markedly improved pain hypersensitivity in reflexive behavior tests which was also evident on microdialysis analysis with increase glutamate concentration during stimulation period. Decreased mean firing and burst rates were evident in VPM thalamic electrophysiological recordings during the stimulation period. Overall, our results suggest the optogenetic activation of vlPAG excitatory neurons in a TN rat model has pain ameliorating effect.ConclusionsThis article presents the prospect of pain modulation in trigeminal pain pathway via optogenetic activation of vlPAG excitatory neurons in rat model. This outlook could potentially assist vlPAG insight and its optogenetic approach in trigeminal neuropathic pain which aid clinicians endeavoring towards enhanced pain relief therapy in trigeminal neuralgia patients.

Protein Kinase‐C Inhibition Within the Periaqueductal Gray Counteracts the Development of Morphine Antinociceptive Tolerance

The ventrolateral periaqueductal gray (vlPAG) is an important midbrain region that regulates the descending pain modulatory system and is implicated in the development of antinociceptive tolerance to opioid analgesics. Protein Kinase-C (PKC) is a Ser/Thr kinase that phosphorylates the μ-opioid receptor (MOPr) following morphine activation. This phosphorylation is responsible for desensitization of the receptor, resulting in the termination of G-Protein signaling. It is speculated that PKC-mediated desensitization of the MOPr does not readily lead to the internalization of the receptor, thus contributing to tolerance. Previous studies have shown that intracerebroventricular administration of PKC inhibitors prevented the development of morphine tolerance in mice. Further, a non-specific PKC/GRK inhibitor was able to block the development of morphine tolerance in the vlPAG. However, the contribution of PKC to morphine tolerance in the vlPAG has not been directly explored. Using immunofluorescence staining, we observed increased phosphorylated PKC that is co-localized with the MOPr at the cell membrane of vlPAG neurons in morphine-tolerant animals. Further, we assessed the development of morphine antinociceptive tolerance following pre-treatment with the PKC inhibitor, Gö-7874, which targets conventional PKC isoforms. Repeated microinjections of the compound into the vlPAG of wild-type mice in combination with morphine led to a reduction in the development of morphine tolerance. Interestingly, repeated microinjections of Gö-7874 alone led to an enhancement of subsequent morphine antinociception. Lastly, morphine-induced changes in intracellular cAMP levels were assessed in-vitro, following pre-treatment with Gö-7874. This study contributes to the understanding of PKC-mediated MOPr desensitization processes leading to morphine tolerance.

Bidirectional control of fear memories by cerebellar neurons projecting to the ventrolateral periaqueductal grey

Fear conditioning is a form of associative learning that is known to involve different brain areas, notably the amygdala, the prefrontal cortex and the periaqueductal grey (PAG). Here, we describe the functional role of pathways that link the cerebellum with the fear network. We found that the cerebellar fastigial nucleus (FN) sends glutamatergic projections to vlPAG that synapse onto glutamatergic and GABAergic vlPAG neurons. Chemogenetic and optogenetic manipulations revealed that the FN-vlPAG pathway controls bi-directionally the strength of the fear memories, indicating an important role in the association of the conditioned and unconditioned stimuli, a function consistent with vlPAG encoding of fear prediction error. Moreover, FN-vlPAG projections also modulate extinction learning. We also found a FN-parafascicular thalamus pathway, which may relay cerebellar influence to the amygdala and modulates anxiety behaviors. Overall, our results reveal multiple contributions of the cerebellum to the emotional system.

Positive allosteric modulation of the cannabinoid type-1 receptor (CB1R) in periaqueductal gray (PAG) antagonizes anti-nociceptive and cellular effects of a mu-opioid receptor agonist in morphine-withdrawn rats.

Opioid drugs are a first-line treatment for severe acute pain and other chronic pain conditions, but long-term opioid drug use produces opioid-induced hyperalgesia (OIH). Co-administration of cannabinoids with opioid receptor agonists produce anti-nociceptive synergy, but cannabinoid receptor agonists may also produce undesirable side effects. Therefore, positive allosteric modulators (PAM) of cannabinoid type-1 receptors (CB1R) may provide an option reducing pain and/or enhancing the anti-hyperalgesic effects of opioids without the side effects, tolerance, and dependence observed with the use of ligands that target the orthosteric binding sites. This study tested GAT211, a PAM of cannabinoid type-1 receptors (CB1R), for its ability to enhance the anti-hyperalgesic effects of the mu-opioid receptor (MOR) agonist DAMGO in rats treated chronically with morphine (or saline) and tested during withdrawal. We tested the effects of intra-periaqueductal gray (PAG) injections of (1) DAMGO, (2) GAT211, or (3) DAMGO + GAT211 on thermal nociception in chronic morphine-treated rats that were hyperalgesic and also in saline-treated control rats. We used slice electrophysiology to test the effects of DAMGO/GAT211 bath application on synaptic transmission in the vlPAG. Intra-PAG DAMGO infusions dose-dependently reversed chronic morphine-induced hyperalgesia, but intra-PAG GAT211 did not alter nociception at the doses we tested. When co-administered into the PAG, GAT211 antagonized the anti-nociceptive effects of DAMGO in morphine-withdrawn rats. DAMGO suppressed synaptic inhibition in the vlPAG of brain slices taken from saline- and morphine-treated rats, and GAT211 attenuated DAMGO-induced suppression of synaptic inhibition in vlPAG neurons via actions at CB1R. These findings show that positive allosteric modulation of CB1R antagonizes the behavioral and cellular effects of a MOR agonist in the PAG of rats.

NMDA receptor partial agonist GLYX-13 alleviates chronic stress-induced depression-like behavior through enhancement of AMPA receptor function in the periaqueductal gray

Depression is a common mental disorder affecting more than 300 million people worldwide and is one of the leading causes of disability among all medical illnesses. The accumulation of preclinical data has fueled the revival of interest in targeting glutamatergic neurotransmission for the treatment of major depressive disorder. GLYX-13, a glutamatergic compound that acts as an N-methyl-d-aspartate (NMDA) modulator with glycine-site partial agonist properties, produces rapid and long-lasting antidepressant effects in both animal models and patients. However, the mechanisms underlying the antidepressant actions of GLYX-13 have not been fully characterized, especially in the midbrain ventrolateral periaqueductal gray (vlPAG), a brain stem area that controls stress-associated depression-like behavior. Here, we use a combination of electrophysiological recordings, behavioral tests, and pharmacological manipulations to study the antidepressant actions of GLYX-13 in the vlPAG. A single intravenous injection of a GLYX-13 rapidly mitigated footshock stress (FS)-induced depression-like behavior in rats. The FS-induced diminished glutamatergic transmission in the vlPAG was also reversed by a single GLYX-13 intravenous injection. Moreover, intra-vlPAG GLYX-13 microinjection produced a long-lasting antidepressant effect; however, this effect was prevented by the intra-vlPAG microinjection of tropomyosin-related kinase B (TrkB) receptor antagonist ANA-12, a selective mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin, and CNQX, an AMPA receptor antagonist. Additionally, a bath application of GLYX-13 enhanced glutamatergic transmission in vlPAG neurons; however, this enhancement effect was blocked by the co-application of ANA-12 and rapamycin. These results demonstrate that BDNF-TrkB-mTORC1 signaling in the vlPAG is required for the sustained antidepressant effects of GLYX-13.

0005 Cataplexy Triggered by Social Cues: A Role for Oxytocin in the Amygdala

Abstract Introduction People with narcolepsy type 1 report that cataplexy is triggered most often by positive social experiences such as laughing with friends, yet the mechanisms through which social interaction promotes cataplexy are unknown. We hypothesize a subpopulation of central amygdala neurons that are sensitive to the prosocial neuropeptide, oxytocin (CeAOTR), respond to positive valence and trigger cataplexy. Methods We have used in vivo calcium imaging, chemogenetic and optogenetic approaches to characterize the activity pattern of these neurons and to manipulate their activity state. Results Cre-dependent anterograde tracing of the CeAOTR neurons of the central amygdala indicate a moderate to dense projection to the REM sleep-regulatory region of the ventral lateral periaqueductal gray (vlPAG). Additionally, Channel Rhodopsin Assisted Circuit Mapping (CRACM) experiments show that CeAOTR neurons inhibit vlPAG neurons that innervate the REM atonia-promoting region, the sublaterodorsal nucleus. Targeted photostimulation (15Hz (10ms) for 20sec every hour) of the CeAOTR fibers in the vlPAG doubled the amount of cataplexy. Preliminary in vivo calcium imaging indicates that the CeAOTR are active just prior to the onset of cataplexy. Chemogenetic and optogenetic activation of CeAOTR neurons increased cataplexy. Conclusion We conclude that the CeAOTR subpopulation is sufficient to promote cataplexy. Our future directions include determining the necessity of these oxytocin sensitive neurons in cataplexy under different conditions of positive valence. Support R01 NS106032 and WakeUp Narcolepsy.

Cerebellar modulation of synaptic input to freezing-related neurons in the periaqueductal gray.

Innate defensive behaviors, such as freezing, are adaptive for avoiding predation. Freezing-related midbrain regions project to the cerebellum, which is known to regulate rapid sensorimotor integration, raising the question of cerebellar contributions to freezing. Here, we find that neurons of the mouse medial (fastigial) cerebellar nuclei (mCbN), which fire spontaneously with wide dynamic ranges, send glutamatergic projections to the ventrolateral periaqueductal gray (vlPAG), which contains diverse cell types. In freely moving mice, optogenetically stimulating glutamatergic vlPAG neurons that express Chx10 reliably induces freezing. In vlPAG slices, mCbN terminals excite ~20% of neurons positive for Chx10 or GAD2 and ~70% of dopaminergic TH-positive neurons. Stimulating either mCbN afferents or TH neurons augments IPSCs and suppresses EPSCs in Chx10 neurons by activating postsynaptic D2 receptors. The results suggest that mCbN activity regulates dopaminergic modulation of the vlPAG, favoring inhibition of Chx10 neurons. Suppression of cerebellar output may therefore facilitate freezing.

Cue-inhibited ventrolateral periaqueductal gray neurons signal fear output and threat probability in male rats.

The ventrolateral periaqueductal gray (vlPAG) is proposed to mediate fear responses to imminent danger. Previously we reported that vlPAG neurons showing short-latency increases in firing to a danger cue - the presumed neural substrate for fear output - signal threat probability in male rats (Wright et al., 2019). Here, we scrutinize the activity vlPAG neurons that decrease firing to danger. One cue-inhibited population flipped danger activity from early inhibition to late excitation: a poor neural substrate for fear output, but a better substrate for threat timing. A second population showed differential firing with greatest inhibition to danger, less to uncertainty and no inhibition to safety. The pattern of differential firing reflected the pattern of fear output, and was observed throughout cue presentation. The results reveal an expected vlPAG signal for fear output in an unexpected, cue-inhibited population.

A Midbrain Circuit that Mediates Headache Aversiveness in Rats

SUMMARYMigraines are a major health burden, but treatment is limited because of inadequate understanding of neural mechanisms underlying headache. Imaging studies of migraine patients demonstrate changes in both pain-modulatory circuits and reward-processing regions, but whether these changes contribute to the experience of headache is unknown. Here, we demonstrate a direct connection between the ventrolateral periaqueductal gray (vlPAG) and the ventral tegmental area (VTA) that contributes to headache aversiveness in rats. Many VTA neurons receive monosynaptic input from the vlPAG, and cranial nociceptive input increases Fos expression in VTA-projecting vlPAG neurons. Activation of PAG inputs to the VTA induces avoidance behavior, while inactivation of these projections induces a place preference only in animals with headache. This work identifies a distinct pathway that mediates cranial nociceptive aversiveness.

(254) Functional and Anatomical Characterization of Descending Periaqueductal Gray (PAG) Projections and their Role in Pain Modulation

Endogenous analgesic pathways embody an alternative target for the development for chronic pain therapies. Previous studies have demonstrated the role of the ventrolateral periaqueductal gray (vlPAG) in descending pain modulation. It has been proposed that tonic GABAergic neurotransmission at the level of the vlPAG serves to inhibit efferent excitatory projections that mediate descending analgesia. Disinhibition of these projection neurons allows subsequent activation of rostral ventromedial medulla (RVM) neurons that inhibit nociception at the level of the spinal cord. However, lack of cell-type specificity in these studies has prevented the determination of the role of specific subsets of vlPAG neurons in analgesia. We have utilized recently developed genetic tools to selectively manipulate the activity of subclasses of vlPAG neurons to identify the circuit components critical for analgesia. Our data suggest that increasing the activity of glutamatergic vlPAG neurons or decreasing the activity of GABAergic vlPAG neurons results in increases in sensory thresholds in uninjured animals, while ameliorating hyperalgesia associated with chronic pain states. To further characterize descending pain circuitry at the level of the vlPAG, we decided to focus on efferent projections to the RVM. We hypothesize that these projections are partially responsible for the observed analgesia in our previous study. Population-specific anatomical tracing revealed that most of these projections are glutamatergic and originate from the ventrolateral columns of the PAG. Optogenetic stimulation of these PAG-RVM neurons results in elevation of thermal thresholds in uninjured animals and seems to reverse thermal hyperalgesia associated with inflammatory states. However, this is not the case in a model of neuropathic pain. In brief, our results provide direct experimental evidence supporting the GABA disinhibition hypothesis, highlighting the role of descending glutamatergic neurotransmission at the level of the PAG-RVM as a key component of endogenous analgesic pathways. This work is supported by NINDS F31NS103472 to JGGR.